U, S tt cytt nttr etyetnet

Dr. U. Satyanardyana
M .Sc..P h D .. F.l .C ..F.A ,CB .

Professor of Biochemistry
Siddhartha M edical Colle ge
(NTR University of Health Sciences)
Vijayawada, A.P., India

BOOf(S AND ALLIED (Pl Lto.

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Biotechnologg
FirstPublished: 2005
Beprinted: 2O06,2007,2008,2009
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d 010

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About Cover Design

Depicts the production of a recombinant DNAmotecule,and its utility in modern Biotechnologywith
reference to microorganisms,plants and animals. (Note : This design is an oversimplification
of the facts. For the sake of clarity,only a few helicesof DNAstructure are shown).

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D.No.48-16-10,
 Biotechnology is an exciting,rapidlydevelopingand revolutionary scientificdiscipline,with its roots
in biologicaland technological sciences. Duringthe pastfew years,the majorityof scientificbreakthroughs
in biologicalscienceshave come from biotechnology, particularlyinvolvinggeneticengineering.Bio-
technologyimpingeson everyone'slife, and is truly regardedas the scientifictechnologyof the twenty-
first century.

THE NECESSITY
FOR A COMPREHENSIVE
EOOK ON BIOTECHNOTOGY
Biotechnology is a subjectlearntby students with differentbackgrounds and post-
at the undergraduate
graduatelevels.lt is truethatthereare somebooksnow availableon biotechnology (by Indianand foreign
authors).Thesebooks,however,deal with one or two specializedareasratherthan cdveringthe broad
spectrumof biotechnology. Consequently, the studentshaveto dependon manybooksfor goodand latest
informationon biotechnology. There is a long-feltneed for a comprehensive book on biotechnology
providingupdatedinformationon the subject.

THE BIRTHOF THE NEW BOOK

It will not be out of placeto mentionhere how and when this book was born. The entirebook was
writtenin the early hours(between2AM and 6AM, when the world aroundis fastasleep),duringwhich
periodlcarry out my intellectualactivities.Aftera soundsleep,a freshmind ppckedwith creativeideas
and innovativethoughts,has largelyhelpedme to write this book in a noveland uniqueway.Truly,each
pageof this book was conceivedin darknessand born at day break!

M A I O N HI GH T IG H T S
OF T H E BOOK ' B I OTE C H N OIOGY '
The presentbook 'BIOTECHNOt-OCy',in colour,with severalnovel features,is comprehensively
written with latestadvancesin the subjectto meet the needsof all categoriesof studentslearning
biotechnology.
'The sectionon 'Basicsto learn Biotechnology'providesthe most essentialinformationneededto
understand biotechnology. Thisis particularly
usefulto students
from diversqbackgrounds who areexposed
to biotechnologyall of a sudden.MolecularBiologyis.dealtwith iri good detail,as it laysthe foundations
for geneticengineeringand modernbiotechnology.
The other sectionsin this book include RecombinantDNA Technology,Medical/Pharmaceutical
Biotechnology,
Microbial/lndustrial
Biotechnology,
Animal Cell/AnimalBiotechnology, Plant/Agricultural
Biotechnologyand Environmental Eachsectionis carefullycraftedto provideextensive,
Biotechnology.
relevantand most recentinformation.
The book is written in a lucid stylewith colour illustrations,
headings,sub-headings,tablesand flou,
charts.Coloursare usefulfor betterunderstanding and easy reproducibilityof the subjectmatter.An
extensiveglossaryis addedto the book to makethe commonlyusedtermsand words in biotechnology
very clear.

(tl
 Besidesthe student community, this book will immensely help the personnelworking in biotechnology
laboratoriesand industries.The languageand contentsof this book are made so simple and easy that even
a lay m an wit h a m inim al k nowled g e o f b i o l o g i c a l s c i e n c e sw i l l b e a b l e t o u n d e r s t a n dt h e e s s e n ceo f
biotechnology.
As the subject of biotechnology is multidisciplinaryi books on the subject are written by authors with
different backgrounds-botany, zoology, genetics,pharmacy, agriculture, engineering,etc, This resultsin
an inevitable bias in the books. For instance, an author with a botany background tends to pay more
attention to planVagriculturalbiotechnolgy.The presentbook is free from such bias. Written by an author
with biochemistry background, covering all aspectsof life sciences, it is well balanced in the treatment
of various branches of biotechnology. However, there is a perceptible bias in the book towards recent
information on the applications of different branches of biotechnology to human healthcare,and for the
ultimate benefit of mankind. This is justifiable from the fact that more than TOohof the budget earmarked
for biotechnologl, vst.tt.^ in developing countries goes towards healthcare research.

AN INVITATIONTO READERS
Thisbook is designed
to caterto the needsof all categories
of studentslearningbiotechnology
in various
disciplines-lifesciences,engineeringand technology,medicine,agricultufe,pharmacy,veterinaryetc.
Thus,this book may be regardedas a buffettablewith somethingfor everyone(in everydiscipline).lt is
ultimatelyfor the readerto do appropriateself-service and enjoy the menu.
I invite the readersfor the wonderfuland excitingworld of biotechnology,
and learnthe subjectto
maximumpossiblewith easeand pleasure.
The subjectof biotechnologyis very vast,thereforeit is not an easyjob to preparea concentrated
capsuleof biotechnology. As this book is intendedto caterto the needsof studentslearningbiotechnologv
f r omma n yd i ffe re ndt i s c i p l i n e sth, e r ei s a l ot of scopeto i mprovethe booki n future.Thi spri mari l ydepends
on the feedbackfrom the facultyand studentswith specificrequestsand ideas.
It is truethat I represent a selectedgroupof individualsauthoringbooks,havingsometime at disposal.
besideshard work, determination and dedication.lconsider myselfas an eternallearnerand a regular
studentof biotechnology. However,it is beyondmy capabilityto keeptrackof the evergrowing advances
in biotechnology due to the exponentialgrowthof the subject,arrdthis makesme nervous.I honestlyadmit
that I have to dependon maturereadersfor subsequent editionsof this book.
I welcomeconstructiveand helpfulsuggestions
to improvethe book.

DR. U. SATYANARAYA\,{

(il1
 I owe a deep debt of gratitudeto my parents,the late Sri U. VenkataSubbaiah,and Smt.Vajramma,
for cultivatingin me the habit of early rising.The writing of this book would neverhave been possible
without this healthyhabit.I am gratefulto Dr. B. S. NarasingaRao (formerDirector,NationalInstituteof
Nutrition,Hyderbad)for discipliningmy professional life, and to my eldestbrother,Dr. U. Cudaru(former
S angl i ),for di sci pl i ni ngmy personall i fe
, a l c h a n dC o l l egeof E ngi neeri ng,
P r of es s oorf Po w e rs y s te msW
My youngerson, U. Amrutpani,has made a significantcontributionat everystagein the preparation
of this book-writing, verification,proof-reading,
etc. My elderson,Dr. U. Chakrapani M.B.B.S,M.S.,has
helpedme with his creativeideasand suggestions
constructive to orientthe book with adequateemphasis
on human healthcare,besidesinvolving himself in the verificationand proof-reading of the book. I
acknowledgethe help of my friend, Dr. P. Ramanujam(Readerin English,Andhra Loyola College,
Vijayawada), for his help at variousstagesof manuscriptpreparation.
lexpress my gratitudeto Mr. ArunabhaSen (ExecutiveDirector,Books& Allied Pvt. Ltd., Kolkata)
for his whole-heartedcooperationin bringing out this book to my satisfaction.I am gratefulto
Mr. Shyamal Bhattacharya for the excellentpage-makingand graphics-workin the book. I thank
Mr. DineshBhunyafor proof-reading. I alsothank Mr. PrasenjitHalderfor the cover designof the book.
Last but not least, I thank my wife, KrishnaKumari,for her constantcooperation,supportand
encouragement.
Interlinks,Vijayawada,
lam gratefulto UppalaAuthor-Publisher and supportingme to
for sponsoring
write this book.

DR. U. SATYANARAYANA

(llrI
 ents in Brief

1l Manipulation in
of CeneExpression
HostCells 137
Introduction to Biotechnology 12 HumanCenomeProiect 148

I of Biotechnology3
TheScope

X{e dic al/Pharrnaceu t i cal

Iliotechnolog;'
Introduction to Nlolecular (Biotecluroloqy in llcalthcare)
Biolog]
13 GeneTherapy 157
2 1 1 14 DNA in Disease
andStructure
DNAandRNA-Composition andMedical
Diagnosis
3 DNA-Replication,
Recombination, Forensics 173
andRepair 23 15 Pharmaceutical of DNA
Products
4 andTranslation 38
Transcription Technology 189
5 CeneExpression 59
Regulation'of 16 Recombinant
Vaccines 199
l7 Monoclonal
Antibcdies
(Hybridoma
Technology) 213
18 Assisted Technology 227
Reproductive
Genetic Engtureering/
Recornbinant. DNA Technolog.v
6 to CeneticEngineering 75
Introduction Ilicr<lbial/ In dustrial
7 BasicTechniques Engineering92
in Cenetic Biotechnology
8 PolymeraseChainReaction 19 Technology 239
Bioprocess/Fermentation
(DNAAmplification) 112 20 DownstreamProcessing 270
9 GeneLibraries 120 2l Enzyme Technology 281
and Protein
Mutagenesis
10 Site-directed 22 Biotransformation306
Engineering 129 23 Microbial
Production
of Oreanic
Solvents
311
( tJ
24 MicrobialProductionof OrganicAcids 318 49 CeneticEngineering pf Plants-
25 MicrobialProductionof Antibiotics 329 Methodology 572
26 MicrobialProductionof AminoAcids 344 50 Applications of PlantTransformation/
27 MicrobidProduction of Vitamins 355 TransgenicPlants 596
28 MicrobialProductionof Foodsand 51 TransgenicPlantsas Bioreactors 622
Beverages 362 52 Growth Promoting in Plants 639
Bacteria
29 Single-CellProtein,
andMushrooms 373
53 MolecularMarker-Aided PlantBreedins648
30 Polysaccharides,
Polyhydroxyalkanoates,
and
Lipids 382
31 Biomass and Bioenergy 393 Envirorunental Biotechnology
32 MicrobialMiningand Metal
Biotechnology 400 54 Environmental - Ceneral 657
Pollution
andControl' 669
55 Air Pollution
56 WaterPollutionand Sewage 679
WaterTreatment 686
SewageAffaste
Anirnal Cell/Anirnal 58 Sludgeand SolidWastes-Treatment and
Disposal 708
Biotechnolosr
59 BiodegradationandBioremediation 718
33 AnimalCellCulture - Fundamentals, Facilities60 ClobalEnvironmental Problems 730
andApplications 407
34 CultureMediafor AnimalCells 415
35 CulturedCells-Biologyand
Characterization 423 Biotechnology and Societ..v
36 Primary Cultureand Cell Lines 437 61 Biotechnology-Society, Risks,Ethics,
37 AnimalCellCulture-Ceneral Considerations Patenting 739
and Scale-up 450
38 CellViabilityandCytotoxicity 459
39 CellTransformationandCell Cloning 463
40 Organand Histotypic Cultures,andTissue Basics to Learn Biotechnolopg'
Engineering 468 62 Cell-The BasicUnit of Life 749
4 l Transgenic
Animals 480 63 Microorganisms 754
64 Bioorganicand BiophysicalChemistry,and
BiochemistrvTools 758
65 Biomolecules 771
Plant/furicultural Biotechnologg' 66 Enzymology 786
42 PlantTissueCulture- General 497 67 Metabolisms 796
43 PlantTissue CultureMedia 517
68 lmmunology 815
69 Cenetics 822
44 ProtoolastCultureandSomatic
Hybridization 523
70 Bioinformatics 827
15 Production of HaoloidPlants 538
i\ppendices
16 Somaclonal Variations 546
17 ClonalPropagation (Micropropagation)552 Clossary 833
48 Cermolasm Conservation and Abbreviations 852
Cryopreservation565 Index 855
(u.)
 ints in Detail

Organization
of DNA in the cell 19
Structure
of RNA 19
Introduction to Biotcchnologv Typesof RNA 21
Catalvtic
RNAs-ribozvmes 22
1 the Scopeof Biotechnology 3
O ld and n e w b i o te c h n o l o g y 3 3 DNA-Replication,
Recombination
Definition(s) of biotechnology 3 and Repair
A
23
Historyof biotechnology a
Replicationof DNA 23
B iot ec hn o l o g- y a m u l ti d i s c i p l i n a ry
growingtree A Replication in prokaryotes 24
Commercial izationof biotechnology 7 Replication in eukaryotes 27
Publicperceptionof biotechnology 7 Processof replicationin eukaryotes )7

lnhibitorsof DNA replication 29

The futureof biotechnology 7
Cellcycleand DNA replication 29
Oreanizationof the book 7
Telomeres andtelomerase 30
Telomere in senescenceand cancer 31
Recombination 31
Homologous recombination 31
Introduction to }lolccular Iliologv
Non-homologous recombination 32
2 DNA and RNA- Composition Damageand repairof DNA ??
and Structure 11 Typesof DNA damages 34
Nucleotides 1l Mutations 34
Structureof nucleotides 11
II Repairof DNA 35
Structureof DNA 1A Defects in DNA reoairandcancer 37
DNAdoublehelix 14
4 Transcription
and Translation 3B
Othertypesof DNA structure 17
Thesizeof DNA molecule-units Cenome 38
of length 17 Transcriotome 3B
Denaturation of DNA strands 1B Proteome 3B

{it
li i l CO NTENTS I N DETAI L

Transcription 39
-franscriptionin prokaryotes 39
Transcription in eukaryotes 4) Genetic Engineering/Recombinant
Post-transcriptionalmodifications +J DNA Technologv
Cellular RNAcontents 45
Reversetranscription +o
6 Introduction
to Genetic 75
Engineering
Translation 46 Briefhistory of recombinant DNA
technology 75
Ceneticcode 46
Molecular toolsof geneticengineering /o
Protein biosynthesis 48
Restriction endonucleases-DNA cuttinB
Requirement of the components 49
enzymes
Activation of aminoacids 50
DNA ligases-DNA joiningenzymes
Proteinsynthesis proper 50
Alkalinephosphatase
lnitiationof translation 51
DNA modifying enzymes
Elongationof translation 53
Host cells- the factoriesof cloning
Termination of translation 53
Prokaryotic hosts
lnhibitorsof proteinsynthesis
Eukaryotic hosts
Chaperones and proteinfolding 55
Vectors- the cloningvehicles
Post-translational modifications
of oroteins 56
Plasmids
Proteintargeting )/
Bacteriophages
Mitochondrial DNA,transcription and
Cosmids
translation 58
Artificialchromosome vectors
5 Regulationof Gene Expression 59 Shuttlevectors
Choiceof a vector
Ceneregulation - general 59
Methodsof genetransfer
Theoperonconcept 60
Transformation
Lactose (lac)operon 60
Conjugation
Tryptophan operon 62
Electroporation
Geneexpression in eukaryotes oz
Liposome-mediated genetransfer
Chromatin andgene
structure
Transduction
expression 63
Enhancers gene Directtransfer of DNA
andtissue-specific
expresston 65 Genecloningstrategies
Combination of DNA elements and proteins Cloning fromgenomic DNAor mRNA?
in geneexpression 65 Geneticengineering of plants
Motifsin proteins andgeneexpression 65 Geneticengineering guidelines
Generegulationin eukaryotes 67 Asilomar recommendations
Methodsto studygeneexpression/regulation 68 NIH guidelines
Ceneanalysisby T-DNAand transposon Thefutureof geneticengineering
tagging 70
Methodsto studyprotein-protein 7 BasicTechniques in
interactions 70 Cenetic Engineering tE
Phage display 70 Agarosegel electrophoresis &:

Yeasttwo-hybrid system 72 PulsedJield PFCi

gel electrophoresis
 Coruterurs rn Derat (iiil

Contour-clamped homogeneous electrical- Reverse transcriptionPCR 115

fieldelectrophoresis(CHEF) 93 Asymmetric PCR 1i5
Polyacrylamide gel electrophoresis Real{imequantitative PCR 116
(PAGE) 93 Random amplifiedpolymorphic DNA
Otherusesof gel electrophoresis 94 (RAPD) 116
lsolationand purificationof nucleicacids 94 Amplifiedfragment lengthpolymorphism
of cellularDNA
Purification 94 (AFLP) 117
of plasmidDNA
Purification 95 Rapidamplification of cDNAends(RACE)118
of mRNA
Purification 96 Applicationsof PCR 118
lsolationof chromosomes 96 PCRin clinicaldiagnosis 118
Fluorescence-activatedcell sorting 96 PCRin DNA sequencing 119
Collectionof chromosomes with PCRin genemanipulation and
identical
sizes 97 exoression studies 119
Nucleicacidblottingtechniques 97 in
PCR comparative studies
of genomes 119
Southern blotting 99 PCRin forensic medicine 119
Northern blotting 99 in
PCR comparison with gene cloning 119
Dot-blotting 100
Westernblotting 100 9 Gene Libraries 120
Autoradiography 100 a genelibrary
Creating 120
Colonyand plaqueblotting 100 PCRas an alternativeto genomiclibrary
DNA sequencing 101 construction 123
MaxamandCilberttechnique 'tot Complementary DNA libraries 123
Dideoxvnucleotide method 102 Screening
strategies 125
Bacteriophage M,, as a cloningand Screeningby DNA hybridization 125
DNA sequencing vector 103 DNA probes 126
DNA sequencing by primerwalking 104 Screeningby colonyhybridization 126
Chromosome walkingin DNA Screeningby PCR 128
sequencing 105 Screeningby immunological assay 128
Automated DNA sequencing 106 Screeningby proteinfunction 128
Alternativemethodsof DNA sequencing 107
Pyrosequencing 107 L0 Site-directedMutagenesisand
"
DNA chips(microarrays) 108 Protein Engineering 129
Chemicalsynthesis of DNA 109 Strategies
to improvein vitroactivitiesof
Thephosphoramidite method 109 enzymes 129
Synthesisof genes 111 Oligonucleotide-directed mutagenesis 130
Casettee mutagenesis 131
8 PolymeraseChain Reaction PCR-ampl ified oligonucleotide-d
irected
(DNA Amplification) 112 mutagenesis 132
Technique of PCR 112 Proteinengineering 133
Sources of DNA polymerase 114 Increasing the stabilityand biological
Keyfactorsfor optimalPCR 114 activityof proteins 133
Variationsof PCR 114 Additionof disulfidebonds 133
NestedPCR It5 Changing asparagineto otheraminoacids 133
Inverse PCR 115 Reducingthe freesulfhydrylgroups 133
Anchored PCR 115 Singleamino acid changes 134
livl CO NTENTS I N DETA I L

lmprovingkineticproperties
of enzymes I 35 Ex vivo genetherapy 158
Proteinengineeringby useof gene Vectorsin genetherapy 159
families 136 Viruses 159
Proteinengineering
throughchemical Humanartificial chromosome 162
modifications 136 Bonemarrowcells 162
Proteinengineering
- an ever Selectedexamplesol ex vivo gene
expanding field 136 therapy 162
Therapy for adenosine deaminase
11 Manipulation of GeneExpression in deficiency 162
HostCells 137 Therapy for familialhyper-
of hostcellsfor geneexpression137
Selection cholesterolemia 164
Manipulation of geneexpression in Therapy for Lesch-Nyhan syndrome 164
prokaryotes 138 Therapy for hemophilia 164
Manipulation of geneexpression in Ex vivogenetherapywith non-
eukaryotes 139 autotogous cells 164
Saccharomyces -the yeastin
cerevisiae In vivo genetherapy 164
expressingclonedgenes 140 Cenedeliveryby viruses 165
Otheryeastexpression systems 141 Cenedeliveryby non-viral systems 166
Insectcell expressionsystems 141 Cenetherapystrategies for cancer 167
Mammalian vectors
cell expression 143 Cenetherapyfor AIDS 169
Geneexpression to produceproteins 143 Antigene and antisense therapy 169
Collectionand purificationof recombinant Antisensetherapyfor cancer 170
proteins 144 Antisensetherapyfor AIDS l.l
Purification
of recombinant oroteins 146 Antisenseoligonucleotides as
therapeutic agents 1-'
1.2 Human Genome Project 148 Chimeric oligonucleotides in gene
Thebirthand activityof humangenome correctron 1-'
project 148 Aptamers as therapeutic agents
Mappingof the humangenome 149 Ribozymes as therapeutic agents
l_
Approaches for genomesequencing 149 Thefutureof genetherapy j -_
Human genome sequence-results
summarised 149 14 ONn in DiseaseDiagnosisand
Cenomes of someotherorganismssequenced
153
of humangenome
Benefits/applications
Medical Forensics
Methodsof DNA assav
i-l
sequencing -53
Nucleicacidhvbridization
andhumangenome
Ethics 154 '- tr
DNA orobes
TheDNA chip-microarray of
geneprobes
of infectious
DNA in the diagnosis
llc dical/Pharmaceutical diseases
Biotecluroloey Tuberculosis
(Biotechnologv in Healthcare) Mal ari a
Chaga'sdisease
13 CeneTherapy 157 A cqui redi mmunodefi ci encvsrndrone
for genetherapy
Approaches 1s7 (AIDS)
 Corurerurs nr Derntl (vl

Humanpapilloma virus 178 Erythropoietin 198

Lymedisease 178 | (DNase
Deoxyribonuclease l) 198
Periodontaldisease 178 Alginate
lyase 198
DNA probesfor otherdiseases 178 Secondgeneration
therapeuticproteins
DNA in the diagnosis of genetic (muteins) 198
diseases 179 Vaccines 198
Cysticfibrosis 179
Sickle-cell
anemia 179 16 Recombinant
Vaccines 199
Duchenne's muscular dystrophy 179 Traditional
vaccines 199
Huntilgton's disease 180 Recombinant vaccines - general 200
FragileX syndrome 180 Subunitvaccines 201
Othertriplerepeatdiseases 180 HepatitisB 201
Alzheimer'sdisease 181 Footand mouthdisease 202
Amyotrophic lateralsclerosis 181 Herpes simplex virus 203
Cancers 181 Tuberculosis 203
Diabetes 182 Meningitis 203
Obesity 183 AIDS(Acquired immunodeficiency
DNAanalysis forotherhumandiseases 183 syndrome) 204
Cenebanks - a novelconcept 184 DNA vaccines(geneticimmunization) 205
DNA analysis for environmental DNAvaccine andimmunity 205
monitoring 184 RNAvaccines 207
Waterqualitytesting 184 Plantsas edible subunitvaccines 207
DNA fingerprinting or DNA profiling 184 Attenuatedrecombinant vaccines 208
DNA markers in diseasediagnosis
and C hol era 208
fingerpri
nting 185
Salmonellaspecies 210
Restrictionfragmentlength
Leishmaniaspecies 210
polymorphisms (RFLPS) 185
Vector recombinantvaccines 211
Variablenumbertandemrepeats
/VNfRs) 186 V acci nesagai nstvi ruses-
vacci ni avi rus 211
Microsatel lites(simpletandem
repeats) 1B g D el i veryof anti gensby bacteri a 212
Singlenucleotidepolymorphisms
/5NPs) 1BB 17 Monoclonal
Antibodies 213
Currenttechnology of DNA Princiolesfor creationof
fingerprinting 1B B hybri domacel l s 213
Productionof monoclonalantibodies 214
15 Pharmaceutical Productsof DNA Large scaleproduction of MAbs 216
Technology 189 H uman monocl onalanti bodi es 216
Humanproteinreplacements 189 Geneticengineeringstrategiesfor the
lnsulin 189 oroductionof human-mouse MAbs 217
Humangrowthhormone 192 Productionof MAbs in E. coli 218
ClottingfactorVIll 194 Secondgenerationmonoclonal
Therapeuticagentsfor humandiseases 194 anti bodi es 218
Tissueplasminogen activator 194 Advantages of monoclonalantibodies 219
Interferons 196 Limitationsof monoclonalantibodies 219
(vil CO NTENTS I N DETAI L

Applicationsof monoclonal antibodies 219 A conventional bioreactor - common

Diagnostic applications 219 features 244
MAbsin biochemica! analysis 219 Operationof a conventional bioreactor 245
MAbsin diagnostic imaging 220 Sterilization 245
Therapeutic applications 222 lnoculation and sampling 245
MAbs as direct therapeuticagents 222 Aeration 245
MAbs as targetingagentsin therapy 223 Control systems 246
Proteinourification 225 Cleaning 246
Miscellaneous applications 226 Solidsubstrate (solid state)fermentation 247
CatalyticMAbs (abzymes) 226 Media(substrates) 248
for industrialfermentation
Antibody fingerprinting 226 Substrates used as carbon sources 249
Substrates usedas nitrogen sources 249
1,8 Assisted Reproductive Technology227 Sources of growth factors 250
Manipulation of reproduction in animals 227 Sterilization of culturemediaafid gases 250
Artificial
insemination 227 Sterilizationof culture rnedia 250
1 t r1
Embryo transfer 228 Sterilizationof air LJ I

ln vitrofertilization 230 lsolationof microorganisms 252

Embryo cloning 23i Preservation of microorganisrns 254
Manipulations of reproduction in humans 231 Microbial metabolic products- low
molecular weightcompounds 254
Causes of infertility
and application
231 Primary metabolites 254
of ART
(lUl) Secondary metabolites 255
lntrauterine insemination 232
Bioconversions 257
In vitrofertilizationand embrvotransfer
(lVFandEl 232 Microbial metabolic products- high
molecularweightcompounds 257
Methodologyof IVF 232
Cenetic improvement (development)
Successratesof IVF 233
of strains 257
The world'spictureof testtube babies 233
Methods of strain development 258
Cameteintrafallopian transfer (CIFT) 233
Mutation 258
ZygoIeintrafallopian transfer(ZIFT) 234 259
Geneticrecombination
lntravaginal culture(lVC) 234
Principles of microbialgrowthand
Cytoplasmic transfer(CT) 234 259
culturesystem
Micromanioulation 234 Batchcultureor batchfermentation 259
Cryopreservation 235 Fedbatchcultureor fed-batch fermentation
261
Assisted hatching (AH) 235 Semi-continuous cultureor semt-
Preimplantation of genetic (PED)236
diagnosis continuous fermentation 261
The negativeaspects of ART 236 Continuous cultureor continuous
fermentation 262
Crowthkinetics of microorganisms 263
Classification of fermentation processes 264
Thefermentation process 265
Ilicrobial/Industrial Biotechnology
Measurement andcontrolof bioprocess
19 Bioprocess/Fermentation oarameters 267
Technology 239 Scale-up 268
Bioreactors/Fermenters 239 Food technology 268
Tvoesof bioreactors 239 Foodoreservation 269
 Corurerurs tru Deretr (viif

270 Opticalbiosensors 301

20 Downstream Processing
biosensors
Piezoelectric 302
processing
in downstream
Stages 270
Whole cell biosensors 302
S olid- liquid s e p a ra ti o n 270
Immunobiosensors 303
Flotation 271
of
Applications biosensors 304
Flocculation 271
271 lmmobilized
enzymes and cells-therapeutic
Filtration
272 applications 304
Centrifugation
Releas of e i n tra c e l l u l aprro d u c ts
:'-". 22 Biotransformation 306
Cell disruption l /4
reactions
Typesof biotransformation 306
Concentration 276
276 and techniquesfor
Sourcesof biocatalysts
Evaporation
biotransformation 306
Liquid-liqui d extractio n 276
Productrecoveryin biotransformation 307
Membranefiltration 277
Examplesof biotransformation 308
Precipitation 277
278 Biotransformationto produce
Adsorption
commercialproducts 308
Purificationby chromatograPhY 278
Biotransformationof steroids 308
F or m ulat io n 27g
Biotransformation of antibiotics 308
Integrationof differentprocesses 280
Biotransformationof arachidonicacid
to prostagl andi ns 3',10
2l EnzymeTechnology 281
Biotransformationfor the productionof
of enzymes
Applications 281
ascorbica'cid 310
Commercial of enzymes
production 281
Biotransformationof glycerolto
The technologyof enzyme 310
dihydroxyacetone
production- generalconsiderations 283
Biotransformationfor the production
l n z y mep ro d u c ti o -n
Regulat ioonf mi c ro b i a e
of i ndi go 310
generalconsiderations 286
Cenet iceng i n e e ri nfo
gr mi c ro b i a l
23 rVticrobial of
Production
enzymeproduction 286
2BB OrganicSolvents 311
lmmobilizationof enzymesand cells
Methodsof immobilization 288 Alcohol 311
Choic eof im m o b i l i z a ti ote n chnique 290 of
Production by
ethanol fermentation 3 12
S t abiliz at i oonf s o l u b l ee n z y me s 290 Acetoneand butanol 31s
lm m obiliz a ti oonf c e l l s 293 Glycerol 316
Effectof immobilizationon enzyme
properties 293 24 rtlicrobial Productionof
lmmobilizedenzymereactors 294 Organic Acids 318
A pplic at io nos f i mmo b i l i z e de n z y m e s Citricacid 318
and c ells 295 Factors in the regulatiorrof citricacid
Manufactureof commercialproducts 2gS production 320
Immobilized enzymesand cells- Production process for citricacid 321
analyticalapplications 296 Surface processes 321
Biosensors 297 Submerged processes 322
Typesof biosensors 298 Production of citricacidfromalkanes 322
Electrochemicalbiosensors 299 Recovery of citricacid 322
Thermornetricbiosensors 300 Gluconicacid 324
(viii! CO NTENTS I N DETA I L

lactic acid 325 L-Tryptophan 353

Aceticacid 326 L-Asparticacid 354
L-Ascorbic acid 327
Itaconicacid 328 27 Microbial Production of Vitamins 355
VitaminBr, 355
25 Microbial Productionof Antibiotics 329 Commercial production
of vitamin8,, 356
Antibiotics
- general 329 Riboflavin 357
Applications of antibiotics 330 Commercial production
of riboflavin 358
Productionof antibiotics - a major p-Carotene 359
pharmaceutical industry 330 Commercial of p-carotene
production 359
Penicillins 331 Cibberellins-plant
growthstimulants 361
Production process of penicillin 332 production
Microbial of gibberellins 361
Production of 6-amino penicillanic
acid 334
Cephalosporins 334 28 MicrobialProduction of
Production process of cephalosporin 334 Foodsand Beverages 362
Production of 7-aminocephalosporanic acid 335 Fermented Foods 362
New B-lactam technology for production 362
Cheese
of 7-ACA JJO
Yoghurt 365
Aminoglycosides 336
Sauekraut 365
Production process of streptomycin 337
Bread 365
Tetracyclines 338
Baker'syeast 365
Production process of chlortetracycline 339
Othervegetarianfoods 366
Production processes
of tetracycline-different 339
Sweeteners 367
Macrolides 340
Thaumatin-a sweetprotein 367
Production process of erythromycin 340
Monellin-a candidate for sugar
Aromaticantibiotics 341
substitute JO/
Chloramphenicol 341
Flavour enhancers 367
Griseofulvin 341
Alcoholicbeverages 367
Nucleoside antibiotics 342
Alcoholicbeverageproduction -
Geneticmanipulations of Streptomyces 342 generalaspects 368
Goodantibioticmanufacturing practices 343 Beer 369
Wines 370
26 Microbial Productionof food products
Other microbially-derived 371
Amino Acids 344 Enzymes in food and beverage
Aminoacid production - general industries 371
considerations 344 Enzymes in the preparation
of
Commercial applicationsof aminoacids 344 fruitjuices 371
Methods for production of aminoacids 345 Diagnostics in foodbiotechnology 372
Straindevelopment for aminoacid Foodbiotechnology -
production 346 publicacceptance 372
L-Glutamicacid 346
L-Lysine 350 29 Single-CellProtein,and Mushrooms373
L-Threonine 3s2 Microorganisms usedfor
and substrates
L-Phenylalanine 353 production of SCP i-r
 Correrurs rru DerarL lixl

of SCPfromhighenergysources375
Production Energy-rich
crops 399
of SCPfrom wastes
Production 378 Thefutureof biomass 399
of SCPfromwooq
Production 379
Production
of SCPfromCO, 379 32 MicrobialMiningand Metal
of SCPfrom sewage
Production 379 Biotechnology 400
Genetically
engineered artificial Bioleaching 400
proteinas animalfeed 380 Commercialprocessof bioleaching 401
Mushrooms 380 Bioleachingof copper 402
Productionof ediblemusnrooms 3Bi Bioleachingof uranium 402
Bioleachingof other metals 403
30 Polysaccharides, Polyhydro- Advantages of bioleaching 403
xyalkanoatesand Lipids 382 Biosorption 403
Polysaccharides 382 Microbial recovery of petroleum 404
Ceneralfeatures of microbial
polysaccharides 383
Xanthan 383
Dextran 385
Animal Cell/Animal Biotechnolos/
Alginate 385
Scleroglucan 385 33 AnimalCellCulture - Fundamentals,
Gellan 386 Facilities and Applications 407
lan
Pollu 386 Facilitiesfor animalcell culture 407
Curdlan 386 Minimalreouirements for cellculture 408
Polyhydroxyalkanoates 386 lnfrastructure 408
PHA-chemistry and properties 386 Equipment 408
Polyhydroxybutyrate(PHB) 3(j/
Culturevesse/s 408
Biosynthesisof otherPHA 389 Useof non-adhesive in
substrates
Biopol- a biodegradable plastic 390 tissueculture 409
Geneticengineering for PHA Contamination, asepticconditions,and
production 390 sterilization 409
Microbiallipids 391 Aseoticconditions 409
Production oils
of single-cell 391 Sterilization 410
Microbialrubber 391 Advantages and limitationsof
Microbialadhesivebiopolymer 392 tissueculture 411
Micobial production of indigo 392 Applications of animalcell cultures 411
Medical/pharmaceutical productsof
3L Biomassand Bioenergy 393 animalcellcultures 412
Biomass 393 Cenetic engineeringof anirnalcells
Sources of biomass
and utilization 394 andtheirapplications 413
Productionof alcoholfrombiomass 395 Risksin a tissueculturelaboratory
Productionof biogasfrom biomass 395 and safety 413
Processof biogasproduction Biohazards 414
(biogas
plant) 397
Hydrogen-a new biofuel 398 34 CultureMedia for Animal Cells 415
Productionof biohydrogen 398 Naturalmedia 415
Ixl CO NTENTS I N DETAI L

Ariificialmedia 415 Techniques for primaryculture +J/

Physicochemical properties of culturemedia 415 Mechanical disaggregation 437

Balanced saltsolutions A1-7
att Enzym atic disaggregation 438
Comolete culturemedia 417 Primaryexplanttechnique 441
Serum 419 Seoaration of viableano
Selection of mediumand serum 420 non-viable cells 441
Supplementation of the mediumwith Medical ethics andsafetymeasures in
trssueextracts 420 culture technioues 441
Serum-free media 421 Cell lines 442
Ad..rantages and disadvantages of Finitecell lines 442
serum-free media 421 Continuous cell lines 443
Develooment of serum-free media 422 Nomenclature of cell lines 443
Commonly usedserum-free media 422 Seleciion of cell lines ' 443
'
Maintenance of cell cultures 444
35 CulturedCells-Biologyand Subculture 445
Characterization 423 Monolayer cultures 445
Characteristics of culturedcells 423 Suspension cultures 446
Celladhension 423 Stem cell cultures 447
Cellproliferation 424 Embryonic stem(ES)cells 447
Celldifferentiation 424 Eoithelial cells 448
Metabolism of cultured cells 425 Maintenance of stemcellsin culture 448
Initiationof cellculture 425 Characterization of stemcells 448
Evolution anddevelooment of cell lines 427 Applications of culturedstemcells 449
Characterization of culturedcells 428
Morphology of cells 428 37 Animal Cell Culture- General
Species of originof cells 429 Considerations and Scale-up 450
Identificationof tissueof oriein 429 Cell culture-general considerations 450
Transformed cells 430 Cellquantitation 450
ldentificationof soecific cell lines 430 Equipment andmedium 451
Measurement of growthparameters of pH and buffersystems 451
culturedcells 432 Oxygen 451
Crowthcycleof culturedcells 432 Crowthkinetics 451
Plating efficiency of cultured cells 433 Typesof cultureprocesses 452
Cellsvnchronization 433 Otherpractical considerations 452
Cellseparation by physicalmeans 433 Scale-up 453
Cellseparation by chemicalblockade 434 Scale-upin suspension 453
Somehighlights of cellsynchronization 435 Factors in scalingup 453
Senescence andapoptosis 435 Stirrerculture 453
Cellularsenescence 435 Continuous flow culture 454
Measurement of senescence 435 Air-liftfermenter culture 454
Apoptosis 435 Othersystems for suspensionculture 455
Measurement of apoptosis 436 Scale-upin monolayer 455
Rollerbottleculture 456
36 Primary Culture and Cell Lines 437 lvlultisurtace culture 456
Primarycell culture 437 Multiarray disks,spiralsandtubes 456
 Conrerurs nr Dernil (xil

culture
Microcarrier 456 Tissue modelling 477
Perfusedmonolaver culture 457 Embryonic stemcell engineering A'7 -7

Monitoringof cell growth EScell cultures to produce

in scale-up 458 differentiatedcells 477
Monitoringof suspensioncultures 458 Humanembryonic stemcell research 477
Monitoringof monolayer cultures 458
4L TransgenicAnimals 480
38 Cell Viability and Cytotoxicity 459 lmportance of transgenic animals - general 480
Limitationsof in vitrocytotoxicitystudies 459 Transgenic mice 481
Assays for cell viabilityand cytotoxicity 460 Retroviralvectormethod 481
Cytotoxicity and viabilityassays 460 Microiniection method 481
Survival assays 461 Embryonic stemcell method 482
Metabolicassays 462 Geneknockout 484
Transformation assays 462 Yeastartificial chromosomein transgenesis485
Inflammation assays 46.2 Transgenicmiceand their applicatiois 485
Thehumanmouse 485
39 Cell Transformationand
TheAlzheimer's mouse 485
Cell Cloning 463
Theoncomouse 486
Transformation of cells +o-)
Theprostate mouse, 486
Geneticinstability 463
Theknockoutmouse 486
lmmortalization 463
Transgenic micefor humandiseases 487
Aberrant groMhcontrol 464
Transgenesisin largeanimals 487
Tumorigenicity 465
Transgenic cattle 4BB
Cell cloning 465
465 Tiansgenic sheepandgoats 488
Dilutioncloning
Transgenic pigs 488
Stimulation of platingefficiency 466
Suspension cloning 467 Transgenic chickens 489
467 Transgenic fish 490
lsolationof clones
Positioneffects 490
Cultures,
40 Organand.Histotypic Animalbioreactors 490
andTissue
Engineering 468 Dolly- the transgenic clone 490
Organotypicmodels 468 Cloningof pet animals 491
Organcultures 468 Transgenic animalsproducedby
Techniquesfor organculture 469 cloningfetal cells 491
Histotypic
cultures 470 Transgenic animalsin xenotransplantation492
Threedimensional cultures 471 Pigsin xenotransplantation? 492
Organotypiccultures 472 Transgenic organisms to interrupt
Tissueengineering 472 diseasecycles 493
Cellsourceandculture 473 Transgenic snails 493
'-- Transgenic mosquitoes 493
Cultureof cells 473
Cellorientation 474 Transgenic tsetseflies 493
Cellsupportmaterials 474 Transgenic bollworms 493
Designand engineeringof tissues 475 Transgenic
medflies 493
(xiil CONTENTS IN DETAIL

of media
Constituents 519
Preparation
of media 522
Plant/furicultural Biotechnolo 95, Selectionof a suitablemedium 522

42 PlantTissueCulture- General 497 44 Protoplast

Cultureand Somatic
Benefitsof plant tissueculture 497 Hybridization 523
Basicstructureand growth of a plant 497 lmportance
of protoplasts
andtheircultures523
Conventionalplant breedingand lsolationof protoplasts 513
p l a n tti s s u ec u l tu re 498 Mechanical method 524
Termsused in tissueculture 498 Enzymatic method 524
Brief historyof plant tissueculture 499 Cultureof protoplasts 526
Typesof culture 499 Culturemedia 526
B a s i cte c h n i q u eo f p l a n tti s s u ecul ture 499 Culturemethods 527
Callusculture 500 Regeneration of protoplasts 527
Cell culture 502 Sub-protoplasts 528
l s o l a ti o no f s i n g l ec e l l s 502 Somatichybridization 528
Suspension cultures- growth and Fusionof protoplasts 529
subculture 502 Selectionof hybridcells 531
Typesof suspension cultures s03 of hybrid(cells)plants
ldentification s33
Synchronization of suspension cultures 504 Chromosome numberin somatichybrids 534
Measurement of growth of cultures 505 Cvbrids 535
Measurement of viabilityof Applications of somatichybridization 536
culturedcells 505 Limitationsof somatichvbridization 537
Culture of isolatedsingle cells
(single cell clones) of HaploidPlants
s05 45 Production 538
Applicationsof plant tissuecultures 506 In vivo and in vitroapproaches 538
Secondarymetabolitesin plant culture 507 Androgenesis 539
Applicationsof secondarymetabolites 508 Antherculture 539
Productionof secondarymetabolites 509 Pollen(microspore) culture 539
Selection of cell lines for high yield Development of androgenichaploids 540
of secondary metabolites 509 Factorsaffectingandrogenesis 540
Largescale (mass)cultivation of Gynogenesis 542
plant cells 511 ldentification of haploids 543
Mediun composition and effect Morphological approach 543
of nutrients 512 Geneticapproach 543
Elicitor-inducedproduction of Diploidizationof haploidplants
secondary metabolites 514 (productionof homozygous plants) 543
Effect of environmental factors 5',t5 Colchicine treatment 543
Biotransformation using plant Endomitosis 544
cell cultures 516 Applications of haploids 544
t r/ c
SeconCarymetabolite release Cropsdeveloped throughantherculture J1J

and analysis 516 limitationsof haploids 545

43 PlantTissue
CultureMedia 517 46 Somaclonal
Variations 546
Major typesof media 517 Basisof somaclonalvariations 546
 Corurerurs rruDEret lxiiil

lsolationof somaclonal variants 547 Vector-mediated genetransfer 573

Withoutin vitroselection J+/ Agrobacter i u m-med iatedgenetransfer 573
With ln vlfroselection 547 Crowngall disease and Ti plasmid 574
Factorsaffectingproductionof Hairyroot disease of A. rhizogenes-
somaclonal variants 549 R, plasmids 577
Applications of somaclonalvariations 549 Ti plasmid-derived vectorsystems 577
Limitationsof somaclonalvariations 550 Planttransformation technique using
Gametoclonal variations ) 51
Agrobacterium 580
Virus-mediatedgene transfer
47 Clonal Propagation (plant virusesas vectors) s81
(Micropropagation) 552 Caulimoviruses as vectors ) dl

Technique of micropropagation 552 Ceminiviruses as vectors 582

Multiplicationby axillary
budsand RNA plant viruses as vectors 582
aoicalshoots 553 Limitations of viralvectorsin genetransfer 583
Multiplicationby adventitious
shoots 556 Direct or vectorlessDNA transfer 583
Factorsaffecti
ng micropropagation 556 Physical genetransfer methods 583
Organogenesis 556 Electroporation 583
Somatic embryogenesis 557 (biol
Particlebombardment istics) 584
Applicationsof micropropagation 5s9 Microinjection 586
Disadvantages of micropropagation 559 Liposome-mediated transformation 586
Productionof disease-freeplants 560 Silicon carbidefibre-medi ated
Methods to eliminatevirusesin plants 560 transformation 587
Chemical gene transfer methods s87
Eliminationof pathogens otherthanviruses 562
Meritsanddemerits of disease-free
olant Polyethy Iene glycoI-mediated
production 562 transformation 587
562 DEAEdextran-mediated transfer 5BB
Embryoculture
Calciumphosphate co-precipitation-
Matureembrvoculture 562
mediatedtransfer 588
Embryorescue 562
DNA imbibition by cells/tissues 588
Nutritional
requirements of embryoculture 563
Markergenesfor plant,transformation 5BB
Applicationsof embryoculture 564 Selectable markergenes 5BB
Antibioticresistance genes 588
48 GermplasmConservationand
Antimetabolite markergenes 589
Cryopreservation J O)
Herbicideresistance genes 589
/n-situconservation 565 Production of marker-free transgenicplants590
Ex-situconservation 565 Cleangenetechnology 590
Cryopreservation 566 Reporter genes 590
Techniquesof cryopreservation 567 Promoters and terminators 592
Cold storage 569 Transgene stabilit,v, expression and gene
low-pressureand low-oxygenstorage 570 silencing 593
Applications
of germplasmstorage 571 Scaffold attachment regions and gene
of germplasm
Limitations storage 571 stability s93
Intronsand geneexpression 593
49 GeneticEngineering
of Genesilencing 593
Plants-Methodology 572 Chloroplast transformation 594
Genetransfermethods 573 Chloroplast engineering 594
{xivf CONTENTS IN DETAIL

50 Applicationsof PlantTransformation/ Coldenrice-the provitamin

A
619
Transgenicplants 596 l1t" , _ L :. ^..^ ^..:1^_:^_
^enri.ched
Resistance
to bioticstresses 596 and mineials 620
Insect(pest)resistance 5l'
genes
Resisrance rrom microorsanism,sgz:,""ffiI1,ffi1J1'ffi::H:"t' 21?
genesfrom higherplants
Resistance 600
/Proteinase(protease)inhibitors 501 51 Transgenic Plants as Bioreactors 622
Lectins 60l Metabolicengineeringof carbohydrates 622
Resistance genesfrom animals 601 Starch 622
lnsectresistancethroughcopy Cyclodextrins 623
naturestrategy 601 Fructans 623
Virus resistance 602 Trehalose 624
Fungaland bacterial drseases 604 Metabolic engineeringof lipids 624
Pathogenesis-related(PR)proteins 605 productionof shorterchain fatty acids 624
Ribosome-inactivating oroteins(RlPs) 605 productionof longerchain fatty acids 625
Phytoalexins 606 productionof fatty acidswith modified
Nematoderesistance 606 degreeof saturation 625
Resistance to abiotic stresses 607 Productionof rare fatty acids 626
H e rb i c i d ere s i s ta n c e 607 B i odegradablpleasti cs(bi opl asti cs) 626
Clyphosateresistance 607 Geneticallyengineeredplantsas protein
Phosohinothricin
resistance 609 factories 628
Sulfonylureas and imidazolinones Approaches for proteinproduction
in plants 628
resistance 610 Oleosinpartitiontechnology 631
Environmental imoactof herbicide- Productionof industrialenzymesin plants 631
' re s i s ta n t
c ro p s 610 P roducti onof l ysosomalenzymesi n pl ant s633
Toleranceto water deficit stresses 610 Productionof antibodies(plantibodies)
Resistance
againstice-nucleating
bacteria 611 in plants 634
fmprovementof crop yield and quality 612 Productionof vaccinesin plants 636
Creen revolution 612 Productiontherapeuticproteinsin plants 637
Ceneticengineering for extended
shelf-lifeof fruits 612 52 Growth-Promoting Bacteria
Biochemica!changesduring tomato in Plants 639
ripening 613 Biological nitrogen fixation 639
Genetic manipulationof fruit ripening 614 Nitrogen cycle 63c
Longer shelf-lifeof fruits and vegetables 615 Nitrogenfixing bacteria 639
Ceneticengineering for preventionof Mechanismof nitrogenfixation 641
discoloration 616 Ceneticmanipulations for nitrogenfixation 641
Ceneticengineering for flower pigrnentation
616 Geneticengineering of nitrogenasegene 642
Ceneticengineering for male sterility 617 Ceneticengineering of hydrogenase gene 611
Transgenic plantswith improved Ceneticengineering of nodulation genes 6'l-{
nutrition 617 Biocontrolof phytopathogens 61+
Amino acidsof seedstorage proteins 617 Siderophores 611
C e n e ti ce n g i n e e ri nfo
g r i m p ro vi ng A nti bi oti cs' 6+- ;
palatabilitvof foods 618 Enzvmes 6-tr
 CorurerursrruDerarr (xvl

Biofertilizers 645 Photosynthesis to reduceatmosphericCO2 664

Symbiotic nitrogenfixers 646 Biologicalcalcificationto reduce
Asymbiotic nitrogen fixers 646 atmospheric CO, 664
Phosphate solubilizing bacteria 646 Sewagetreatment by bacteriaand algae 664
Organicfertilizers 647 Eutrophicationand phosphorus pollution 665
Benefits of biofertilizers 647 Biologicalremovalof phosphorus 666
Limitations of biofertil
izers 647 Management of metalpollution 666
Bioscavengers of metals 666
53 MolecularMarker-Aided Mechanism of metalscavenging 666
Plant Breeding 648 lmmobilized cellsin the management
Molecularmarkers 648 of pollution 667
Markers basedon DNA hybridization 649
55 Rir Pollutionand control 669
Markers basedon PCRamplification
Molecularmarkerassisted selection
Molecular breeding
Quantitativetraitloci
Arid and semi-aridplant biotechnology
Greenhouse and greenhome technology
for gaseous
Controldevices pollutants 675
Controldevices
for volatileorganic
compounds (VOCs) 678
Enr.ironmental Biotechl oloE
56 Water Pollutionand Sewage 679
54 EnvironmentalPollution- General 657 Dissolved oxygen 679
Environment-basic concepts 657 Waterpollution 680
Atmosphere 657 Natureof wateroollutants 680
Hydrosphere 657 Organicpollutants 680
Lithosphere 658 Inorganic pollutants 680
Biosohere 658 Microbiological pollutants 680
Environmental pollution-sourcesand nature 658 Radioactive pollutants 681
Sources of pollution 658 Wastewater and sewage 682
Natureof pollutants 658 Composition of sewage 682
Pollutionmonitoring/measurement 659 Typesof sewage 682
Biotechnological methods for measurement Measurement of water pollution 6B3
of pollution 659 Measurement of organicmatterof sewage683
Criteriafor biomonitoringof pollution 659 Detection of pathogenicorganisms
Bioassays in environmentalmonitoring 650 of sewage 684
Cell biologyin environmental monitoring 661 Laboratorymethodsfor detectionof
Molecularbiologyin environmental coliformorganisms 684
monitoring 662 Techniques fecalfrom
to distinguish
Biosensors in environmentalmonitoring 663 non-fecal bacteria 685
Biotechnological methodsfor management
of pollution 663 57 Sewage/WasteWater Treatment 686
Atmospheric CO" reduction 664 Classification
of treatment
Drocesses 686
(xvil CONTENTS IN DETAIL

Preliminarytreatment 687 58 Sludgeand Solid Wastes-Treatment

Primarytreatment 688 and Disposal 708
Secondary or biologicaltreatment 689 708
Sources and characteristics of sludge
Aerobic suspended-growth treatment 708
Preliminaryoperations
processes 690
Sludgethickening (concentration) 709
Activated sludgeprocess 690
Sludge stabilization 710
Aeratedlagoons 691
Composting 711
Sequencing batchreactor 691
Mechanism of composting 712
Aerobicdigestion 692
Methods of composting 712
Aerobicattached-growthtreatmentprocesses692
692 Vermicomposting 714
Tricklingfilters
Roughing filters 693 Conditioning of sludge 714
Rotating biological contactors (RBC) 693 Disinfection of sludge 714
11A
Packed-bed reactors 693 Dewatering
(fluidizedbed reactors) Heatdrying 715
Anaerobicsuspended-growth treatment Thermalreduction of sludge 715
Processes 694 Ultimate disposal of sludge 715
Anaerobic digestion 695 Landfilling / lJ

Anaerobiccontactprocess 696 Lagoon ing 716

Anaerobicattached-growthtreatment Septage and septage disPosal 716
processes 696 Treatment and disposalof
Anaerobic filterprocess 696 solidwastes 716
Expanded-bed process 696 Separation and composting plants 717
Pond treatmentprocesses 696
Aerobicoonds 696 59 niodegradation 718
and Bioremediation
Anaerobic oonds 697 Pseudomonas- the predominant
Facultative ponds 697 microorganismfor bioremediation 718
Tertiarytreatment 698 xenobiotics
Recalcitrant 720
Solidsremoval 699 Typesof bioremediation 720
Biological nitrogenremoval 700 Metabolic of microorganisms
effects on
Biological phosphorus removal 701 xenobiotics 721
Disinfection 703 in bioremediation
Typesof reactions 721
Sewage/waste watertreatment - of hydrocarbons
Biodegradation 722
a summary 703 of pesticides
Biodegradation and
Wastewatertreatmentof someindustries 703 herbicides 722
Wastewatertreatment for dairies 703 of someotherimportant
Biodegradation
Wastewatertreatment for distillery 704 comoounds 721
Wastewatertreatment for tannery 704 for more efficient
Ceneticengineering
Wastewatertreatment for sugarindustry 705 bioremediation 721
Treatment in wastewater
of antibiotics 705 Geneticallyengineered microorganisms
Waterrecycling 705 (CEMs) in bioremediation 726
Smallsewage/waste watertreatmentsystems706 Bioremediationof contaminatedsoilsand
Oxidationditch 706 wastelands 72-
Septictanks 706 Technioues of soil bioremediation ;:-
lmhofftanks 707 Bioremediation of groundwater :tc
 Coxrerurs tru Deratl (xviif

60 ClobalEnvironmental Problems 730

Greenhouseeffectandglobalwarming 730
730 Basics to Learn
Biotechnolog,v
Creen housegases
Measuresto control green houseeffect 732 62 Cell- The BasicUnit Life 749
The problem of ozone / )/- Prokaryotic cells
andeukaryotic 749
Depletionof ozone / J)
cell
Eukaryotic 750
Ozone hole / JJ
Animalcell 750
Effectsof ozone dePletion / t)
cell
Plant 752
Measuresto control ozone depletion 734
Acid rain /J + 63 Microorganisms 754
Developmentof acid rain 734 Bacteria 754
Effectsof acid rain /J +
of bacteria
Characteristics 754
Measuresto control acid rain lmportanceof bacteria 755
Environmentalsustainabilityand Controlof pathogenicbacteria /5b

biotechnology 735 Fungi /)o

of fungi
Characteristics 756
lmportance of fungi 756
Controlof fungi 757
Biotechnologf' and Socief' Viruses / J/

of viruses
Characteristics / J/
6l Biotechnology Risks,
- SocietY,
Biologicalstatusof viruses 757
Ethics,Patenting 739
lmportance of viruses 757
of biotechnologY
Benefits 739
I bio te c h n o l o g y 739
E LS of 64 Bioorganic and Biophysical
Recombinant therapeuticproductsfor Chemistry, Tools758
and Biochemistry
humanhealthcare 740
chemistry
to bioorganic
Introduction 758
andfoodconsumption
Ceneticmodifications 740
Most common orqaniccompoundsfound
Recombinantfoodsand religiousbeliefs 742 in livingsystem 758
Are GM foodssafe? 742 grouPsin
Commonfunctional
Releaseof geneticallyengineeredorganisms 742 biochemistry 758
of humangeneticrDNA research
Applications 743 Commonringstructures in
Humanembryonic stemcell research 744 biochemistry 758
Cloninghumans? 744 lsomerism 759
inventions
biotechnology
Patenting 744 Overviewof biophysicalchemistry 762
BiotechnologyProducts 744 Acidsand bases 762
Biotechnology 744 Buffers 762
Processes
Whatis a patent? 744 Solutions 762
propertyrights
Intellectual 744 Colloidal state /b3

Theprocess of Patenting 745 Diffusion 763

746 Osmosis /b4
Plantbreeders'rights
research 746 Viscosity 764
Patentingand biotechnology
Surface tension 765
and the develoPing
Biotechnology
746 lsotopes 765
countries
 (rviiil CO NTENTS IN DETAIL

Toolsof Biochemistry 766 Enzyme inhibition 790

Chromatography 766 Enzyme specificity 791
Electrophoresis 767 Coenzymes 792
Photometry- colorimerand Mechanism of enzymeaction 793
spectrophotometer 767 Diagnostic importance of enzymes 795
Ultracentrifugation 768 lsoenzymes 795
Radioimmunoassay 769
Enzyme-linkedimmunosorbant
assay 770 67 Metabolisms 796
lntroduction to metabolism 796
65 Biomolecules 771 Metabolism of carbohydrates 798
Chemical molecules of life 771 Clycolysis 798
Carbohydrates 772 Conversion of acetylCoA to pyruvate 798
Classificationof carbohydrates 772 Citricacidcycle 799
Monosaccharides 773 Cluconeogenesis 800
Clycosides 773 Glycogen metabolism 800
Derivativesof monosaccharides 773 Glycogenesis 800
Disaccharides 774 Glycogenolysis 800
Polysaccharides 774 Hexosemonophosphate shunt 800
Homopolysaccharides 774 Clyoxylate cycle 803
Heteropolysaccharides 775 Photosynthesis 803
tipids 775 Metabolism of lipids 804
Classification of lipids 775 Fattyacidoxidation 804
Fattyacids 776 Biosynthesis of fattyacids 806
Triacylglycerols 776 Metabolism of cholesterol 806
Phospholipids 776 Cholesterolbiosynthesi s 806
Lipoproteins 777 Degradationof cholesterol 806
Steroids 777 Metabolism of Aminoacids 806
Proteins and aminoacids 777 Metabolismof amino acids-
Amino acids '7'7 Q generalaspects BOB
Structureof proteins 781 Transamination 809
Primarystructure of protein 781 Deamination 809
Secondary structure of protein 782 Ureacycle 809
Tertiarystructure of protein 783 Metabolism of individual amino acids 809
Quaternary structure of protein 783 Clycine 809
Classificationof proteins 783 Phenylalanine and tyrosine 809
lsoprenoidsand pigments 784 Tryptophan 811
lsoprenoids 785 Sulfur amino acids 811
Pigments 7g5 Clutamate and glutamine 8l;
Fate of carbon skeletonof amino acids 81
66 Enzymology 786 Integrationof metabofism Bt-
Nomenclature and classification 786 Energy demandand supply o._
Chemical natureof enzymes 787 Integrationof majormetabolic
Factorsaffecting
enzymeactivity 787 pathways andenergymetabolism ci
Activesite 789 Regulation of metabolic pathways
 CorururutsrruDetnrr (xi xl

68 Inirnunology 816 69 Genetics 822

Innateimmunity 816 Briefhistoryanddevelopment of genetics 822
The immunesystem 816 Basicprinciples
of heredity
in humans 823
Organization of immunesystem 817 Patternsof inheritance 823
Cellsof the immunesystem 917 Ceneticdiseasesin humans 824
lmmunoglobulins 817 Eugenics 825
of immunoglobuli
Structure ns P ,17
::^ 70 Bioinformatics 827
of
Classes immunoglobulins Etb
Iins
of immunoglobu
Synthesis
complex
Majorhistocompatibility
Theimmuneresponse
Cytokines
lmmunityin healthand disease
Autoimmune diseases 821 Append
ices 831
Organtransplantation 821 Clossary 833
Cancers B2j Abbreviations 852

AIDS 821 Index 855

 The ScoPeof BiotechnologY

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BiotechnologY[1]
J he term biotechnology representsa fusion or an
I alliance between biology and technology.
Fran klyspe ak ing,biot ec hnologyis as old as hu m a n
civilizatio n, and is an int egr al par t of hum an l i f e .
Thus, biotechnology is a newly discovered
discipline for age-old practices. There are records
th at win e an d beer wer e pr epar ed in as ear l y a s
6 00 0 8 .C., bre ad and c ur d in 4000 B. C. Toda y ,w e
know that all these are the processesbased on the
n atu ral ca pa bilit iesof m ic r oor ganis m s .
OL D AND NEW BI O TEG HNO LO G Y
Manv authors prefer to use the term old or
traditional biotechnology to the natural processes
that have been in use for many centuriesto produce
beer, wine, curd, cheese and many other foods
The new or modern biotechnology embraces all
the genetic manipulations, cell fusion techniques
a nd th e im pr ov em ent s m ade in t he o l d
biotechnological processes.
We have to accept that the present day
bio techn olo gy is not s om et hing new bu t i t
representsa series of technologies, some of them
d atin g b ack to t hous andsof y ear s e. g. pr odu c t i o n
of foods, beverages, modification of plants and
an imals with des ir edt r ac t s .lt is only in r ec ent y e a r s
that these traditional practices are being subjected
to scientific scrutiny, understood and improved, at
least in some instances.
DEFTNTTTON(SI OF BTOTECHNOLOGY
T h e t e r m b i o t e c h n o l o g yw a s i n t r o d u c e d i n 1 9 1 7
b y a H u n g a r i a n e n g i n e e r ,K a r l E r e k y . H e u s e d t h e
term for large-scale production of pigs by using
sugar beets as the source of food. Ereky defined
biotechnology as 'all lines of work by which
products are produced from raw materials with
the aid of living thingt This definition was almost
ignored for many years. For most people,
biotechnology represented two aspects of
engineering-industrial fermentation and study of
the efficiencv at work olace.
T h e f a c t t h a t b i o t e c h n o l o g yi s i n t e r d i s c i p l i n a r yr n
nature, with a wide range of applications has
createdsome confusion with regardto its definition.
This is mainly because scientists from each
d i s c i o l i n e h a v e d e s c r i b e dt h e t e r m f r o m t h e i r o w n
perspective Around a dozen of the selected
d e f i n i t i o n so f b i o t e c h n o l o g ya r e g i v e n i n T a b l e 1 , 1.
T h e E u r o p e a nF e d e r a t i o no f B i o t e c h n o l o g y( E F B )
b r o a d l y c o n s i d e r sb i o t e c h n o l o g ya s " t h e i n t e g r a t i o n
of natural sciences and organisms, cells, parts
thereof, and molecular analoguesfor products ancl
s e r vi c e s " .
In whichever way the term biotechnology has
been defined, it essentially represents the use of
microbial, animal or plant cells or enzymes to
synthesize, breakdown or transform materials.
4 B IOTE C HNO LO CY

fermentation, (between 1857-1876) as the father of

biotechnology.
The development of biotechnology, in the first
1 . Theapplications
of scientific
andengineeringprinciples half of twentieth century is associated with the
to the processing
of materials agentst0
by biological f i e l d s o f a p p l i e d m i c r o b i o l o g y a n d i n d u str i a l
provide goodsandservices. f e r m e n tations (production of penicillin, organlc
etc.)
Theapplications
of biological
organisms, and solvents
systems
processes
to manufacturingandservice
induStries. The development of modern biotechnology is
agentssuch as c l o s e l y l i n k e d w i t h t h e a d v a n c e sm a d e i n mo l e cu l a r
The controlleduse of biological
microorganisms
or cellular
componentsfor beneiicialbiology. A selectedlist of historicalfoundationsthat
purp0ses. contributed to the advancementof biotechnology is
given in Table 1.2.
Theintegrated microbiology
useof biochemistry, and
engineeringsciences technological The biotechnologyrevolution began in the 1970s
in orderto achieve
applicationof the capabilities and early 1980s when the scientistsunderstoodthe
of microorganisms,
cultured andpartsthereof.
tissues/cells genetic constitution of living organisms. A strong
.
5. Theuseof livingorganisms andtheircomponentsin foundation of genetic engineering and modern
food,andotherindustries.
agriculture, biotechnology was laid down by Cohen and Boyer
in 1973 when they could successfullyintroduce the
6 . Theuseof biological organisms or theirconstituents
desired genes of one organism into another, and
for the transformation of inoutsinto commercial
clone the new genes(For more details,referChapter
0ut0uts.
6). lt is an acknowledged fact that of all the
A technology usingbiologicalphenomena forcopying scientific development, related recombinant DNA
andmanufacturing variouskindsof use{ul substances,technology (rDNA technology) triggend the most
8 . Controlledand deliberateapplicationof simple significant and profound advancements in
biological
agents-living
or deador cellcomponents-inbiotechnology. Thus, rDNA technology laid firm
technicallyuseful operations, eitherof productive foundations for genetic engineering.
manufacture or as serviceoperation.
Theuseof livingorganisms in systems or processes BIOTECHNOLOGY-A MULTIDISCIPLINARY
for manufacture of usefulproducts, lt may involve GROWING TREE
algae,fungi,yeast,cellsof higherplantsor
bacteria,
B i o t e c h n o l o g y i s a n i n t e r d i s c i p l i n a r y p u r su i t
animalsor subsystems of any of theseor isolated
w l t h m u l t i d l s c i p l i n a r y a p p l i c a t i o n s ,a n d it m a y b e
componenls fromlivingmatter.
represented as a growing biotechnology tree
10.Theuseof livingorganisms to solveproblems or make (Fig. 1.1). This figure gives an overview of
usefuloroducts. biotechnology with special reference to the
11. The industrial productionof goodsand servicesby f u n d a m e n t a l p r i n c i p l e s a n d s c i e n t i f i c f o un d a ti o n s,
processes usingbiological organisms, systems and b i o t e c h n o l o g i c a l t o o l s a n d a p p l i c a ti o n s o f
pr0cesses. biotechnology.
tz. A setof techniques andprocesses involving biological
malerials. Scientific foundations of biotechnology

T h e r e i s a l m o s t n o d i s c i p l i n e a m o n g t he sci e n ce
subjects that has not contributed either directly or
HISTORY OF BIOTECHNOLOGY indirectly for the growth of biotechnology.About a
dozen specialized branches of science that have
From the h istorical perspective, the
predominantlyprovided the inputsfor biotechnology
biotechnology datesbackto the time (around6000
are shown These may be appropriatelyregardedas
BC)when the yeastwas first usedto producebeer
the roots of biotechnology, and include
and wine, and bacteriawere first usedto prepare
biochemistry,genetics,molecular biology, chemical
yogurt.
engineeringand bioinformatics.A large number of
Some researchersconsider Louis Pasteur, scientists working in these specialities have
w h o i d e n ti fi e dth e ro l e o f mi crooreani sms
i n contributed to the development of biotechnology.
Chaot er1 : T H E S C OP EOF BIOT E C H N OLOC \

Year Development
Tlsrr 1.2 A selected list of historical
foundationsfor the developmentof 1977 Firstgenome
(ofbacteriophage
QX174)
biotechnology sequenced.

Development 1978 Production

ol human
insulin
in E coli
1980 mutagenesis
Site-directed byGillam
et a/.
6000Bc Winepreparation (using yeast)
4000Bc Bread making (employing yeast) 1980 U.S.SupremeCourlrulesthatgenetically
engineeredmicroorganisms can be
1670-1680 Useof microorganisms forcopper
patented
(thecasewasfoughtby Anand
minrng.
Chakrabarty).
1865 ol genetic
Inheritance characters of
Gregor Mendel. 1981 Firstdiagnostic
kitsbasedon monoclonal
antibodies
approved
in U.S
1876 LouisPasteur identified role
microorganisms intermentation 1981 DNAsynthesizers
Firstautomated sold.
1897 Extraction
of enzymes lromyeastby 1982 U.S.approved (human
humulin insulin),
the
Edward Buchner. first pharmaceutical
productof rDNA
191
0 Sewage purificationby employing forhuman
technology, use.
microorganisms established. 1982 givenin Europe
Approval fortheuseof first
1914 Productionol industrial chemicals animal vaccineproducedby rDNA
(acetone,butanol, glycerol) by using technology.
bacteria. UseofTi plasmids
1983 to genetically
transform
1917 Thetermbiotechnology wascoined by plants,
KarlEreky.
1987 bybiolistlc
Genetranster transformation.
1928 Discoveryof penicillinbyAlexander
Flaming. 1988 ofpolymerase
Development chainreaction.
1943 production
lndustrial of penicillin. 1988 U.S. patent grantedto genetically
1944 of DNAasthegenetic
ldentification engineered
mouse (susceptible
to cancer).
material(Avery, Macleod andMcOarty). 1990 granted
Approval in U.S.lortrallof human
1953 Determination of DNAstructure by cellgenetherapy.
somatic
Watson andCrick. 1990 of humangenome
Officiallaunching
1958 Semiconservative replicationof DNAby project.
Messelson andStahl.
1992 (ofyeast)
Firstchromosome sequenced
1961 Lacoperon model forgeneregulation,
proposed byJacob andMonod. 1994-95 Geneticand physical
mapsof human
elucidated.
chromosomes
1961 Launching of theJournal ol
Biotechnology andBioengineering. 1996 Firsteukaryotic (Saccharomyces
organism
1962 Microbialmining of uranium. cerevisiael
sequenced.
1962-66 Entrregenetic codedeciphered. 1997 The first mammalian sheep, Doily
1970- of thefirstrestriction
lsolation by nuclear
developed cloning.
endonuclease enzyme. 2000 Firstplantgenome
(ofArabidopsis
thaliana)
1972 Synthesisof IRNAgeneby Khorana ef a/. sequenced.
1973 Establishment of recombinant DNA 2001 Humangenome,
the first mammalian
technologyby Boyer andGohen. genome,
sequenced.
1975 Productionof monoclonal antibodies
by genome
2002 Firstcropplant(rice,Oryzasatival
Kohler andMilstein.
sequenced.
1976 NationalInstituteof Health, USAissued
firstguidelinesforrDNAresearch. 2003 Mouse(Mus nusculislgenome,the
animal
experimental model
closest to man,
1976 Sanger andGilbert developed
sequenced.
techniquesto sequence DNA.
Table 1.2 contd. hext column
6 B IOTE C H NO LO G Y

Foods
Diagnostics
Therapeutics

Pollution
Vaccines control

Cropyield

Applications of
biotechnology

Environmental
ENVIRON- monitoring
Food quality MENTAL
AGRICUL-
TURAL
MEDICAL
AND
HEALTH

Bioremediation
Animal ffff
/ / //
health
.Protein engineering
. Bioorocessfermentation
technology
. Biosensortechnology
.Monoclonalantibody
technology
GROWINGPLANTWITH Biotechnological
.Cell and tissueculture
STEMS,BRANCHESetc. tools
technology
.Transgenesistechnology
.Antisensetechnology
.DNA chip technology
.lmmunotechnology
. Metabolicengineering

," l,,rv1 l V,' ,V,

V1 ' ;r,r' ,),1I tV1 ,r,\,,,
\f ry.l rr,' ),,v
' ffff \, / r ryrA(q,/ /r,!,lnqJrtl(\,
/ r ryr A([, // r,{1'/,
/
/ / //
ROOTS Scientific
foundations of
biotechnology

Material
, sclence
Biochemical ooo
engrneenng 1a"; scl€nC€

lmmunology
biology biology

Fig. 1.1 : The biotechnology tree.

 I : T H E S C OP EO F BIOT E C H N OLOC Y
ChA O t Cr

Biotechnological tools States and Europe. The biotechnology-based

industries can be profitably run by brains than
Several methods, techniques or procedures
muscles. lt is predicted that the twenty-firstcentury
wh ic h may be c o l l e c ti v e l y c a l l ed as
will be dominated by biotechnology driven
brotechnological tools have been developedfor
industries which may be considered as new money
t r ans f or m i n g th e s c i e n ti fi c fo u n d a ti ons i nto
plants.
biot ec hno l o g i c a pl p l i c a ti o n sT.h e s eto o l s i ncl ude
protein engineering, bioprocessffermentatron
PUBLIC PERCEPTION OF
t ec hnolog y c, e l l a n d ti s s u e c u l tu re te c hnol ogy,
BIOTECHNOLOGY
transgenesis and antisense technology.
Humans are the ultimate beneficieries of
Applications of biotechnology biotechnology. This may be through healthcare,
t r a n s g e n i cp l a n t s a n d a n i m a l s ,p e s t i c i d e s f, e r t i l i z e r s ,
The fruits of biotechnologicalresearchhave
in vitro cultures etc. The public perceptions of
wide rangeof applications. In fact,thereis no other
biotechnology will significantly influence the rate
br anc hof s c i e n c ew h i c h h a sa s ma n ya p p l i cati ons
and direction of future growth of biotechnology.
as biotechnology.
The use of recombinant DNA technology has
Biotechnology hasbenefitedmedicaland health raised
safety concerns. The public attitudes to
s c ienc es ( d i a g n o s ti c s ,v a c c i n e s , th e ra peuti cs, biotechnology are mostly related to matters
of
foods),agriculturalsciences(improvedcrop yield, i m a g i n a r y
d a n g e r s o f g e n e t i c m a n i p u l a t i o n s .S o m e
f ood qual i ty , i mp ro v e d a n i ma l h e a l th) and p e o p l e
argue against genetic engineering, and
env ir onm e n ta l s c i e n c e s (p o l l u ti o n c ontrol , m a n y t i m e s ,
the public and politiciansare misled.
env ir onm e n tamo l n i to ri n gb, i o re m e d i a ti o n). There is a need for the biotechnology community
It is desirableto describeat leastone exampleof t o f r e q u e n t l y i n t e r a c t w i t h t h e m e d i a a n d p u b l i c t o
biotechnological achievementthat has helpedthe clear the unwarranted fears about the genetic
m ank ind.P ri o r to 1 9 8 2 , i n s u l i n re q u i re dfor the e n g i n e e r i n g a n d b i o t e c h n o l o g y .
treatmentof diabeticswas obtainedfrom pig and
cow pancreases. The procedurewas tedious,and T H E F U T U R E O F B I O T E C H N O L O G Y
t he us eof a n i m a li n s u l i nw a s fre q u e n tl a
y s soci ated
Biotechnology,with much fanfare, has become
wit h c om p l i c a ti o n sT. h e n th e h u m a n g ene for
a comprehensive scientific venture from the point
ins ulin was i s o l a te d ,c l o n e d a n d e x p ressedrn
o f a c a d e m i c a n d c o m m e r c i a l a n g l e s ,w i t h i n a s h o r t
microorganisms for the large scale productionof
time with the sequencingof human genome and
insulin. lnsulin was the first pharmaceutical
genomes of some other important organisms.The
product of recombinant DNA technologyapproved
future developments in biotechnology will be
for humanuse.Millionsof diabeticsworldoverare
exciting. lt may be rather difficult to make any
benef it ed b y th e b i o te c h n o l o g y o f i nsul i n
specific predictions, since new technical
pr oduc t ion .
innovations are rapidly replacing the existing
technologies.
CO M M E RC IA L IZ AT ION O F
BIOTECHNOLOGY It is expected that the development in
biotechnology will lead to a new scientific
The progress of biotechnology, to a greatextent, r e v o l u t i o n t h a t c o u l d c h a n g e t h e l i v e s a n d f u t u r e o f
is dr iv enby e c o n o mi c sT. h i si s s o s i n c eth e ul ti mate t h e p e o p l e . l t h a s h a p p e n d t h r o u g h i n d u s t r i a l
objectiveof biotechnologyis the developmentof r e v o l u t i o n a n d c o m p u t e r r e v o l u t i o n A n d n o w , i t i s
commercial products. Due to high stakes in the turn of biotechnology revolution that promises
biotechnology,the businessand research are major changes in many aspects of modern life.
closely associated. lt is a fact that many
biotechnology companies(besides the government-
ORGANIZATION OF THE BOOK
r un ins t it u ti o n sh)a v e s i g n i fi c a n tl cy o n tri b utedto
the developmentof presentday biotechnology. As the subject of biotechnology rs
Most of the commercial developments of m u l t i d i s c i p l i n a r yi n n a t u r ew i t h s e v e r a li n p u t s ,t o o l s
biotechnologyhave been centeredin the United and outputs (see Fig. t.t), the organization of the
8 B IOTE CHNO LO CY

book is as complicatedas writing of the book. the fermentation technology, downstream

However,the complex subject of biotechnology processing, enzyme technology, microbial
has been made simpler and understandable by production of organic solvents,organic acids,
g e b o o k i n to 1 0 Secti ons
o rg a n i z i nth 70 anti bi oti cs,ami no aci ds, vi tami ns, fo ods and
compri si ng
Chapters.Eachsectioncan be read independently beverages,and polysaccharides.Biomass and
a s i t i s a rra n g e di n th e h e i rarchi calorder of bi oenergy,besi des mi crobi al mi ni ng ar e also
learning. lt may however, be noted that each descri bedi n thi s secti on.
section or even a chapter cannot be treated in
isolation as the subject of biotechnology is Section Vl (Chapters 33-411
integrated,and interdependenton many other This section deals with animal cell,/animal
sections/chapters. biotechnology.The animal cell cultures-media,
biology, characterization,cell-lines, scale-up,
Section | (Ghapter 1l
primarycultures,tissueengineering, and transgenic
This section deals with the history, scope, animalsare describedin this section.
relevanceand importanceof biotechnology.
Section Vll (Chapters 42-53)
Sectian ll lGhapters 2-51
This section gives details on biotechnology
The most important information on the directly concernedwith planfsand agriculture.The
molecular biology to equip the readerfor a good plant tissue cultures, production of haploid
understanding of modernbiotechnologyis given in plants, somaclonalvariations,micropropagation,
this section.The chemistryand functionsof nucleic cryopreservation,methodology in the genetic
acids (DNA and RNA) and gene regulationare engineeringof plants, applicationsof transgenic
described. plantsetc., are described.

Section lll (Chapters 6-121 Section Vlll (Chapters 54-6Ol

T h i s s e c ti o np ri ma ri l yd e a lsw i th the basi cand The information on biotechnology with
applied aspects of genetic engineering. The reference to environmenf is furnished in this
methodology,polymerasechain reaction, gene section. The variqus aspectsof environmental
Iibraries,site-directed mutagenesis, manipulationof pollution (air/water),and treatment modalities,
gene expression,and human genomeproject are besidesthe glilbal environmentalproblems are
discussedin good detail. given in good detail.

Section lV {Ghapters 13-18f Section lX (Chapter 611

This section is directly concerned with The direct relevanceo[ biotechnology to rhe
biotechnologyin human healthcarei.e. medical/ societywith particularreference to risks,ethicsand
pharmaceutical biotechnology. The advances In patentingare described.
genetherapy,DNA in diseasediagnosisand finger-
printing,specializedproductsof DNA technology, Section X {Chapters 62-70l
and assisted reproductive technologyare dealtwith
This section provides basic information on
i n th i s s e c ti o n .
living systems-cell, microorganisms, basic
chemistry,biomolecu les,enzymologyr metabolisms,
Section V (Chapters 19-321
immunology,genetics,and bioinformatics. lt makes
The information on microbial/industrial the fundamentalsclear and helps the readerto
biotechnology is givenin thissection.This includes easilyunderstandall aspectsof biotechnology
2 DNA and RNA-ComPositionand
Structure 11
3 DNA-RePlication,Recombination,
and RePair 23
and Translation 38
4 Transcription
of Cene ExPression5t
5 Regulation
J
he re a re t wo t y pes of nuc leic ac ids , n a m e l y
I deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA) . Pr im ar ily , nuc leic ac ids s e r v e a s
re po sito riesand t r ans m it t er sof genet ic inf or m a t i o n .
Brief histor y
DNA wa s dis c ov er ed in 1869 by Jo h a n n
Fried rich M ies c her , a Swis s r es ear c he r . T h e
de mon stra tion t hat DNA c ont ained g e n e t i c
information was first made in 1944, by Avery,
Macleod and MacCary.
Fun ctio ns of nuc leic ac ids
DNA is the c hem ic al bas is of her edit y an d m a y
be regarded as the reserve bank of genetic
in forma tion . DNA is ex c lus iv elv r es oonsi b l e f o r
main tain ing t he ident it y of dif f er ent s pe c i e s o f
o rga nisms o v er m illions of y ear s . Fur t her , e v e r y
aspe ct of cellular f unc t ion is under t he c on t r o l o f
DNA. The DNA is organized into genes, the
fundamental units of genetic information. fhe
genes control the protein synthesis through the
med iatio n o f RNA, as s hown below
----J RNA-------|Protein
G^
The inte r r elat ions hioof t hes e t hr ee c las s e so f
bio mole cu les ( DNA, RNA and pr ot eins )c ons t i t u t e s
the central dogma of molecular biology or more
commonly rhe central dogma of life.
C omponents of nucl ei c aci ds
Nucleic acids are the polymers of nucleotides
(pol ynucl eoti des)
hel d by 3' and 5' phosphate
bri dgesIn otherw ords,nucl ei caci dsare bui l t up
by the monomeri cuni ts-nucl eoti des(l t may be
recal l edthat protei ni s a pol ymerof ami noaci ds).
Nucleotides are composed of a nitrogenous
base,a pentosesugar and a phosphate.Nucleo-
tides perform a wide variety of functionsin the
Ii vi ng cel l s,besi desbei ng the bui l di ngbl ocksor
monomeri cuni ts i n the nucl ei c aci d (D N A and
R N A ) structure These i ncl ude thei r rol e as
structural componentsof some coenzymes of
B -compl exvi tami ns (e.9. FA D , N A D + ), i n the
energy reactions of cells (ATP is the energy
currency), and i n the controlof metabol i creacti ons.
S TR U C TU R E OF N U C LE OTID E S
A s al ready stated,the nucl eoti deessenti al l y
consistsoI base, sugar and phosphate.The term
nucleosiderefersto base+ sugar.Thus,nucleotide
+ phosphate.
i s nucl eosi de
P uri nes and pyri mi di nes
The ni trogenousbasesfound i n nucl eoti des
(and, therefore, nucleic acids) are aromatic
heterocyclic compounds.The bases are of two
lf
12 B IOTE CHNO LO CY

H o
I
(A) N/"C'
,_ao
*,
H H
Adenine (A) Guanine(G)
H (6-aminopurine) (2-amino6-oxypurine)
I
(B) *tt"-"n
rll
""\ *'tt
I
Fig. 2.1 : General structure of nitrogen bases H
(A) Purine (B) Pyrimidine (The positions are Cytosine (C)
numbered according to the international system). (2-oxy4-aminopyrimidine)
n
tl
ty p e s -p u ri n e s a n d p y ri m i di nes.Thei r general HN)
structuresare depicted in Fig. 2.1. Purinesare l" | , l
n u mb e re di n th e a n ti c l o c kw i sedi recti on w hi l e o4N/
p y ri m i d i n e s a re n u m b e re d i n the cl ockw i se I
direction.And this is an internationally accepted H
systemto represent the structureof bases. Thymine(T) U racil( U)
methylpyrimidine) (2,4-dioxypyrimidine)
(2,4-dioxy-5
Major bases in nucleic acids
Fig. 2.2 : Structures of major purines (A, G) and
T h e s tru c tu reosf m a j o rp u ri nesand pyri mi di nes pyrimidines (C, T, U) found in nucleic acids.
found in nucleicacidsare shown in Fig.2.2. DNA
a n d R N Ac o n ta i nth e s a m ep u ri nesnamel yadeni ne
(A) a n d g u a n i n e (C ). F u rther,the pyri mi di ne The puri ne-guani neand pyri mi di n es- cyt osine,
cytosine (C) is found in both DNA and RNA. The lact am
thymi neand uraci lexhi bi ttautomeri sm.
However,the nucleic acids differ with respectto and lactim forms of cytosineare representedtn
the secondpyrimidinebase.DNA containsthymine Fig. 2.3.
(T) whereas RNA contains uracil (U). As is
At physiologicalpH, the lactam (keto) tauto-
observedin the Fig. 2.2, thymine and uracil differ meric forms are predominantlypresent.
in structureby the presence (in T) or absence(in U)
of a methylgroup. Minor basesfound in nucleicacids: Besides
the
basesdescribedabove,severalminor and unusual
Tautomeric forms of purines bases are often found in DNA and RNA. These
and pyrimidines
The existenceof a moleculein a keto (lactam)
NHa NH,
and enol (lactim) form is known as tautomerism.
c n g so f p u ri nesand pyri mi di nes
T h e h e te ro c y c l i ri tC u,C -
- c c
to\ N'/ N'-
till r t lll
with oxo \-C-l functionalgroupsexhibit tauto-
//
.c-ccc r ,/ \tt
me ri s ma s s i mp l i fi e db e l o w . - x tr r r\
o' Ho
H
OH OH
Lactam form Lactim form
lll I
I

-c+l- Fig. 2,3 : The tautomeric forms of cytosine.

- - l I-
Lactamform Lactim form
 2 : D N A AN D R N A -C OMP O S ITION
ChA P I CT A N D S TR U C TU R E
'3

The pentosesare bound to nitrogenousbasesby

B-N-glycosidic bonds. The Ne of a purine ring
binds with C',,',,, of a pentose sugar to form a
c o v a l e n t b o n d i n t h e o u r i n e n u c l e o s i d e .I n c a s e o f
p y r i m i d i n e n u c l e o s i d e s ,t h e g l y c o s i d i c l i n k a g e i s
between Nl of a pyrimidine and Ci of a pentose.

The hydroxyl groups of adenosine are esterified

Fig. 2.4 : Structuresof sugarspresentin nucleic with phosphates to produce 5'- or 3'-mono-
acids (ribose is found in RNAand deoxyribosein p h o s p h a t e s . 5 '- H y d r o x y l i s t h e m o s t c o m m o n l y
DNA; Notethe structuraldifferenceat Cr).
e s t e r i f i e d ,h e n c e 5 ' i s u s u a l l y o m i t t e d w h i l e w r i t i n g
nucleotide names. Thus AMP reoresentsadenosine
5'-monophosphate. However, for adenosine
include 5-methylcytosine, N4-acetylcytosine, N6-
3'-monophosphate,the abbreviation3'-AMP is used.
m et hy lade n i n eN, 6 , N 6 -d i m e th y l a d e n i ne, pseudo-
ur ac ilet c . l t i s b e l i e v e dth a t th e u n u s u a bases
l rn The structures of two selected nucleotides
nuc leicac id sw i l l h e l pi n th e re c o g n i ti oonf speci fi c namely AMP and TMP are depicted in Fig. 2.5.
enzymes.

S ugar s of n u c l e i c a c i d s
The five carbonmonosaccharides (pentoses) are
f ound in t h e n u c l e i ca c i d s tru c tu reR. N A contai ns
D-ribose while DNA contains D-deoxyribose.
Riboseand deoxyribosediffer in structureat C -2'
Deoxyribosehas one oxygenlessat C, compared
to ribose Gig. 2.a).

Nomenclature of nucleotides
The additionof a pentosesugarto baseproduces
a nuc leos i d el f. th e s u g a ri s ri b o s e ri
, b o n u cl eosi des
ar e f or m e d .Ad e n o s i n eg, u a n o s i n ec, y ti di ne and
ur idinear e th e ri b o n u c l e o s i d eo sf A , C , C and U
respectively. lf the sugaris a deoxyribose, deoxyribo-
nuc leos id easre p ro d u c e d .
T het er m mo n o n u c l e o ti di e
s u s e dw h e n a si ngl e
phos phat e m o i e tyi s a d d e dto a n u c l e o s i de.Thus
adenosinemonophosphate (AMP)containsadenine
+ ribose+ phosphate.
o
T he pr in c i p abl a s e sth, e i rre s p e c ti vneu cl eosi des
o--P
tl
and nuc le o ti d efo s u n d i n th e s tru c tu reo f nucl erc
acidsaregivenin Table2.1. Notethatthe prefix'd'
I
o-
(e.g.
is us edt o i n d i c a tei f th e s u g a ri s d e o x y ri bose
dA M P ) . OHH
TMP
The binding of nucleotide components
Fig. 2.5 : The structures of adenosine
T he at omsi n th e p u ri n eri n g a re n u m b e redas 1
S'-monophosphate (AMP) and thymidine
to 9 and for pyrimidineas 1 to 6 (Fig.2.1). The
S'-monophosphate (TMP) [*-Addition ol second or
carbons of sugars are representedwith an
third phosphate gives adenosine diphosphate (ADP)
associatedprime (') for differentiation.Thus the and adefiasine triphosphate (ATF) respective,lyl.
oentosecarbonsare 1' to 5'.
14 BIOTECHNOLOCY

Tasrr 2.1 Principal bases,nucleosidesand nucleotides

Base Ribonucleoside Ribonucleotide (5'-monophosphate) Abbreviation

(A)
Adenine Adenosine Adenosine or adenylate
5'-monophosphate AMP
(G)
Guanine Guanosine Guanosine or guanylate
5'-monophosphate GMP
(C)
Cytosine Cytidine Cytidine or cytidylate
5'-monophosphate CM P
(U)
Uracil Uridine 5'-monophosphate
Uridine or uridylate UMP

Base Deoxvribonucleoside Deoxyri bonucleotide (5' -monophosphate) Abbreviation

(A)
Adenine Deoxyadenosine dAMP
DeoxyadenosineS'-monophosphateordeoxyadenylate
(G)
Guanine Deoxyguanosine dGMP
DeoxyguanosineS'-monophosphateordeoxyguanylate
(C)
Cytosine Deoxycytidine Deoxycytidine or deoxycytidylate
Slmonophosphate dCMP
(T)
Thymine Deoxythymidine Deoxythymidine ordeoxylhymidylate dTMP
Slmonophosphate

Nucleoside di. and triphosphates l i ne i s C y phosphate

l i nkagew hi l e at the ot herend
of the line is Cy phosphatelinkage(Fig.2.6).
Nucleosidemonophosphates possessonly one
phosphatemoiety (AMB TMP). The addition of
Chargaff's rule of DNA composition
secondor third phosphates to the nucleosideresults
i n n u c l e o s i d e d i p h o s p h ate (e.9. A D P ) or .l
Erwin Chargaff in late 940s quantitatively
triphosphate (e.9. ATP),respectively. analysed the DNA hydrolysatesfrom different
in all the specieshe
T h e a n i o n i c p ro p e rti e sof nucl eoti desand species.He observedthat
studi edD N A had equal numbersof adenineand
nucleic acids are due to the negativecharges
contributedby phosphategroups. thymine residues(A = T) and equal numbersof
guani neand cytosi neresi dues(C = C) . This is
known as Chargaff'srule of molar equivalence
between the purines and pyrimidines in DNA
structure.The significanceof Chargaff'srule was
not i mmedi atel yreal i sed.The doub le helical
structureof DNA derivesitsstrengthfrom Chargaff's
DNA is a polymerof deoxyribonucleotides (or
rule (discussed later).
s i mp l y d e o x y n u c l e o ti d e s l).t i s composed of
monomericunits namely deoxyadenylate (dAMP), Single-stranded DNA, and RNAs which are
deoxyguanylate (dCMP), deoxycytidylate(dCMP) usuallysingle-stranded, rule.
do not obeyChargaff's
and deoxythymidylate (dTMP) (lt may be noted However, double-strandedRNA which is the
here that some authors prefer to use TMP for genetic material in certain viruses satisfies
s i,n c ei t i s f ound onl y i n D N A ). Chargaff'srule.
d e o x y th y m i d y l a te
The detailsof the nucleotidestructureare siven
above. D N A D OU B LE H E LIX
The doubl e hel i cal structureof DNA was
Schematic representation of
proposed by lames Watson and Francis Crick in
polynucleotides
1953 (N obel P ri ze, 1962). The el uc idat ionot
T h e m o n o m e ri cd e o x y n u cl eoti des
i n D N A are DNA structureis consideredas a milestonein
held together by 3', 5'-phosphodiester bridges the era o( modern biology. The structure o;
(Fig. 2.6). DNA (or RNA) structure is often DNA double helix is comparableto a twistec
reoresented in a short-handform. The horizontal ladder.The salientfeaturesof Watson-Crick mode
line indicatesthe carbonchain of sugarwith base of DNA (now known as B-DNA) are given belou
attachedto Cr,: Near the middle of the horizontal (Fig. 2.V.
Chapt er2 : D N A A N D R N A -C OMP O S I TIONA N D S TR U C TU R E 15

5' end 3. The width (or diameted of a double helix ls

20 A" (2 nm).
I
4. Each turn (pitch) of the helix is 34 A'
J
( 3 4 n m ) w i t h 1 0 p a i r s o f n u c l e o t i d e s ,e a c h p a r r
Adenine placed at a distance of about 3.4 A".

OH
o = P-o-
o
G uanine

HH

Major
groove
o = P-o-
l 0.34 nm
o
I
3' end

c
-r l' .^

P
s',

(B)
5' end

3'
Fig. 2.6 : Sttucture of a polydeoxyribonucleotide ^' '/P
J!_

/ tln

segment held by phosphodiester bonds. On the lower

part is the representation of shorl hand form of
oligonucleotides.

1. The D NA is a r ight handed double h e l i x . l t ,/ .1'

/
consists oI two polydeoxyribonucleotide chains J-t - - - - - - - - - t- v ==:- u

(strands)twisted around each other on a common ,/

A X IS .
OH
2. The two strands a(e antiparallel, i e, one 3' end
stran d ru ns in t he 5' t o 3' dir ec t ion while t h e o t h e r
in 3' to 5' dir ec t ion. This is c om par able t o t w o
parallel adjacent roads carrying traffic in opposite Fig. 2.7 : (A) Watson-Crick model of DNA helix
(B) Complementary base pairing in DNA helix.
d ire ctio n.
16 B IOTE C HNO LO CY

10. The genetic information resides on one of

the two strands known as template strand or sense
' - H\ strand. The opposite strand is antisense strand
The double helix has (wide) major grooves and
N -H
( n a r r o w ) m i n o r g r o o v e s a l o n g t h e p h o s p ho d i e ste r
backbone. Proteins interact with DNA at these
g r o o v e s , w i t h o u t d i s r u p t i n g t h e b a s e p ai r s a n d
double helix

Adenine To
Chain Conformations of DNA double helix

H V a r i a t i o n i n t h e c o n f o r m a t i o n o f t h e n u cl e o ti d e s
I of DNA is associatedwith conformational variants
*-". o f D N A . T h e d o u b l e h e l i c a l s t r u c t u r eo f D NA e xi sts
(B)
in at least 6 different forms-A to E and Z. Among
these, B, A and Z forms are important (Table 2.2).
fhe B-form of DNA double helix, described by
Watson and Crick (discussedabove), is the most
predominant Iorm under physiological conditions.
E a c h t u r n o f t h e B - f o r m h a s 1 0 b a s e p a i r s sp a n n i n g
a d i s t a n c eo f 3 . 4 n m . T h e w i d t h o f t h e d o u bl e h e l i x
H
' To
Gu ani ne Chain is 2 nm.

Fig. 2.8 : Complementary base pairing in DNA T h e A - f o r m i s a l s o a r i g h t - h a n d e d h e l i x. l t

(A) Thymine pairs with adenine by 2 hydrogen bonds c o n t a i n s 1 1 b a s e p a i r s p e r t u r n . T h e r e i s a ti l ti n g
(B) Cytosine pairs with guanine by 3 hydrogen bonds. of the base pairs by 20o away from the central axis.
The Z-form (Z-DNA) is a left-handed helix and
.l
contains 2 base pairs per turn. The polynucleotide
5 . Ea c h s tra n d o f D N A h as a hydrophi l i c strands of DNA move in a somewhat 'zig zag'
deoxyribosephosphatebackbone(3'-5' phospho- f a s h i o n , h e n c e t h e n a m e Z - D N A .
diesterbonds) on the outside (periphery)of the
m o l e c u l ew h i l e th e h y d ro p h o b i bases
c are stacked
in s i d e(c o re ).
Tmu 2.2 Conparlsonof str[ctural featuresof
6 . T h e tw o p o l y n u c l e o ti dechai ns are not different confolmationsof DNAdoublehelix
identicalbut complementary to each other due to
b a s ep a i ri n g . Feature B .D N A A-DNA I Z-DNA
7 . The two strands are held together by Helixtype i Right"handed
Right-handed Left-handed
hydrogen bonds formed by complementarybase
Helical
pairs(Fig.2.8).fhe A-T pair has2 hydrogenbonds
diameter(nm) 2.37 2.55 184
w h i l e C -C p a i r h a s 3 h y d ro g e nbonds.The C = C
is strongerby about 50% than A = T. per
Distance
eachcomplete
B. The hydrogenbonds are formed betweena turn(nm)
p u ri n e a n d a p y ri mi d i n eo n l y. l f tw o puri nes
Riseperbase
face each other, they would not fit into the pair(nm) 0.34 0.29 0.37
a l l o w a b l e s p a c e . A n d tw o p yri mi di nesw oul d
be too far to form hydrogenbonds.The only base Number
of base
percomplete;
pairs
arrangementpossible in DNA structure,from
tern 10 j 11 t1
spacialconsiderations is A-T, T-A, C-C and C-C.
Basepairtilt +19o i -1.2' -9'
9 . T h e c o m p l e me n ta ry b a s e pai ri ng i n D N A ; (variable)
helix provesChargaff'srule. f he contentof adenine
Helixaxisrotation Majorgroove: Through
basei Minorgroove
e q u a l sto th a t o f th y mi n e (A = T) and guani ne
; pairs(variable)
i
equalsto that of cytosine(C = C).
 Chaot er2 : D N A AN D R N A -C OMP O S IT I ONA N D S TR U C TU R E 17

It is believed that transition between different

h elical fo rms of DNA play s a s ignif ic ant r o l e i n
regulating gene expression.

OTHER TYPES OF DNA STRUCTURE

It is now recognizedthat besidesdoublehelical

s t r uc t ur e,DN A a l s o e x i s ts i n c e rta i n u n usual
structures.It is believedthat such structuresare
importantfor molecularrecognitionof DNA by
proteinsand enzymes.This is in fact neededfor the
DNA to dischargeits functionsin an appropriate
manner.Someselectedunusualstructuresof DNA
are brieflydescribed.

Bent DNA

In general,adeninebasecontainingDNA tracts
are rigid and straight.Bent conformationof DNA
occurswhen A-tractsare replacedby otherbasesor
a collapseof the helix into the minor grooveof A-
t r ac t . B endin gi n D N A s tru c tu reh a s a l s o been
reported due to photochemical damage or
mispairingof bases. Ftg. 2.9 : An outline of Hoogsteen triple helical
r ru g s(e .9 .c i s p l a ti np) ro duce
Cer t ainan ti tu mo d structureof DNA.
bentstructurein DNA. Suchchangedstructurecan
take up proteinsthat damagethe DNA.
The ends of eukaryoticchromosomesnamely
telomeresare rich in guanine,and thereforeform
Triple-stranded DNA
C-tetraplexes.In recent years, telomeres have
Triple-stranded DNA formationmay occur due becomethe targetsfor anticancerchemotherapies.
to additionalhydrogenbonds betweenthe bases. C-tetraplexeshave been implicated in the
Thus, a thymine can selectively form two recombi nati on
of i mmunogl obul i n
genes,and i n
Hoogsteenhydrogenbonds to the adenineof A-T dimerizationof double-strandedgenomic RNA of
pair to form I-A-f. Likewise,a protonatedcytosine
vi rus(H l V ).
the humani mmunodefi ci ency
can alsoform two hydrogenbondswith guanineof
C-C pairs that resultsin C+-C-C. An outline of THE SIZE OF DNA MOLECULE.UNITS
Hoogsteentriple helix is depictedin Fig. 2.9. OF LENGTH
Triple-helical
structureis lessstablethan double D N A mol ecul esare huge i n si ze. On an
helix. This is due to the fact that the three average/a pair of B-DNA with a thicknessof 0.34
negativelychargedbackbonestrandsin triple helix nm has a molecularweight of 660 daltons.
resultsin an increasedelectrostaticrepulsion.
For the measurement of lengths,DNA double-
Four-stranded DNA stranded structure is considered,and expresssed in
the form of base pairs (bp). A kilobase pair (kb) is
Polynucleotideswith very high contents of 103 bp, and a megabase pair (Mb) is 106 bp and a
guanine can form a novel tetramericstructure gi gabase pai r (C b) i s 10e bp. The kb, Mb and C b
called G-guarfefs.Thesestructures are planarand relationsmay be surpmarized as follows :
are connected by Hoogsteenhydrogen bonds 1 kb = 1000 bp
(Fig. 2.10A). Antiparaller four-strandedDNA .l
structures,referred to as G-tetraplexshave also Mb = 1000 kb = 1,000,000bp
been reported(Fig. 2.108). 1 C b = 1000 Mb = 1,000,000,000 bp

Biotechnology
[2]
l8 B IOTE CHNO LO CY

(A) D N A i n a cel l . S omeexampl esof orga nismwit

s h
bp and contour lengthsare listed.
. l , phagevi rus- 4.8 x l oa bp-contour lengt h
16.5 mm.

o E. coli- 4.6 x 106bp - contourlength'l .5 mm.

. D i pl oi d human cel l (46 chromosomes)

- 6. 0 x
109 bp-contour l ength2 meters.

It may be noted that the genomicDNA size is

usual l ymuch l argerthe si zeof the cel l o r nucleus
contai ni ngi t. For i nsi ance,i n humans,a 2- m et er
l ong D N A i s packedcompactl yi n a nucleusof
about 1Oumdi ameter.

Thegenomi cD N A mayexi sti n l i nearor cir cular

forms. Most DNAs in bacteria exist as closed
ci rcl es. Thi s i ncl udes the D N A of bact er ial
chromosomes and the extrachromosomal DNA of
(B) pl asmi ds. Mi tochondri a and chl oroplast sof
3', s' eukaryoti ccel l sal socontai nci rcul arD NA.

tl t1 C hromosomalD N A s i n hi gher organism sar e

G- G
mostl y l i near. Indi vi dual human chrom osom es
G-G II
u- u contai n a si ngl e D N A mol ecul e w i th var iable
tl ll I G---f-G si zes compactl y packed. Thus the sm allest
9t l -9 9t t -9 )^/ A/ |
u- u I chromosome contai ns34 Mb w hi l e the lar sestone
G _G G _G
has 263 Mb.
tl
tl II
tl
G _G G _G DENATURATION OF DNA STRANDS
II lu-'u i
The two strandsof DNA helix are held together
G _G G-G by hydrogenbonds.Disruptionof hydrogenbonds
G _G II (by changein pH or increasein temperature)results
in the separationof polynucleotidestrands.This
tl ohenomenonof loss of helical structure of DNA is
3', s', 3', s', known as denaturation(Fig. 2.11). fhe phospho-
q' e, diesterbondsare not brokenby denaturation. Loss
of helical structurecan be measuredby increase
Fig. 2.10 : Four-stranded DNA structure (A) Parallel in absorbance at 260 nm (in a spectrophotometer).
G-quartets (B) Antiparallel G-tetraplex.
Melting temperature (Im) is defined as the
temperature at which half of the helicalstructureof
D N A i s l ost.S i nceC -C basepai rsare m or e st able
It may be noted here that the lengthsof RNA
(due to 3 hydrogenbonds)than A-T base parrs
m o l e c u l e s (l i k e D N A m o lecul es)cannot be
(2 hydrogenbonds),the Tm is greaterfor DNAs
expressed in bp, sincemostof the RNAsare single-
w i th hi gherC -C content.Thus,the Tm is 65oCf or
stranded.
35% C-C contentwhile it is 70"C for 50"/' C-C
The length of DNA varies from speciesto content.Formamide destabilizeshydrogenbondsoi
species,and is usuallyexpressed in termsof base basepairsand,therefore,lowersTm. Thischemical
pair compositionand contour length. Contour compoundis effectivelyusedin recombinantDNA
length representsthe total lengthof the genomic exoenments.
Chaot er2 : D N A AN D R N A-C O MP O S IT IO NA N D S TR U C TU R E 19

two turnsof DNA wrappedround it (approximately

with 150 bp) is termedas a nucleosome, the basic
unit of chromatin.Nucleosomes are separatedby
spacer D N A to w hi ch hi stone H , i s attached
(Fi g.2.13) Thi sconti nuous stri ngof nucl eosomes,
representing beads-on-a stringform of chromatinis
termedas 10 nm fiber.The lengthof the DNA rs
consi derablreduced
y by the formati onof 10 nm
) fi ber.Thi s 10-nmfi ber i s furthercoi l edto oroduce
Twostrands 30-nmfi berw hi ch hasa sol enoi dstructure w i th si x
DNAhelix
separated nucleosomes to everyturn. These30-nmfibersare
furtherorganizedinto loopsby anchoringthe fiber
Fig. 2.11 : Diagrammatic representation of at Afi-rich regions namely scafold-associated
denaturation and renaturation of DNA. regi ons (S A R S )to a protei n scafol d. D uri ng
the course of mitosis. the looos are further
coiled, the chromosomescondenseand become
Renaturation(or reannealing) is the processIn vi si bl e.
x hic h t he s ep a ra tecdo mp l e m e n ta ry
D N A s tr ands
c an f or m a do u b l eh e l i x .

R N A i s a pol ymer of ri bonucl eoti des hel d

togetherby 3', 5'-phosphodiester bridges.Although
R N A has certai nsi mi l ari ti es
w i th D N A structure.
As already stated,the double-stranded DNA they have severalspecificdifferences
helix in eac h c h ro m o s o meh a s a l e n g thth a t i s
1 Pentose: The sugar in RNA is ribose in
t hous ands t ime sth e d i a me te ro f th e n u c l e u s.For
contrastto deoxyribosein DNA.
in hu m a n sa, 2 -me te lro n gD N A i s p a cked
ins t anc e,
in a nuc leusof a b o u t1 0 u m d i a m e te r!
T h i si s m aoe 2. P yri mi di ne: R N A contai nsthe pyri mi di ne
pos s ibleby a c o mp a c ta n d m a v e l l o u sp a c k a gi nB , uraci l i n pl aceof thymi ne(i n D N A ).
and or ganiz a ti oonf D N A i n s i d ei n c e l l .
3. Singlestrand: RNA is usuallya single-stranded
prokaryotic polynucleotide.However,this strandmay fold at
Organization of DNA
certainplacesto give a double-stranded structure,
In prokaryoticcells,the DNA is organizedas a if complementary basepairsare in closeproximity.
s ingle c hr om o s o mei n th e fo rm o f a d o ubl e-
strandedcircle. Thesebacterialchromosomes are 4. Chargaff's rule-not obeyed : Due to the
packed in the form of nucleoids,by interaction single-stranded nature,there is no specificrelation
rr ith proteinsand certaincations(polyamines). betw eenpuri neand pyri mi di necontents. Thusthe
guani necontenti s not equalto cytosi ne(asi s the
casei n D N A ).
Organization of eukaryotic DNA
In the eukaryoticcells, the DNA is associated 5. Susceptibility to alkali hydrolysis: Alkali can
ir ith variousoroteinsto form chromatinwhich then hydrol yse R N A to 2' , 3' -cycl i cdi esters.Thi s i s
gers organized into compact structures namely possibledue to the presence of a hydroxylgroupat
chromosomes(Fig. 2.12). 2' posi ti on.D N A cannot be subj ectedto al kal i
s to l ack of thi s group.
hydrol ysi due
T he DNA d o u b l e h e l i x i s w ra p p e da ro u n dthe
c or e pr ot einsn a m e l yh i s to n e sw h i c h a re b a si ci n 6. Orcinol colour reaction : RNAs can De
nature.The core is comoosedof two moleculesof hi stol ogi cal liydenti fi edby orci nolcol our reacti on
histones(H2A, H2B, H3 and H4). Eachcore with due to the presenceof ribose.
20 B IOTE C HNO LO CY

t
Naked DNA l 2nm
doublehelix

'Beads-on-a-string'
form of chromatin
I,.",
30-nm chromalin
fibre composedof
nucreosomes

Chromosomein an
extendedform
(non-condensed
loops)

Condensedform of
cnromosome

J'*0..
Metaphase
chromosome

Fig, 2.12 : Organization of eukaryotic DNA structure in the form of chromatin and chromosomes.

T
11nm
J

lntemucleosome

Fig, 2.13 : Structureof nucleosomes.

Chapt er2 : D N A AN D R N A-C O MP O S IT I ONA N D S TR U C TU R E 21

Tlelr 2.3 Cellular RNAsand their function(s)

Typeof RNA Abbreviation Function(st

Messenger
FNA mRNA genetic
Transfers fromgenes
information to
ribosomes proteins.
to synthesize
nuclearRNA
Heterogeneous hnRNA Serves
asorecursorformRNA andotherRNAs.
TransferRNA tRNA Transfers
amino acidto mRNA forprotein
biosynthesis.
Ribosomal
RNA rRNA Provides framework
structural forribosomes.
nuclear
Small RNA snRNA Involved
in mRNA processing.
Smallnucleolar
RNA snoRNA Playsa keyrolein theprocessingof rRNA
molecules.
RNA
Smallcytoplasmic scRNA Involved of proteins
in theselection forexport.
RNA
Transfer-messenger tmRNA Mostlypresent Addsshortpeptide
in bacteria.
tagsto proteinsto facilitate
thedegradation
ol
incorrectly ptoteins.
synthesized

TYPES OF RNA hydrolysisof mRNA by 5'-exonucleases. Further,

T he t hr e e ma j o r ty p e s o f R N As w i th thei r the cap may be also involved in the recognition of
mR N A for protei nsvnthesi s.
r es pec t ivcee l l u l a rc o m p o s i ti o na re g i v e n b e l ow
1. MessengerRNA (mRNA) : 5-10ok The 3' -termi nalend of mR N A contai ns a
polymer of adenylateresidues(20-250nucleotides)
2. TransferRNA (tRNA) : 10-20oh
which is known as poly (A) tail. This tail may
3 RibosomalRNA (rRNA) : 50-80% provi destabi l i tyto mR N A , besi despreventi ngi t
Besidesthe three RNAs referredabove, other from the attackof 3'-exonucleases.
R\ A s ar e al s o p re s e n itn th e c e l l s .T h e s ei ncl ude mRNAmoleculesoftencontaincertainmodifieo
. r et er ogene o u ns u c l e a r R N A (h n R N A), smal l
basessuch as 6-methyladenylates in the internal
auc lear RN A (s n R N A),s ma l l n u c l e o l a r R N A structure.
s noRNAan ) d s ma l lc y to p l a s miR c N A(s c R NA ). The
nr ajor f unc ti o n s o f th e s e R N A s a re g i ven In Transfer RNA (tRNAl
Table2.3.
TransferRNA (solubleRNA) moleculecontains
The RNAsare synthesized from DNA, and are 71-80 nucl eoti des(mostl y75) w i th a mol ecul ar
pr im ar ily in v o l v e d i n th e p ro c e s s o f protei n weight of about 25,000. There are at least 20
bios y nt hes i(C s h a p te4r ). T h e R N Asv a ry i n thei r speci esof tR N A s correspondi ngto 20 ami no
structureand function.A brief descriptionon the acids presentin protein structure.The structure
m ajor RNA si s g i v e n . of tR N A (for al ani ne)w as fi rst el uci datedby
H ol l ey.
M es s enger R N A (mR N A f
The structureof IRNA depicted in Fig. 2.14
T he m RN A i s s y n th e s i z e idn th e n u c l e us(i n
resembles thatof a cloverleaf.IRNAcontainsmainly
eukaryotes) as heterogeneous nuclear RNA
four arms,each arm with a basepairedstem.
hnRNA ) . hn R N A , o n p ro c e s s i n gl,-i b e ra testhe
f unc t ionalm R N A w h i c h e n te rsth e c y to p l asmto 1. The acceptorarm : Thi sarm i s cappedw i th
participatein protein synthesis.mRNA has high a sequenceC C A (5' to 3' ). The ami no aci d i s
m olec ularwe i g h tw i th a s h o rth a l f-l i fe . attachedto the acceptorarm.
The eukaryotic mRNA is capped at the 2. The anticodonarm : This arm, with the three
e n d b y 7 -me th y l g u a n o s itri
5' - t er m inal n ep h o sphate. specificnucleotidebases(anticodon), is responsible
It is believedthat this cap helps to preventthe for the recognitionof triplet codon of mRNA.The
22 B IOTE CHNO LO CY

3,+-Amino acid The anticodon arm 5bp

The D arm 4bp

Ribosomal RNA (rRNA|

Acceptorarm
The ribosomesare the factories of protein
synthesis. The eukaryoticribosomesare composed
of two majornucleoprotein complexes-60S subunit
and 40S subuni t.The 605 subuni tcon t ains2BS
D arm TyC arm rR N A , 55 rR N A and 5.B S rR N A w hi l e t he 40S
Complementary Variablearm .l
base pairs subuni tcontai ns B SrR N A .The functi o nof r RNAs
Anticodonarm i n ri bosomesi s not cl earl yknow n. l t i s believed
rol e i n the b indingof
that they pl ay a si gni fi cant
mRNA to ribosomesand proteinsvnthesis.

Anticodon
Other R N A s
Fig. 2.14 : Structure of transfer RNA. The variousother RNAsand their functionsare
summarisedin Table2.3.

codon and anticodon are complementary to each C A TA LY TIG R N A s-R IB OZY ME S

other.
In certaininstances, the RNA componentof a
3. The D ar m : lt is s o n a m e d d u e t o t n e (R
ri bonucl eoprotei n N Ai n associ ati on
w it h pr ot ein)
pr es enc eof dihy dr our idine.
is catalyticallyactive. Such RNAs are termed as
4. The TYC arm : This arm contains a sequence ribozymes. At leastfive distinctspeciesof RNAthat
of T, pseudouridine (representedby psi, Y) and C. act as catalystshave been identified.Three are
5. The variable arm : This arm is the most involvedin the self processings reactionsof RNAs
v ar iable in I RNA. Bas ed on t h i s v a r i a b i l i t y , t R N A s while the other two are regarded as true catalysts
(R N aseP and rR N A ).
ar e c las s if iedint o 2 c at egor ie s:
(a) Class I tRNAs : The most predominant RibonucleaseP (RNase P) is a ribozyme
(about75"h) form with 3-5 base pairs length. contai ni ng protei nand R N A componen tI.t cleaves
IRNA precursors to generate mature tRNA
(b) Class lt tRNAs : They contain 13-20 base
mol ecul es.
pair long ar m .
Base pairs in IRNA : The structure of IRNA is RNA moleculesare known to adapt tertiary
m aint aineddue t o t he c om plem e n t a r y b a s e p a i r i n g structure just like proteins (i.e. enzymes).The
in the arms. The four arms with their respective specificconformationof RNA may be responsible
base pairs are given below for its function as biocatalyst.lt is believedthat
ribozymes (RNAs) were functioning as catalysts
The acceptor arm 7bp before the occurrence of protein enzymes, during
The TYC arm 5bp the courseof evol uti on.
lI

- - eo xyribo nuc leic ac id ( DNA) is a m ac r om o l e -

,
i- J
- cule tha t c ar r ies genet ic inf or m at ion f r o m
:=^=-ation to generation lt is responsibleto preserve
--= :centitvof the speciesover millionsof years DNA
----, be regarded as a reserve bank of genetic
information or a memory bank.
Fig. 3.1 : The central dogma of life
\ sin gle ma m m alian f et al c ell c ont ains on l y a
':,r p ico gra ms( 10- 12 g) of DNA. lt is s ur pr is ingt h a t
.^ s little qu an t it y of DNA s t or es inf or m at ion t h a t
p a s s e dd o w n a c c u r a t e l yt o t h e d a u g h t e rc e l l s T h r e e
.', d ete rmine t he dif f er ent iat ion and e v e r y
--^ ction of a n adult anim al. d i s t i n c t p r o c e s s e sa r e d e s i g n e d f o r t h i s p L r r p o s e
fhe 'three Rs' of DNA-replication, recombination,
a n d r e p a i r ,a r e d e a l t w i t h i n t h i s c h a p t e r T h e r e a r e
i'.;r,; *rfi DIrEA evrylve as ge*letic
certain common features between the three Rs.
i. +-,eyial?
. T h e y a c t o n t h e s a m e s u b s t r a t e( D N A ) .
R\A mo lecules , in pr inc iple, c an per f or m t h e
-ellu lar fun ctio ns t hat ar e c ar r ied out by DNA . I n . They are primarily concerned with the making
':ct, ma ny vir us es c ont ain RNA as t he gen e t i c and breaking of phosphodiester bonds (the
-.rteria l. Ch em ic ally , DNA is m or e s t able t h a n backbone of DNA structure).
R\ \ Hen ce , d ur ing t he c our s e of ev olut ion, D N A
o Enzymes used in the three processesare mostly
: l)re ferre da s a m or e s uit able m olec ule f or lo n g -
similar/comparable.
rerm re po sito r yof genet ic inf or m at ion.

T'. + ce ntra l dogm a of lif r e

fhe biological information flows from DNA to
RNA and from there to proteins This is the central
:ro gma o f life ( Fig. 3. t ) . lt is ult im at ely t he D N A
trat co ntro ls ev er y f unc t ion of t he c ell t hr o u g h
Dro tein syn the s is .
,\s th e carri er of genet ic inf or m at ion, DNA i n a
ce ll mu st b e du plic at ed ( r eplic at ed)m
, aint aineda n d
REPLICATIONOF DNA
D N A i s the geneti cmateri al .W hen the cel l
di vi des,the daughtercel l s recei vean i denti cal
copy of geneticinformationfrom the parentcell.
Replicationis a processin which DNA copies
itself to produce identical daughter molecules of
D N A . R epl i cati on
i s carri edout w i th hi gh fi del i ty

23
 24 B IOTECHNO LO CY

specificprotein called dna A (20-50 monomers)

binds with the site of origin for replication.This
causesthe double-stranded DNA to separate.

Replication bubbles
The two complementary strands of DNA
separate at the siteof replicationto form a bubble?
Mul ti pl e repl i cati on bubbl es ar e f or m ed I n
eukaryoticDNA molecules,which is essential for a
rapid replicationprocess(Fig.3.3).

RNA primer
For the synthesis of new DNA, a shortfragment
pf RNA (about 5-50 nucleotides,variable with
speci es)i s requi redas a pri mer.This pr im er is
synthesized on the DNA templateby a specificRNA
polymerasecalledprimase.A constantsynthesisand
suppl yof R N A pri mersshoul doccuron t he lagging
Daughter DNA Parent DNA Daughter DNA strandof DNA. This is in contrastto the leading
strandw hi ch has al mosta si ngl eRNA pr im er '
Fig, 3.2 : DNA teplication-semiconservative
DNA synthesis is semidiscontinuous
and bidirectional
which is essential for the survival of the species.
The reolicationof DNA occursin 5' to 3' direc-
Synthesisof a new DNA molecule is a complex
tions,simultaneously, on both the strandsof DNA.
process involving a series of steps.
On one strand,the leading (continuous or forward)
The salient featuresof replication in prokaryotes strand-the DNA synthesisis continuous. On the
are described first. This is followed by some recent other strand, the kqCing (discontinuous or
information on the eukaryotic replication. retrograde) strand-the synthesisof DNA is discon'
tinuous.Shortpiecesof DNA (15-250nucleotides)
REPLICATION IN PROKARYOTES are producedon the laggingstrand.
Replication is semiconselvative
The parent DNA has two strands complemen-
tary to each other. Both the strands undergo
simultaneous replication to produce two daughter
molecules. Each one of the newly synthesized DNA
has one-half of the parental DNA (one strand from
original) and one-half of new DNA (fA. 3.2). This
type of replication is known as semiconservative J
since half of the original DNA is conserved in the
daughter DNA. The first experimental evidence for
the semiconservative DNA reolication was
provided by Meselson and Stahl (195S).
J
Initiation of replication
The initiation of DNA synthesis occurs at a
site called origin of replication. In case of prokaryotes,
there is a single site whereas in eukaryotes, there
Fig. 3,3 : Schematic representation of multiple
are multiple sites of origin. These sites mostly
replicatian bubbles in DNA replication.
consist of a short sequence of A-T base pairs. A
 AN
3 : D N A-R EP L IC A T IO N R E C OMB IN A TION
ChA O t Cr , D R E P A IR 25

I n t he r e p l i c a ti o nb u b b l e ,th e D N A synthesi s p- dna A protein

I

occurs in both the directions(bidirectional) from v -v

t he point o f o ri g i n .
Native DNA
Replication fork and DNA synthesis
The seoaration of the two strandsof parentDNA ;-dna A protein
o, ------J
,/---\Sf \'\)-- E,
resultsin the formationof a replicationfork. The
activesynthesis of DNA occursin this region.The E'_ a _a,

replicationfork movesalongthe parentDNA as the

daughterDNA moleculesare synthesized. Replication bubble

DNA helicases: Theseenzvmesbind to both the

DNA strandsat the replicationfork.Helicases
move '.1gf,ltnl'*s1?i#
| 3\)l- 5'
along the DNA helix and separatethe strands.
Their function is comparablewith a zip opener. 5
' '-Leading\-l-l
^ : t-3'
Lagging-
Helicasesare dependenton ATPfor energysupply. strand ---j+y strand
Single-strandedDNA binding (SSB)proteins :
Theseare also known as DNA helix-destabilizing
proteins.They possessno enzyme activity. SSB
proteins bind only to single-strandedDNA
(separatedby helicases),keep the two strands
separateand provide the template for new DNA
lt is believedthatSSBproteinsalsoprotect
synthesis.
the single-stranded DNA degradationby nucleases. Newly
synthesized
DNA
DNA synthesis catalysed
by DNA polymerase lll DNA helicase

The synthesisof a new DNA strand,catalysed

by DNA polymerase lll, occursin 5'-;3' direction. Okazaki pieces
This is antiparallelto the parent template DNA
Repllcation fork
strand. The presence of all the four deoxy-
ribonucleosidetriphosphates(dATP,dCTP, dCTP Fig. 3,4 : Overview of DNA replication process
and dTTP)is an essential prerequisite
for replication (SSB-Srng/e-stranded binding p rotein).
to take place.
The synthesis"of two new DNA strands,
simultaneously,takes place in the opposite by sequentialadditionof new nucleotides. The other
direction-one is in a direction(5'+3') towardstheDNA strand(laggingstrand)with 5'-end presents
t
some problem, as there is no DNA polymerase
replicationfork which is continuous,the other in a
direction (5'-+3') away from the replicationfork enzyme (in any organism)that can catalysethe
which is discontinuous (Fig.3.4. additionof nucleotidesto the 5' end (i.e. 3'-+5'
direction) of the growing chain. This problem
The incomingdeoxyribonucleotides are added
howeveris solvedby synthesizing this strandas a
one after another,to 3' end of the growing DNA
(PPi) seriesof smallfragments. These piecesare made in
chain (Fig.3.t. A moleculeof pyrophosphate
the normal5'-+3'direction,and laterjoinedtogether.
is removedwith the additionof each nucleotioe.
The templateDNA strand(the parent)determines Okazaki pieces : The small fragmentsof the
the base sequence of the newly synthesized discontinuously synthesized DNA are called
complementary DNA. Okazakipieces.Theseare producedon the lagging
strandof the oarentDNA. Okazakipiecesare later
Polarity problem loined to form a continuousstrandof DNA. DNA
The DNA strand(leadingstrand)with its 3'-end polymeraseI and DNA ligaseare responsible for
(3'-OH)orientedtowardsthe fork can be elongated this process(detailsgiven later).
26 B IOTE C HNO LO CY

E
o

z CTG AACTG
o
ilr il ill il il ilt il lll ll
ilt il lll ll Lt ill ll ll l1
G ACUTG AC
o

(g

o
E

RNAprimer GrowingDNA

Fig. 3.5 : DNA replication with a growing complementary strand.

Froof.reading function of Supercoils and DNA topoisomerases

DNA polymerase lll
Fidelit y of r eplic at ion is t he m o s t i m p o r t a n t f o r
the very existenceof an organism. Besidesits 5'-+3'
dir ec t ed c at aly t ic f unc t ion, DN A p o l y m e r a s e l l l
also has a proof-reading activity. lt checks the
inc om ing nuc leot idesand allow s o n l y t h e c o r r e c t l y
matched bases (i.e. complementary bases) to be
added to the growing DNA strand. Further, DNA
polymerase edits its mistakes (if any) and removes
t he wr ongly plac ed nuc leot ide b a s e s .
Replacement of RNA primer by DNA
The s y nt hes isof new DNA s t r a n d c o n t i n u e s t i l l
it is in c los e pr ox im it y t o RNA p r i m e r . N o w t h e
DNA polymerase I comes into picture. lt removes
t he RNA pr im er and t ak es i t s p o s i t i o n . D N A
polymeraseI catalysesthe synthesis(5'+3' direction)
of a fragment of DNA that replaces RNA primer
(Fig. 3.6).
As the doublehelix of DNA separates from one
sideand replicationproceeds, supercoils areformed
at the otherside.The formationof supercoils can be
better understoodby comparingDNA helix with
two twistedropestied at one end. Hold the ropesat
the tied end in a fixed position.And let your friend
pull the ropes apart from the other side. The
formationof supercoilsis clearlyobserved.
The problemof supercoils that comesin the way
of DNA replicationis solvedby a groupof enzymes
called DNA topoisomerases.Type I DNA
cutsthe si ngl eD N A strand(nuclease
topoi somerase
o + 5' NewlysynthesizedDNA

The enzyme DNA ligase catalyses the forma-

tion of a phosphodiester linkage between the
3'
DNA s y nt hes iz edby DNA poly m e r a s e l l l a n d t h e I
s m all f r agm ent s of DNA pr o d u c e d b y D N A I 1--Nick sealed
| | by DNA ligase
poly m er as e L This pr oc es s - nic k s e a l i n g - r e q u i r e s
3'--------------- -5'DaughterDNA
energy, provided by the breakdown of ATP to AMP
and PPi. 5' 3' ParentDNA

Another enzyme-DNA polymerase ll-has

Fig. 3.6 : Overview of the action of DNA
been isolated. lt participates in the DNA repair
polymercse I and DNA ligase.
oroceSs.
Chapt er3 : D N A-R EP L IC A T IO N ,R E C OMB IN A TION
AN, D R E P A IR 27

activity)to overcomethe problemof supercoils and of eukaryotes.This is depicted in Fig. 3.7, and
thenreseals the strand(ligaseactivity).Typell DNA brieflydescribedhereunder.
topoisomerase (also known as DNA gyrase)cuts
The parentalstrandsof DNA are separatedby
both strandsand resealsthem to overcometne the enzyme hel i case.A
si ngl e-stranded
DNA
pr oblemof s u p e rc o i l s .
binding protein called replicationprotein A (RPA)
REPLICATION IN EUKARYOTES bindsto the exposedsingle-stranded template.This
strandhas been openedup by the replicationfork
Replic at io no f D N A i n e u k a ry o te sc l o sel y (a previouslyformed Okazaki fragmentwith an
resembles that of prokaryotes. Certaindifferences, RNA primer is also shown in Fig. 3.4).
howev erex , ist.Mu l ti p l eo ri g i n so f re p l i c a ti o ni s a
The enzyme primase forms a complex with
characteristic featureof eukaryoticcell. Further,at
DNA polymerase ocwhich initiatesthe synthesis of
least frve distinct DNA polymerasesare known in
Okazaki fragments.The primaseactivity of pol
eukaryotes. Creek lettersare usedto numberthese
u-pri masecompl exi s capabl eof produci ng1O-bp
enz y m es .
RNA primer.The enzymeactivityis then switched
1. DNA polymerased is responsiblefor the from pri mase to D N A pol ymeraseG w hi ch
of R N A p ri me rfo r b o th th e l e a d i n gand elongatesthe primer by the addition of 20-30
s y nt hes is
laggings t r an d so f D N A . deoxyribonucleotides. Thus, by the action of pol
2. DNA po l y m e ra spe i s i n v o l v e di n th e repai r c,-primase complex,shortstretchof DNA attached
of DNA . lt s fu n c ti o n i s c o m o a ra b l ew i th DN A to RNA is formed. And now the comolex
polymeraseI found in prokarybtes. dissociatesfrom the DNA.
3. DNA polymeraseTthis enzymeparticipates The nextstepis the bindingof replicationfactor
in t he r eplic a ti o no f m i to c h o n d ri aDl N A. C (RFC)to the elongatedprimer(shortRNA-DNA).
RFC servesas a clamp loader,and catalysesthe
4. DNA polymerased is responsiblefor the
replicationon the leadingstrandof DNA. lt also assembl yof prol i ferati ngcel l nucl ear anti gen
(PCNA)molecules. The DNA polymerase 6 bindsto
possesses proof-reading activity.
the sl i di ng cl amp and el ongatesthe .Okazaki
5. DNA polymerasee is involved in DNA fragmentto a final lengthof about 150-200 bp.
synthesison the laggingstrandand proof-reading B y thi s el ongati on,the repl i cati on compl ex
f unc t ion. approachesthe RNA primer of the previous
The differences in the DNA replicationbetween Okazakifragment.
bacteria and human cells, attributed to the The R N A pri merremovali s carri edout by a pai r
enz y m es , ' ares u c c e s s fu l l uy s e d i n a n ti b a cteri al of enzymes namely RNase H and flap
therapy to target pathogen(bacterial)replication endonuclease | (FENI).This gap createdby RNA
and s par et he h o s t(h u ma n )c e l l s . removal is filled by continuedelongationof the
new Okazakifragment(carriedout by polymerase
PBOCESs OF NEPL'CAT'ON IN 6, descri bed above).The smal lni ck that remai nsi s
EUKANYOTES fi nal l yseal edby D N A l i gase.

T he r eplic a ti o no n th e l e a d i n g (c o n ti n uous) EukaryoticDNA is tightly bound to histones

(basic proteins)to form nucleosomeswhich, in
stand of DNA is rather simple, involving DNA
polymerase 6 and a sliding clamp called turn, organi ze i nto chromosomes.D uri ng the
proliferating cell nuclear antigen (PCNA). PCNA is courseof replication,the chromosomes are relaxed
so namedas it was first detectedas an antigenin and the nucleosomes get loosened. The DNA
c e l l s .PC N Afo rm sa ri ng
t he nuc leiof r e p l i c a ti n g strands separate for replication,and the parental
ar oundDNA t o w h i c h D N A p o l y m e ra s6e b i nds. histones associatewith one of the parentalstrands.
Formationof this ring also requiresanotherfactor As the synthesis of new DNA strand proceeds,
namely replication factor C (RFQ. histonesare also producedsimultaneously, on the
parentstrand.At the end of replication,of the two
The replication on the l^gging (discontinuous) daughterchromosomalDNAsformed,one contains
strand in eukaryotes is more complex when the parentalhistoneswhile the otherhasthe newly
comparedto prokaryotes or eventhe leadingstrand synthesized histones.
2A B IOTE C H NO LO CY

o, Previous
- Okazakifragment
5' Templatetor
lagging
strand

DNA polymerasect-
primasecomplex

RNaseH

DNA ligase

s',

Fig. 3.7 : An outline of DNA replication on the lagging strand in eukaryotes (RPA-Replication protein A;
PCNA-Proliferating cett nuclear antigen; RFC-Reptication factor C; RNase H-Ribonuclease H; FENI-Flap
endonuclease l; Note : Leading sttand not shown)'
 AN
Chaot er3 : D N A -R E PL IC AT ION ,R E C OMB IN A TION , D R E P A IR 29

,NH'BTTOBS OF DNA REPL'CA|TON Detectionof

damaged
Bacteriacontaina specifictype ll topoisomerase DNA
namelygyrase.This enzymecuts and resealsthe
circular.DNA(of bacteria), and thusovercomesthe
problemof supercoils. Bacterialgyraseis inhibited Detectionof
incomplete
by t he ant i b i o ti c sc i p ro fl o x a c i nn, o v o b i o ci nand replication
nalidix ic a c i d . T h e s e a re w i d e l y u sed as
antibacterial agentssincethey can effectivelyblock
n f D N A a n d mu l ti p l i c a ti oonf cel l s.
t he r eplic ati o o
Theseantibacterial agentshavealmostno effecton DamagedDNA
detected
humanenzymes.

Certain compounds that inhibit human Fig. 3,8 : The cell cycle of a mammalian cell
(M-Mitotic phase; Gr-Gapl phase; G6Dormant phase;
topoisomerases are used as anticanceragentse.g.
S phase-Period of replication; Gp-Gap 2 phase).
adriamycin,etoposide, doxorubicin.The nucleotide
analogst hat i n h i b i tD N A re p l i c a ti o n
a re a l soused
as ant ic an c e r d ru g s € ,8 . 6 -me rc a p t opuri ne,
C ycl i nsand cycl i n-dependent ki nases(C D K 1,
5- fluor our a c i l .
CDK2,CDK4,CDK6)are intimatelyconnectedwith
the progression of cell cycle.For instance, cyclin D
CELL CYCLE AND DNA REPLICATION
levelsrise in late C1 phasewhich activateCDK4
The cell cycle consistsof four distinctphasesin and C D K 6.Thi s resul tsi n the assembl of y nucl ear
higher organisms-mitotic,Cr, S and C, phases proteins in a complex form in late C1 phase.
(Fig.3.A. When the cell is not growing,it existsin
a dor m anto r u n d i v i d i n gp h a s e(C !. C -' p h asei s Gell cycle check points
by activeproteinsynthesis.
characterized A s depi cted i n Fi g.3.B , there occurs a
continuous monitoringof the cell cyclewith respect
of DNA occursonly once in S-phase
Replication
to DNA replication,chromosomesegregation and
and the chromosomesget doubled i.e. diploid
genomegets convertedinto tetraploid.The entire integrity.lf any ddmageto DNA is detectedeither
phaseof the cycle, or if there is a
processof new DNA synthesistakes place in in G1 or C2
formation of defective spindle (i.e. incomplete
about B-10 hours and a large number of DNA
polymerases (500-1,000) are simultaneously chromosomal segregation),
the cell cycle will not
progressuntil appropriatelycorrected.lf it is not
involved in this process. lt is believed that
possibleto repair the damage done, the cells
methylationof DNA servesas a markerto inhibit
undergo apoptosis(programmedcell death).
reolication.

The G, phaseis characterized by enlargement Gancer and cell cycle

of cytoplasmand this is followedby the actualcell
Cancerrepresentsan excessivedivision of cells.
divisionthat occursin the mitotic phase.
In cancer,a largequantityof cells are in mitosis
and most of them in S-phase.
Gyclins and cell cycle
Majority of the drugs used for cancertherapy
Cyclinsare a groupof proteinsthat are closely
are designedto block DNA replicationor inhibit
associatedwith the transition of one phaseof cell
the enzymesthat participatein replication(directly
cycle to another, hence they are so named. The
. Methotrexate(inhibitsdihydrofolate
or indirectly)
most importantcyclinsare cyclin A, B, D and E.
reductase)and 1-fluorouracil (inhibitsthymidylate
The concentrations of cvclinsincreaseor decrease
synthase)block nucleotidesynthesis.
during the courseof cell cycle. Thesecyclins act
on cyclin-dependent kinases (CDKs) that ln recent years, topoisomerase inhibitors are
phosphorylate certainsubstances for
essential the being used.They block the unwindingof parental
transitionof one cycle to another. DNA strandsand preventreplication.
30 B IOTE C HNO LO CY

TE!.OMERES AND TELOMERASE (A) s', 3' ParentalDNA strand

5' LaggingDNA strand
Ther e ar e c er t ain dif f ic ult iesin t h e r e o l i c a t i o no f t
linear DNAs ( or c hr om os om es )o f e u k a r y o t i c c e l l s . RNAprimer
Okazakifragment--f
T he leading s t r and of DNA c a n b e c o m p l e t e l y
t^.
[ . Pnmerremoveo
synthesizedto the very end of its template. This is
not pos s ible wih t he lagging s t r a n d , s i n c e t h e +
5' --3' ,
r em ov al of t he pr im er RNA le a v e s a s m a l l g a p LTelomere
3,-5,
which cannot be filled (Fig. 3.9A). Consequently,
t he daught er c hr om os om es wil l h a v e s h o r t e n e d
D NA m olec ules . This bec om e s s i g n i f i c a n t a f t e r
s ev er al c ell c y c les inv olv in g r e p l i c a t i o n o f
chromosomes. The result is that over a oeriod of
time, the chromosomes may lose certain essential
genes and the cell dies. This is however, avoided to
a large extent.
s'-TTAGGGTTAGGG 3'
Telomeres are the special structures that
3'-.CAAUCCCAAUC\
prevent the continuous loss of DNA at the end of e"
------

the chromosomes during the course of replication. " \"I-:,u-_)r'

-'------ '/;'

Thus, they protect the ends of the chromosomes, I

and ar e als o r es pons ible t o p r e v e n t t h e I Extensionof 3'end
DNAsynthesis)
chromosomes from fusing with each other. l(New
Telomeres are many repeat sequences of srx
nucleotides present at the ends of eukaryotic 3'-
(CAAUCCCAAUC)
c hr om os om es .Hum an t elom er esc o n t a i n t h o u s a n d s 3', s',
oI repeat TTAGGG sequences,which can be up to I
a lengt h of 1500 bp. JTranslocation
s/-TTAGGGTTAGGGTTAG 3'
Role of telomerase 3'-
(CAAUCCCAAUC)
Telomeres are maintained bv the enzvme s', 5',
telomerase, also called as telomere terminal I, NewDNAsynthesis
transferase.Telomeraseis an unusual enzyme as it l(Extension)
is composed of both protein and RNA. In case of
+
5/-TTAGGGTTAGGGTTAGGGfiAG 3,
h um ans , t he RNA c om ponent is 4 5 0 n u c l e o t i d e sr n
3'-
l engt h, and at t he 5' - t er m inal a n d i t c o n t a i n s t h e ( c A A U C C C A AU C )
a/ 5',
sequence 5'-CUAACCCUAAC-3'. lt may be noted
t hat t he c ent r al r egion of th i s s e q u e n c e i s Extensionand translocation
reoealedseveraltimes
complementary to the telomere repeat sequence
Bindingof DNA polymerase
5'-TTACCC-3'. The telomerase RNA seouence can
crand primase
be used as a temolate for extension of telomeres
(Fig. 3.98).
s',
The telomeraseRNA base bairs to the end of the
D NA m olec ule wit h t elom er es a n d e x t e n d s t o a
s m all dis t anc e. Then t r ans loc a t i o n o f t e l o m e r a s e
occurs and a fresh extension of DNA takes olace. 5',
This pr oc es sof DNA s y nt hes isa n d t r a n s l o c a t i o ni s CompletedDNA strands
repeated several times until the chromosome gets
Fig, 3.9 : Replication of DNA with telomeres
sufficiently extended. The extension process gets
(A) Formation of telomere (B) Role of telomerase in
completed through the participation of DNA
the replication of telomere (Note : Only 23 repeat
p oly m er as e and pr im as e c om pl e x a n d s e a l i n g o f
seguences of telomere shown for clarifi).
the new DNA formed
Cr apt er3 : D N A -R E PL IC AT ION , R EC O MB IN A TION
AN, D R E P A IR 31

It may be noted here that as such the telomeres

ABCDEFGHIJKLKNOP
do not encode proteins. Hence, when extended by
telomerase,they need not have to remain the same +
len gth , an d s om e s hor t ening will not pos e a n y abcdefghijklmnop
problem. During the course of repeatedcell cycles,
there occurs progressive shortening of telomeres, Parentalchromosomes
and this has to be prevented, which is appropriately
carried out by telomerase. I Horotogor.
Jrecombination
TELOMEBE SEVESCENCE AND CANCEB
''V ABCDEFGHljklmnop
Evidence is now forthcoming that telomerase ts
no t a ctive in all t he m am m alian c ells . T h i s r s
ma inly b ecaus e c ells t hat hav e unde r g o n e abcdefgh iJKLMNOP
differentiation no longer divide or divide only to a
Chromosomeswith
limited e xte nt . Telom er as eis highly ac t iv e i n t h e DNA lrom both parents
early embryo, and after birth it is active in the
re pro du ctive and s t em c ells . St em c ells d i v i d e Fig. 3.10 : A diagrammatic representation of
co ntin uo uslyt hr oughoutt he lif et im e. ofan or g a n i s m homologous recombination.
to produce new cells. These cells in turn are
re sp on sib let o t is s uesand or gans in t he f unc t i o n a l .l
. Homologousrecombination: This is also
statee.g. hematopoietic stem cells of bone marrow.
called as general recombination, and occurs
Many biologists linft the process of telomere betw eeni denti calor nearl yi denti calchromosomes
shortening with cell senescence (i.e. cell death). (D N A sequences).The best exampl e i s the
This is mainly based on the observationsmade in recombinationbetweenthe paternaland maternar
the in vitro mammalian cell cultures. However, chromosomalpais (Fig.3.lO).
some researchersquestion this relation between
2 Non-homologousrecombination: This is
te lome re sh or t ening and s enes c enc e
regarded as illegitimate recombinafion and does
Can ce rou sc ells ar e able t o div ide c ont inu o u s l y . not requi reany speci alhomol ogoussequences.
There is a strong evidence to suggest that the Transposition is a good example of non-
absence of senescencein cancer cells is linked to homologousrecombination. Randomintegrationof
the activation of the enzyme telomerase. Thus, outsi degenes i nto mammal i anchromosomes is
te lome re le ngt h is m aint ained t hr oughout m u l t i p l e anotherexamol e.
ce ll divisio ns . lt is howev er , not c lear wh e t h e r
telomerase activation is a cause or an effect of H OMOLOGOU S R E C OMB IN A TTON
cancer. There is however, evidence to suggestthat It is a known fact that the chromosomes
are not
telomeraseactivation is in fact the cause of certarn passedon intact from generationto generation.
cancers e.g. dyskeratosis congenita due to a Instead,they are inheritedfrom both the parents.
mutation in the gene responsible for the RNA Thi si s possi bl e
due to homol ogousrecombi nati on.
component of telomerase. Three models have been put forth to explain
The enzyme telomerase is an attractive target homologousrecombinations.
for cancer chemo.therapy. The drugs have been
. H ol l i daymodel
designedto inactivatetelomerase,and consequently
in du ce se ne s c enc ein t he c anc er c ells . This i n t u r n . Mesel son-R addimodel
ng
preventsthe rapid cell proliferation. . Double-strand
breakmodel.

Holliday model
H ol l i daymodel(proposed by H ol l i dayi n 1964)
Recomb inat ionbas ic ally inv olv es t he ex ch a n g e i s the si mpl est among the homol ogous
of genetic information. There are mainly two types recombi nati on model s.l t i s depi ctedi n Fi g. 3.11,
of recombinations. and brieflyexplainedin the next page.
o2 B IOTE C H N O LO CY

The two homologous chromosomescome

closer,get properlyaligned,and form single-strand
breaks.Thi s resul tsi n tw o al i gnedD N A duplexes.
Now the strandsof eachduplexpartlyunwind and
ab invade in the oppositedirection to form a two
Twohomologous DNAmolecules strandscrossbetweenthe DNA molecules.
withsingle-strand
breaks
J There occurs si mul taneousunw i ndi ng and
AB rew i ndi ngof the dupl exesi n sucha w ay thatther e
i s no net changei n the amountof basepai ri ng,but
the positionof crossovermoves This phenomenon
-.r/-+-
-----l!)- referred ro as branch migration, results in the
formationof heteroduplexDNA. The enzymeDNA
CrossDNAstrands l i gaseseal sthe ni ck. The tw o D N A dupl exes( 4
strandsof DNA),joined by a singlecrossover point
oJB
can rotate to create a four-standed Holliday
iunction.Now the DNA moleculesare subjectedto
symmetricalcuts in either of the two directions,
and the cut ends are resealedby ligase.
ao
Hoteroduplex
sealedby DNAligase The D N A exchange i s determi nedby t he
lcrtz di recti onof the cuts,w hi ch coul d be hori zont alor
+, vertical.lf the corssstrandsarecut horizontally(cut
BiA
1), the fl anki ng genes (or markers,i .e. AB/ ab)
remain intact, and no recombinationoccurs.On
the other hand, if the parentalstrandsare cut
vertically(cut 2), the flankinggenesget exchanged
(i.e.Ab/aB)due to recombination.

N ON .I{ OMOLOGOU S R E C OMB IN A TION

The recombinationprocesswithout any special
homologoussequences of DNA is regardedas non-
homol ogous
recombinatron.

Transposition
Transposition primarily involvesthe movement
of specific pieces of DNA in the genome. The
mobilesegments of DNA are called transposonsor
transposableelements.They were first discovered
by B arbaraMcC l i ntock(i n 1950) i n mai ze ,and
thei r si gni fi cancew as i gnored for about t wo
decadesby other workers.
Transposonsare mobileand can movealmostto
any placein the targetchromosome. Thereare two
modesof transposition. One that involvesan RNA
a i.........' ...r b intermediate,and the otherwhich doesnot involve
Recombined
daughter Recombined
daughter RNA intermediate.
DNAstrands DNAstrands
Retrotransposition: Transpositioninvolving
Fig. 3.11 : Hollidaymodelfor homologous
RNA intermediate representsretrotransposition
recombination(Note : Heteroduplexregionsare
(Fig.3.12).By the normalprocessof transcription,
shown in dotted boxes).
a copy of RNA formed from a transposon(also
 AN
c hapt er3 : D N A -R E PL IC AT ION ,R EC O MB IN A TION , D R E P A IR 33

Transooson and the targetsite to resultin copying of the donor

element.
ln case of conservative transposition, the
transposonis excised and reintegratedat a new
sife.
DNA transposition is less common than
retrotransposition
in caseof eukaryotes.
However,
in caseof prokaryotes,
DNA transposonsare more
importantthan RNA transposons.

Significance of transposition
It is now widely acceptedthat a largefractionof
the human genome has resulted due to the
Transposon copy
(retrotransposon)accumulation of transposons.Shorf intetspersed
elements(SlNEs)are repeatsof DNA sequences
which are presentin about 500,000 copies per
rcpreaentation
Fig. 3,12 : A diagrammatic ot haploid humangenomee.g. Alu seguences,
Long interspersed elements (LrNfs) are also
repeatedDNA sequences and are presehtin about
called as retrotransposon).Then by the enzyme 50,000 copi es i n the human genome e.g. L1
reversetranscriptase,DNA is copiedfrom the RNA. elements.
The newly formed DNA which is a copy of the
Someof the diseasescausedbv mutationsare
transposongets integratedinto the genome.This
due to insertionof transponsinto a genes.
integrationmay occur randomly on the same
chromosomeor, on a differentchromosome. As a
there are now two
resultof the retrotransposition,
copiesof the transposon,at differentpointson the
genome.
DNA transposition: Some transposonsare Being the carrier of genetic information,the
capableof direct transpositionof DNA to DNA. cel l ul ar D N A must be repl i cated(dupl i cated),
This may occur either by replicativetransposition maintained,and passeddown to the daughtercells
or conservative
transposition (Fig.3.13). Both the accurately. In general,the accuracyof replicationis
mechanisms requireenzymesthat are mostlycoded extremely high. However, there do occur
by the geneswithin the transposons. replicationerrors.lt is estimated
that approximately
ln the replicative transposition, a direct one erroris introducedper billion basepairsduring
interactionoccurs betweenthe donor transposon each cycle of replication.The cells do possesthe
capabilityto repair damagesdone to DNA to a
largeextent.

Gonsequences of DNA damage

Despite an efficient repair system for the
damagedDNA, replicationerrorsdo accumulate
Conservative that ultimately result in mutations.The human
body possesses 1014 nucleatedcells, each with
3 x 109 base pairs of DNA. lt is estimatedthat
about 1016cel l di vi si onsoccur i n a l i feti me.l f
- 10-10mutationsper basepair per cell generation
Fig. 3.13 : A diagrammatic representation of DNA escaperepair,this resultsin aboutone mutationper
106 basepai rsi n genome.

Biotechnology [3]
34 B IOTE CHNO LO CY

Bes ides t he pos s ible er r or s i n r e p l i c a t i o n , t h e

DNA is constantly subjected to attack by both Trau 3.1 Dlajor types of DlU| danages
phy s ic al and c hem ic al age n t s . T h e s e i n c l u d e
Category Types
r adiat ion, f r ee r adic als ,c hem i c a l s e t c . , w h i c h a l s o
r es ult in m ut at ions . Single-basealteration Deamination
(C-+U;A-+hVpoxanthine)
It is fortunate that a great majority of the
m ut at ionspr obablv oc c ur in t h e D N A t h a t d o e s n o r
Depurination
enc ode pr ot eins , and c ons eq u e n t l y w i l l n o t h a v e Basealkylation
any s er ious im pac t on t he o r g a n i s m . T h i s i s n o t , Insertion
or deletion
of
howev er , all t he t im e t r ue, s i n c e m u t a t i o n s d o nucleotides
oc c ur in t he c oding r egionsof D N A a l s o . T h e r e a r e Incorporation
of baseanalogue
s it uat ionsin whic h t he c hange i n a s i n g l e b a s e p a r r Two-basealteration UVlightinduced pyrimidine
in t he hum an genom e c an c a u s e a s e r i o u sd i s e a s e dimeralteration(T-T)
e. g. s ic k le- c ell anem ia. Chain breaks freeradical
Oxidative formation
radiation
lonizing
TYPES OF DNA DAMAGES Cross-linkage Between basesin thesameor
The dam age done t o DNA b y p h y s i c a l ,c h e m i c a l uPPUJlttr
^^^^ail^ )U
^ + r^ adlruJ
i^

and env ir onm ent alagent sm ay b e b r o a d l y c l a s s i f i e d the DNAandprotein

into four categorieswith different types (Table 3.1).
The DNA damage may occur due to srhgle-base
alterations (e.g. depurination, deamin ation), two-
base alterations (e.g. pyrimidine diamer) charn
breaks (e.g. ionizing radiation) and cross-linkages
(e.g. between bases).Some selected DNA damages
are briefly described.
The oc c ur r enc e of s pont a n e o u s d e a m i n a t r o n
bases in aqueous solution at 37"C is well known.
Cytosine gets deaminated to form uracil while
adenine f or m s hy pox ant hine.
Spont aneousdepur inat ion , d u e t o c l e a v a g e o f
gly c os y l bonds ( t hat c onne c t p u r i n e s t o t h e
backbone) also occurs. lt is estimated that
2000- 1 0, 000 pur ines m ay be l o s t p e r m a m m a l i a n
c ell in 24 hour s . The depur ina t e ds i t e sa r e c a l l e d a s
abasic sites. Originally, they were detected rn
purines, and called apurinic sites (AP sifes) which
representlack of purine. Now, the term AP sites is
generally used to represent any base lacking in
DNA.
The production of reactive oxygen species is are of two sub-types.
often associated with alteration of bases e.g.
f or m at ion of B- hy dr ox y gu a n i n e . F r e e r a d i c a l
formation and oxidative damage to DNA increases
with advancement of age.
Ult r av iolet r adiat ions r es u l t i n t h e f o r m a t i o n o f
c ov alent link s bet ween adjac e n t p y r i m i d i n e s a l o n g
the DNA strand to form pyrimidine dimers. DNA
c hain br eak s c an be c aus ed b y i o n i z i n g r a d i a t i o n s from the DNA, respectively,causing insertion or
( e. 9. X- r ay s ) .
Between
m0lecules
MUTATIONS
The genetic macromolecule DNA is highly
stable with regard to its base compositioh and
sequence. However, DNA is not totally exempt
from gradual change. A general picture of DNA
d a m a g e a n d i t s r e p a i r i s a l s o d e s c r i b e d i n th i s
chapter.
Mutation refers to a change in the DNA structure
of a gene. The substances(chemicals) which can
induce mutations are collectively known as mutagens.
T h e c h a n g e s t h a t o c c u r i n D N A o n m u ta ti o n
are reflected in replication, transcription and
translation.
Types of mutations
Mutations are mainly of two major types-point
mutations, frameshift mutations (Fig. 3.1a)
1 . Point mutations : The replacement of one
b a s e p a i r b y a n o t h e r r e s u l t si n p o i n t m u ta ti o n . Th e y
( a ) T r a n s i t i o n s : I n t h i s c a s e , a p ur i n e ( o r a
pyrimidine) is replaced by another.
(b) Transversions: These are characterized by
r e p l a c e m e n to f a p u r i n e b y a p y r i m i d i n e o r
vtce versa.
2 Frameshift mutations : These occur when
one or more base pairs are inserted in or deleted
deletion mutations.
 AN
Chapt er3 : D N A -R E PL IC AT ION ,R EC O MB IN A TION , D R E P A IR 35

(A) genetic code. Therefore,there are no detectable

-c-G-A-G- -c-G-G-G- effectsin silentmutation.
ttrl ttll 2. Missense mutation: In this case,the changed
-G-C-T-C- -G-C-C-C- base mav code for a differentamino acid. For
example,UCA codesfor serinewhile ACA cooes
-9-9-+-$- -9-9-T-9-
rransversion for threoni ne.The mi staken(or mi ssense) ami no
|||I - - - ___ _ _ _ .i | l | | l acid may be acceptable, partially acceptable or
-G-C-T-C- -G-C-A-C-
unacceptablewith regardto the function of protern
molecule.Sickle-cellanemiais a classicalexample
(B)
of missensemutation.
-c-G-A-T-G-
tttl 3. Nonsensemutation : Sometimes, the codon
-c-T-A-C- with the alteredbasemay becomea termination(or
-c-G-A-G- nonsense)codon. For instance,change in the
ttll secondbaseof serinecodon (UCA) may resultrn
-G-C-T-C- UAA. The alteredcodon acts as a stop signaland
_c_c_
causes terminationof proteinsynthesis,
at that point.
-G-C-G-
Gonsequences of frameshift mutations

Fig. 3.14 : An illustration of mutations (A)-Point The insertion or deletion of a base in a

gene results in an altered reading frame of the
mRNA (hencethe nameframeshift). The machinery
of mRNA (containingcodons)does not recognize
Gonsequences of point mutations that a basewas missingor a new basewas added.
Sincethere are no punctuationsin the readingof
The change in a single base sequence in codons,translationcontinues.The resultis that the
point mutation may cause one of the following proteinsynthesized will haveseveralalteredamino
( F ig. 3. 1s ) . acidsand/orprematurelyterminatedprotein.
l. Silent mutation : The codon (of mRNA)
Mutations and cancet
containingthe changed base may code for the
same amino acid. For instance,UCA codes for Mutationsare permanentalterationsin DNA
serineand change in the third base (UCU,)still structure,which have been implicated in the
codesfor serine.This is due to degeneracy
of the etiopathogenesis
of cancer.

REPAIR OF DNA
UCU As alreadystated,damageto DNA causedby
(codonfor Ser)
replicationerrorsor mutationsmay have serious
Tsitent consequences. The cell possesses
an inbuiltsystem
I to repairthe damagedDNA. This may be achieved
rnutation, by four distinctmechanisms (Table3.2).
I
UCA 1. B aseexci si on-repai r
(codonfor Ser)
2. N ucl eoti deexci si on-reoai r
3. Mismatchrepair
4. Double-strand
breakrepair.

@cn UAA Base excision-repair

(codonfor Thr) (terminationcodon)
The basescytosi ne,adeni neand guani necan
Fig. 3.15 : An illustration of point mutations
(represented by a codon of nRNA).
undergospontaneous depurinationto respectively
form uraci l , hypoxanthi neand xanthi ne.These
36 B IOTE C HNO LO G Y

Tmu 3.2 Major nechanisns of DNA repair

Mechanism Damage to DNA DNA repair

Base excision-repair Damageto a singlebasedueto Removalol thebaseby N-glycosylase;
spontaneous or by
alteration abasicsugarremoval,
replacement.
chemical
or radiation
means.
Nucleotideexcision-repair Damage of DNAby
to a segment Removal (= 30 nt
of theDNAfragment
spontaneous, or radiation
chemical length)andreplacement.
means.
Mismatchrepair Damage errors
dueto copying Removal of thestrand(byexonuclease
(1-5baseunpaired
loops). andreplacement.
digestion)
Double-strand
breakrepair Damage
causedby ionizing andligation.
alignment
radiations, Unwinding,
freeradicals, etc.
chemotherapy

altered basesdo not exist in the normal DNA, and TCCT

therefore need to be removed. This is carried out Itl
by base excision repai (Fig. 3.16). AGGA
NormalDNA
A def ec t iv e DNA in wh i c h c y t o s i n e r s II
deaminated to uracil is acted upon by the enzyme I
ur ac il DNA gly c os y las e.This r esu l t si n t h e r e m o v a l
of the defective base uracil. An endonucleasecuts
J
TC U T
the backbone of DNA strand near the defect ano
removes a few bases.The gap so created is filled
llll
AGGA
up by the action of repair DNA polymerase and DefectiveDNA
DNA ligas e.
I
I UracilDNA
Nucleotide excision-repair Uy'lglvcosvlase
The DNA dam age due t o u l t r a v i o l e t l i g h t ,
+
ioniz ing r adiat ion and ot h e r e n v i r o n m e n t a l TCXT
factors often results in the modification of ||
A GGA
certain bases, strand breaks, cross-linkages etc.
Nuc leot ide ex c is ion- r epairis ide a l l y s u i t e d f o r s u c h Ii _
large-scaledefects in DNA. After the identification I tsnoonucreases
I
of the defective piece of the DNA, the DNA double
helix is unwound t o ex pos e t he d a m a g e d p a r t . A n
+
excision nuclease (exinuclease) cuts the DNA on
either side (upstream and downstream) of the AGGA
damaged DNA. This defective piece is degraded.
The gap c r eat ed by t he nuc leot i d e e x c i s i o n i s f i l l e d
up by DNA polymerasewhich gets ligated by DNA
ligase (Fig. 3.17).

Xeroderma pigmentosum (XP) is a rare TCCT

autosomal recessivedisease.The affected patients I ll
are photosensitiveand susceptibleto skin cancers. AGGA
It is now recognized that XP is due to a defect rn
Fig. 3.16 : A diagrammatic representation of
t he nuc leot ide ex c is ion r epai r o f t h e d a m a g e o
base excision-repair of DNA.
DNA.
 f f inm AN
I : D N A-R EP L IC A T IO N ,R EC O MB IN A TION , D R E P A IR 37

9Hs 9Hs

I Singlestrand
cutbVGATCendonuclease
I
9Hg
{' QHe

eronrr,""""
Cuttingat twosites f
to removedefective
oligonucleotide

I DNApolymerase
+
CHa
9Ht t-

Degradationof
defectiveDNA
Resynthesisand
religation | ,,n"."
9H. + ?H.

Fig. 3.17 : A diagrammatic representation of Fig, 3.18 : A diagrammatic representation ot

nueleatide excision+epair af DNA. mismatchrepair of DNA.

Iismatch repair Double.strand break repair

defectsdo
Despitehigh accuracyin replication, Double-strandbreaks (DSBs) in DNA are
occurwhenthe DNA is copied.Forinstance,cytosine dangerous.They result in genetic recombination
(insteadof thymine!could be incorporated
opposite which rnay lead to chromosomaltranslocation,
to adenine. Mismatch repair corrects a single brokenchromosomes, and finally cell death.DSBs
mismatchbasepair e.g.C to A, insteadof T to A. can be repairedby homologousrecombinationor
non-homol ogous end j oi ni ng. H omol ogous
The templatestrand of the DNA exists in a
strand recombination occursin yeastswhile in mammals,
methylatedform,while the newlysynthesized
non-homol and j oi ni ngdomi nates.
ogous
is not methylated.This difference allows the
recognitionof the new strands.The enzymeGATC
endonucleasecuts the strand at an adjacent DEFECTS IN DNA REPAIR AND CANCER
methylatedCATC sequence(Fig, 3.18). This is Cancer develops when certdin genes
followedby an exonuclease of thedefective that regulate normal cell division fail or are
digestion
strand,and thus its removal.A new DNA strandis altered. Defectsin the genes encoding proteins
now synthesized to replacethe damagedone. involved in nucleotide-excision repair,mismatch
colon cancer(HNPCC)
Hereditary nonpolyposis repair and recombinationalrepair are linked to
is one of the mostcommon inheritedcancers.This human cancers.For instance,as alreadyreferred
a cancer is now linked with faulty mismatchrepair . above, HNPCC is due to a defect in mismatch
of defectiveDNA. reparr.
J he c onv ent ion. r l c onc ept o f c e n t r a l d o g m a o f
I lif e whic h in es s enc e is " D N A m a k e s R N A
( Replication)
makes protein" is an oversimplification of \..........-.-...-_/
m olec ular biology . W it h t h e a d v a n c e s i n c e l l
-IGENOMEI^
biology and r apid dev elopm e n t si n b i o i n f o r m a t i c s , t_
r ranscflpron
the terms genome, transcriptome and proteomes J
ar e in c ur r ent us e t o r epr es e n tt h e c e n t r a l d o g m a TRANSCRIPTOME
of m olec ular biology ( Fig. a. l \ . S o m e r n f o r m a t i o n t_
I lranslauon transtation
on t he new c onc ept s and te r m i n o l o g y i s g i v e n
her eunder .
+ f
tPnjrErNl
G ENO M E Conventional concept Currentconcept
(pre-bioinformatics
era) (bioinformatics
era)
The t ot al DNA ( genet ic in f o r m a t r o n )c o n t a i n e d
in an or ganis mor a c ell is r eg a r d e da s t h e g e n o m e . Fig. 4.1 : The central dogma of life (or molecular
Thus , t he genom e is t he s t or e h o u s eo f b i o l o g i c a l biology) represented in the form of conventional
inf or m at ion. lt inc ludes t he c h r o m o s o m e s i n t h e and current conceDts.
nuc leus and t he DNA in m i t o c h o n d r i a , a n o
c hlor oplas t s .
Transcriptomics : The study of transcriptome
Genomics : The study of the structure and
f unc t ion of genom e is ge n o m i c s . T h e t e r m that involves ail the RNA molecules made by a
functional genomics is used to represent the gene c e l l , t i s s u e o r a n o r g a n i s m i s t r a n s c r i p to m i cs.
ex pr es s ion and r elat ions hip o f g e n e s w i t h g e n e
PROTEOME
products, Structural genomics refers to the
s t r uc t ur al m ot if s and c om ple t e p r o t e i n s t r u c t u r e s The cell's repertoire (repository/storehouse) of
Comparative genomics involves the study of p r o t e i n s w i t h t h e i r n a t u r e a n d b i o l o g i c al fu n cti o n s
c om par at iv e gene f unc t ion an d p h y l o g e n y is regardedas proteome.Thus, proteome represents
t h e e n t i r e r a n g e o f p r o t e i n s a n d t h e i r b i o l o g i ca l
TRANSCRI PTO M E
functions in a cell
The RNA c opies of t he a c t i v e p r o t e i n c o d i n g
Proteomics : The study of the proteome.
Benesr epr es entt r ans c r ipt om e .T h u s , t r a n s c r i p t o m e
is t he init ial pr oduc t of gen e e x p r e s s i o n w h i c h Metabolomics : The use of genome sequence
dir ec t s t he s y nt hes isof pr ot ei n s . a n a l y s i s f o r d e t e r m i n i n g t h e c a p a b i l i t y o f a ce l l ,

38
 AND TRANSLATION
Chaoter4 : TRANSCRIPTION 39

smallmolecules
tissueor an organismto synthesize
(metabolites)is metabolomics.
Whetherthe centraldogmaof life is represented
in the conventional or more recent form, o-
r eplic at iont r, a n s c ri p ti oann dtra n s l a ti oanrethe key
or core processesthat ultimately control life.
Replicationof DNA hasbeendescribedin Chapter
2, while transcription and translationare discussed
in this chapter.
Core enzyme Sigmafactor

s a p ro c e s si n w h i c h ri b o nucl ei c
T r ans c r i p ti oi n
acid (RNA) is synthesized from DNA. The word
Fig. 4.2 : RNA polymerase of E coli.
gene referslo the functional unit of the DNA that
can be transcribed. Thus,the geneticinformation
storedin DNA is expressed throughRNA. For this
modificationsetc.) commonly known as post-
purpose,one of the two strandsof DNA servesas
transcriptionalmodifications,to producefunctionally
a template(non-codingstrandor sensestrand)and
acti veR N A mol ecul es.
producesworking copiesof RNA molecules.The
other DNA strandwhich does not participatein Thereexistcertaindifferences in the transcription
transcriptionis referredto as coding strand or between prokaryotesand eukaryotes.The RNA
antisensestrand(frequentlyreferredto as coding synthesisin prokaryotesis given in some detail.
strandsincewith the exceptionof T for U, primary Thi si s fol l ow edby a bri efdi scussi on
on eukaryoti c
m RNA c onta i n s c o d o n s w i th th e s a m e base transcri pti on.
sequence).
TRANSCRIPTION IN PROKARYOTES
Transcription is selective A single enzyme-DNA dependent RNA
The entiremoleculeof DNA is not expressed in polymerase or simply RNA polymerase-
RNAsare synthesized
transcription. only for some synthesizesall the RNAs in prokaryotes.RNA
selectedregionsof DNA. For certainother regions polymeraseof E. coli is a complex holoenzyme
of DNA, theremay not be any transcription at all. (mol wt. 465 kDa)with five polypeptidesubunits-
The exact reasonfor the selectivetranscriptionis 2u, 1p and 1B' and one sigma(o) factor(Fig.4.2).
not k nown.Th i sma y b e d u e to s o mei n b u i l ts i gnal s The enzymewithout sigmafactor is referredto as
in t he DNA mo l e c u l e . core enzyme(orpB'1.
The productformed in transcriptionis referredto An overview of RNA synthesisis depicted in
as primary transcript.Most often, the primary RNA Fig. 4.3. Transcriptioninvolves three different
transcriptsare inactive. They undergo certatn stages-i ni ti ati on, el ongati on and termi nati on
alt er at ions( s p l i c i n g , te rmi n a l a d d i ti o n s , base (Fig. 4.4).

Codingstrand

t- RNApotymerase
Template
strand

Fig. 4,3 : An overview of transcription.

 D
Transcription Termination o
unit site

DNA template

5, M 3'
Newly synthesized
RNA
Termination

A
Rho factor

--mt
o
I
z
a\
-
Fig. 4.4 : Synthesis of RNA trom DNA template (transcription)' -o
Chapt er4 : T R A N S C R IPT ION
AN D T R A NS LA TION 41

-3 5 Pribnow
Sequence oox
Coding 5'- TTGACA TATAAT
strand
Template
strand

Start of
transcription

Fig. 4.5 : Promoterrcgionsof DNAin prokaryotes.

lnitiation The sequenceof nucleotidebasesin the mRNA

The bindingof the enzymeRNA polymerase to is complementary to the templateDNA strand.lt is
DNA is the prerequisitefor the transcriptionto however, identicalto that of coding strandexcept
start.The specificregionon the DNA where the that R N A contai ns U i n pl ace of T i n D N A
enzymebinds is known as promoterregion.fhere Gig. a.6).
are two basesequenceson the coding DNA strand RNA polymerasediffersfrom DNA polymerase
which the sigma factor of RNA polymerasecan in two aspects.No primer is requiredfor RNA
recognizefor initiationof transcription(Fig.4.5). polymeraseand, further, this enzyme does not
1. Pribnow box (TATAbox) : This consistsof 6 possess endo-or exonuclease activity.Due to lack
nucleotidebases(TATAAT),locatedon the left side of the latterfunction(proof-reading activity),RNA
about 10 basesaway (upstream)from the starting polymerase has no abilityto repairthe mistakesin
point of transcription. the RNA synthesized. This is in contrastto DNA
repl i cati on
w hi ch i s carri edour w i th hi ghfi del i ty.l t
2. The '-35' sequence: This is the second is, however. fortunate that mistakes in RNA
recognitionsite in the promoterregionof DNA. lt synthesisare less dangerous,since they are not
contains a base sequenceTTCACA, which is transmittedto the daughtercells.
locatedabout35 bases(upstream, hence-35) away
on the left side from the site of transcriptionstart. The doubl ehel i calstructure of D N A unw i ndsas
the transcriptiongoes on, resultingin supercoils.
Elongation The problem of supercoils is overcome by
topoisomerases (moredetailsgivenunderreplication).
As the holoenzyme,RNA polymerase recognizes
the promoterregibn,the sigmafactor is released Termination
and transcriptionproceeds.RNA is synthesized
The processof transcription stopsby termination
from 5' end to 3' end (5'-+3') antiparallelto the
si gnal s.Tw o typesof termi nati onare i denti fi ed.
DNA template. RNA polymerase utilizes
ribonucleotidetriphosphates (ATB CTB CTP and 1. Rho (p) dependenttermination: A specific
UTP)for the formationof RNA. Forthe additionof protein,namedp factor,bindsto the growingRNA
each nucleotide to the growing chain, a (and not to RNA polymerase) or weakly to DNA,
pyrophosphate moiety is released. and in the bound state it acts as ATPaseand

|_s'_A T G C A T G G C A 3' Codingstrand

DNA--
L _ 3 /_ T A C G T A C C G T 5' Templatestrand

RNA____-------+5'........AU G C A U G G C A........3'

Fig. 4.6 : Promoter regions of DNA in prokaryotes.

42 B IOTE C H NO LO CY

s',
DNA
c

t
RNApolymerase
ll RNApolymerase
tll
'ly

J I
J

Ribosomal Messenger RNA

Transfer
RNAs RNA

Fig. 4,7 : An overviewof Iranscriptionin euKaryotes.

R N A . The p
s n s c ri p ti oann d re l e ases
t e rm i n a tetra 3. RNA polymerase lll participatesin the
factor is also responsiblefor the dissociationof formati onof tR N A sand smal lri bosomalRNAs.
RNA polymerase from DNA.
Besidesthe threeRNA polymerasesfound in the
2. Rho(p) independent termination: The termi- nucleus,there also exists a mitochondrialRNA
nation in this case is brought about by the polymerase in eukaryotes.The latter resembles
formationof hairpinsof newly synthesizedRNA. prokaryotic RNA polymerase in structure and
This occursdue to the presenceoI palindromes.A function.
palindromeis a word that readsalike forwardand
backward e.g. madam, rotor. The presenceof Promoter sites
p a l i n d ro meisn th e b a s es e q u e n ce
of D N A templ ate In eukaryotes, a sequence of DNA bases-which
(same when read in opposite direction) in the is almostidenticalto pribnowbox of prokaryotes-
terminationregionis known.As a resultof this,the is identified(Fig. a.0. This sequence,known as
newly synthesized RNA foldsto form hairpins(due Hognessbox (or TATA box), is located on the left
t o c o mp l e m e n ta ryb a s e p a i ri ng) that cause about 25 nucleotidesaway (upstream)from the
o f tra n s c ri p ti o n .
t e rmi n a ti o n startingsite of mRNA synthesis. There also exists
another site of recognitionbetween 70 and 80
TRANSCRIPTTON IN EUKARYOTES nucleotides upstreamfrom the startof transcription.
This secondsite is referredto as CAATbox. One of
RNA synthesisin eukaryotesis a much more
these two sites (or sometimesboth) helps RNA
complicated process than the transcription
polymerasell to recognizethe requisitesequence
describedabove for prokaryotes. As such, all the
on D N A for transcri pti on.
d e ta i l s o f e u k d ry o ti ctra n s c ri pti on(parti cul arl y
a b o u t te rm i n a ti o n a
) re n o t c l e arl y know n. The
lnitiation of transcription
salientfeaturesof availableinformationare siven
here. The moleculareventsrequiredfor the initiation
of transcriptionin eukaryotesare complex, and
RNA polymerases broadlyinvolvethree stages.
The nuclei of eukaryoticcells possessthree
1. Chromatincontainingthe promotersequence
distinctRNA polymerases (Fig.4.71.
made accessible to the transcriptionmachinery.
1. RNA polymeraseI is responsiblefor the
synthesis of precursors for the largeribosomalRNAs. 2. Bindingof transcription factors(TFs)to DNA
sequences in the promoter region.
2. RNA polymerase ll synthesizes the
o re c u rs o rsfo r m R N Asa n d s ma l lnucl earR N A s. 3. S ti mul ati on by enhancer s.
of transcri pti on
 43
--)^to r a T R AN SC R IP T IOANN D T R AN SLA TION

Hogness
CAAT box box
Non-coding5i.- GGCCAATC ATATAA
strancl
Coding
strand
-70 bases i -25 bases

Start of
transcription

Fig. 4.8 ; Promoterregionsof DNAin eukaryotes'

A large number of transcription.factorsinteract POST.TRANSCRIPTIONALMODIFICATIONS

*'ith eukaryoticpromoterregions'ln humans,about The R N A s producedduri ng transcri pti onare
factorshavebeenidentified(TFIID,
six transcription They undergomany
cal l ed pri mary transcri pts.
TFIIA, TFIIB,TFIIF,TFIIE,TFIIH). lt is postulated alterations-t erminal base additions, base
that the TFsbind to each other,and in turn to the modifications,splicing etc., which are collectively
enzymeRNA PolYmerase. referredto as post-transcriptional modifications'
Enhancer canincrease gene expression by about This process is required to convert the RNAs into
p o s s i b l eb y b i n d i ng to the active forms. A group of enzymes, namely
100 f old. T h i s i s m a d e
to transcription factors to torm ribonucleases, are responsible for the processingof
enhancers
lt is believedthat the chromatinformsa tRNAs and rRNAs of both prokaryotes and
activators.
loop that allowsthe promoterand enhancerto be eukaryotes.
closetogetherin spaceto facilitatetranscription'
The prokaryoticmRNA synthesizedin trans-
cri pti oni s al mostsi mi l arto the functi onalmR N A '
Heterogeneous nuclear RNA (hnRNAl
ln contrast,eukaryoticmRNA (i.e' hnRNA)under-
fhe primary mRNA transcriptproducedby RNA goesextensivepost-transcriptional changes'
polymerase ll in eukaryotes is often referred to as
het er ogene o u n su c l e a rR N A (h n R N A )'T h i s i s then An outline of the post-transcriptional
processedto produce mRNA neededfor protein modifications is given in Fig, 4,9, and some
synthesis. hi ghl i ghtsare descri bed.

hnRNA(preRNA)

End
modifications

-c-#
o-_-=----fl lntrons Cutpieces New chemicalgrouPsadded
cap Poly(A)tail
removeo
nuclearRNl).
Fig. 4'g : An a||ttingof pagt.trensc|iponal mdllieabn$Q| RNA(lnlNA.Heterogeneous
44 B IOTE C H NO LO CY

for protein synthesis. The splicingand excisionof

mRNA
intronsis illustratedin Fig, 4.10. The removalof
Exon2
i ntrons i s promoted by smal l nucl ear r ibo-
nucl eoprotei n parti cl es (snR N P s). s nRNPs,
ATP (pronouncedas snurps)in turn, are formed by the
of smal l nucl earR N A (snR NA)wit h
40 associ ati on
oroteins.

AD P + P i The term spliceosomeis used to representthe

w i th hnR N Aat the exon- int r on
snR N Passoci ati on
l uncti on.
modifications of mRNA
Post-transcriptional
occursin the nucleus.The matureRNA then enters
the cytosolto performits function(translation).
A d iagrammatic representation of the
relationship between eukaryotic chromosomal
D N A and mR N A i s depi ctedi n Fi g. 4.11.

Different mRNAs produced by

alternate splicing
Alternatepatternsof hnRNA splicing result rn
Excised different mRNA moleculeswhich can produce
intron

Fig. 4.10 : Formation of mature RNA from eukaryotic

nRNA (SnRNPs-Small nuclear ribonucleoprotein
particles).

1 . 5x 1 0 8b p
Messenger RNA

T h ep ri ma rytra n s c ri potf mR NAi s the hnR N Ai n

eukaryotes,which is subjectedto many changes
beforefunctionalmRNA is produced. Genecluster 1 . 5x 1 0 6b p
(-1 6 genes)
1 . T h e 5 ' c a p p i n g: T h e 5 ' end of mR N A i s
c a p p e d w i th 7 -me th y l g u a n o si ne by an unusual
5 ' + 5 ' tri p h o s p h a te
l i n k a g eS
. -Adenosyl methi oni ne
is th e d o n o ro f m e th y lg ro u p .T h i scap i s requi red
m
Onegene(with8 exonsand 2 . 5 x 1 0 4b p
for translation,besidesstabilizingthe structureof 7 introns)I
mR N A .
J
2. Poly-Atail : A large number of eukaryotic m8x1o3nt
m R N Asp o s s e sasn a d e n i n en u c l e oti de chai nat the Primary
transcript
3 ' -e n d .T h i s p o l y -Ata i l , a s s u c h ,i s not produced
du ri n g tra n s c ri p ti o nl t. i s l a te r addedto stabi l i ze J
mRNA. However,poly-Achain getsreducedas the I 2x103nt
mRNA enterscvtosol. mRNA

3. Introns and their removal : Intronsare the

Fig, 4.11 : A diagrammatic representation of the
in te rv e n i n n
g u c l e o ti d es e q u e n c es
i n mR N A w hi ch
relationship between eukaryotic chromosomal DNA
do not codefor proteins.On the otherhand,exons
and nRNA (bp-Base pair; nt-Nucleotides).
of nRNA possessgenetic code and are responsible
 AND TRANSLATION
Chapter4 : TRANSCRIPTION 45

oinerent proteins. Alternate splicing results in Actinomycin D : This is also known as

:nR\A heterogeneity. In fact, the processingof dactinomycin.lt is synthesizedby Streptomyces.
hnRNAmoleculesbecomesa sitefor the regulation ActinomycinD binds with DNA templatestrand
or geneexpression. and blocksthe movementof RNA polymerase. This
was the very first antibioticusedfor the treatment
Faultysplicingcan causediseases: Splicingof
of tumors.
hnRNA has to be performedwith precisionto
produce functional mRNA. Faulty splicing may Rifampin: lt is an antibioticwidely usedfor the
resultin diseases. A good exampleis one type of treatmentof tuberculosisand leprosy.Rifampin
binds with the p-subunit of prokaryotic RNA
ftthalassemiain humans.This is due to a mutation
that resultsin a nucleotidechange at an exon- polymeraseand inhibitsits activity.
intronjunction.This leadsto diminishedor lack of cr-Amanitin : lt is a toxin produced by
svnthesis of B-chain of hemoglobin, and mushroom,Amanita phalloides.This mushroomis
consequently the diseasep-thalassemia. deliciousin tastebut poisonousdue to the toxin
c-amanitinwhich tightlybindswith RNApolymerase
Transfer RNA ll of eukarvotes and inhibitstranscriotion.
All the tRNAs of prokaryotesand eukaryotes
CELLULAR RNA CONTENTS
undergo post-transcriptionalmodification.These
includetrimming,convertingthe existingbasesinto A typical bacterium normally contains
unusualones,and additionof CCA nucleotidesto 0.05-0.10pg of RNA which contributesto about
3' terminalend of tRNAs. 6% of the total weight. A mammaliancell, being
larger in size, contains20-30 pg RNA, and this
Ribosomal RNA represents only 1% of the cell weight.
Transcriptome, representing the RNA derivedfrom
The preribosomalRNAs originallysynthesized
proteincoding genesactuallyconstitutesonly 4o/",
are convertedto ribosomalRNAs by a seriesof
w hi l e the remai ni ng96/" i s the non-codi ngR N A
post-transcriptional
changes.
ffig. aJ). The different non-codingRNAs are
ribosomalRNA, transferRNA, small nuclearRNA,
Inhibitors of transcription
smal l nucl eol arR N A and smal lcytopl asmi Rc NA.
The synthesisof RNA is inhibited by certain The functionsof differentRNAs are describedin
antibioticsand toxins. Chapter2 (Table2.J).

Fig. 4.12 : A diagrammatic representation of RNA content of a cell (Note : RNAs represented in
black are found in all organisms; RNAs in colour and exclusively present in eukaryotes only;
. hnRNA-Heterogeneous nuclear RNA; rFNA-Ribosomal RNA: tBNA-Transfer HNA;
snRNA-Small nuclear RNA; snoBNA-Small nucleolar RNA; scRNA-Small cytoplasmic RNA).
46 B IOTE C H N OLO CY

3', 5, ViratRNA Variability of cells in translation

T h e r e a r e w i d e v a r i a t i o n s i n t h e c e l l s w i th
Primer
respect to the quality and quantity of proteins
s y n t h e s i z e d .T h i s l a r g e l y d e p e n d s o n t h e n e e d a n d
ability of the cells. Erythrocytes (red blood cells)
l a c k t h e m a c h i n e r y f o r t r a n s l a t i o n , a n d t h e r e fo r e
3', 5' RNA cannot synthesize proteins.
c 3' DNA I n g e n e r a l , t h e g r o w i n g a n d d i v i d i n g ce l l s
produce larger quantities of proteins. Some of the
Fig. 4.13 : Reverse transcription of RNA virus c e l l s c o n t i n u o u s l y s y n t h e s i z e p r o t e i n s f o r e xp o r t.
F o r i n s t a n c e ,l i v e r c e l l s p r o d u c e a l b u m i n a n d bl o o d
clotting factors for export into the blood for
c i r c u l a t i o n . T h e n o r m a l l i v e r c e l l s a r e v e r y r i ch i n
t h e p r o t e i n b i o s y n t h e t i c m a c h i n e r y , a n d t h u s th e
Iiver may be regarded as the protein factory in the
human body.
Some of the viruses-known as retroviruses--
poss e sR s N A a s th e g e n e ti cma te ri a lThese
. vi ruses
GENETIC CODE
c au s e c a n c e rs i n a n i ma l s , h e nce know n as
oncogenic. They are actually found in the The three nucleotide (triplet) base sequences in
t r an s fo rmecde l l so f th e tu mo rs . nRNA that act as code words for amino acids in
protein constitute the genetic code or simply
t N A pol ymerase codons. The genetic code may be regarded as a
T h e e n z y m eR N A d e p e n d e nD
-or simply reversetranscriptase-isresponsible d i c t i o n a r y o f n u c l e o t i d e b a s e s( A , G , C a n d U ) th a t
for the formation of DNA from RNA (Fig, a.13). d e t e r m i n e st h e s e q u e n c eo f a m i n o a c i d s i n p r o te i n s.
(c D N A)to vi ral R N A
T his D N A i s c o mp l e m e n ta ry
The codons are composed of the four nucleotide
and can be transmittedinto host DNA.
bases, namely the purines-adenine (A) and
Synthesisof cDNA from mRNA : As already g u a n i n e ( C ) , a n d t h e p y r i m i d i n e s - c y t o s i n e ( C ) a n d
des c ri b e d , th e D N A e x p re s s esthe geneti c uracil (U). These four bases produce 64 different
inf o rm a ti o ni n th e fo rm o f R N A . A nd the mR N A
combinations (43)of three base codons, as depicted
det e rm i n eth
s e a mi n o a c i d s e q u e n cei n a protei n.
in Table 4.1 . The nucleotide sequenceof the cooon
T he mR N A c a n b e u ti l i z e da s a te mpl atefor the
synthesisof double-strandedcomplementaryDNA o n m R N A i s w r i t t e n f r o m t h e 5 '- e n d t o 3 'e n d . Si xty
(cDNA)by usingthe enzymereversetranscriptase. n e c o d o n s c o d e f o r t h e 2 0 a m i n o a c i d s f o u nd i n
o
T hisc D N A c a n b e u s e da s a p ro b eto i denti fythe p r o t e i n .
of DNA in genes.
s eq u e n c e The three codons UAA, UAG and UGA do not
code for amino acids. They act as stop signals in
orotein svnthesis. These three codons are
collectively known as termination codons or non-
s e n s e c o d o n s . T h e c o d o n s U A C , U A A a n d UGA
are often referred to, respectively, as amber, ochre
The genetic information stored in DNA is passect
and opal codons.
on to RNA (through transcription), and ultimately
expressed in the language of proteins The The codons AUG-and, sometimes. GUC-are
biosynthesis of a protein or a polypeptide in a the chain initiating codons.
Iiving cell is referred to as translation. The term
Other characteristics of genetic code
tra ns lat ion is us ed t o r epr es ent t he b i o c h e m i c a l
translation of four-letter language information from T h e , g e n e t i c c o d e i s u n i v e r s a l , s p e c i f i c , no n -
nu cl eic ac ids ( DNA and t hen RNA ) t o 2 0 l e t r e r overlapping and degenerate.
lan guageof pr ot eins .The s equenc eo f a m i n o a c i d s I . Universality : The same codons are used to
in the protein synthesized is determined by the c o d e f o r t h e s a m e a m i n o a c i d s i n a l l t h e l i vi n g
n ucleot ide bas e s equenc e of m RNA . organisms. Thus, the genetic code has been
-t;rr:'i A N D T R AN S LA TION
: T R A N S C R IPT ION 47

::f:-."eci Cur ing t he c our s e of ev olut ion. H e n c e of one or two bases will radically change the
r,:"-F:r: :Ce is appropriately regardedas universal. message sequence in mRNA. And the protein
-*r+.= :'e however, a few exceptions. For instance, synthesized from such mRNA will be totally
r - r : :f e c odon f or m et hionine in m it oc h o n d r i a . different. This is encountered in frameshift
-rr: i;Te codon (AUA) codes for isoleucine rn mutations which cause an alteration in the readine
: ,r:: ;s'n. W it h s om e ex c ept ions not e d , t h e frame of mRNA.
: co de is univ er s al.
-:.n : 4. Degenerate : Most of the amino acids have
-" Specificity : A particular codon always codes more than one codon. The codon is degenerateor
'r:':-e redundant,since there are 61 codons available to
sam e am ino ac id, henc e t he genet ic c o d e r s
-:-',. spe c if ic or unam biguous e. g. UCC i s t h e c o d e f o r o n l y 2 0 a m i n o a c i d s . F o r i n s t a n c e ,g l y c i n e
::.:or tbr tryptophan. has four codons. The codons that designate the
same amino acid are called synonyms. Most of the
-1. Non-overlapping : The genetic code is read s y n o n y m s d i f f e r o n l y i n t h e t h i r d ( 3 ' e n d ) b a s e o f
---- a fixed point as a c ont inuous bas e s eq u e n c e .
the codon.
: : n on -over lapping,c om m ales s and wit ho u t a n y
:-^ ciua tion s . For ins t anc e,UUUCUUACAC C C i s The Wobble hypothesisexplainscodon degeneracy
-=ad as UUU/CUUIACA/CCC. Addition or deletion (described later).

Tlslr 4.1 The genetic code along with respective anlno acids

First base Second base (middle one) Third base

5'end 3'end
-l -I
UUUI UCU i unu UGU tl

I Phe r lrry Icvs

uucl ucc i UACI uccI
-I S eri -
UUA UCA i unn stop UGA Stop
ILeu
uucI UCG UAG Stop UGG Trp

CUU
CUC
CXJA
Leu
ccu

ccA
Pro i
i
i
'i
cAU
cnc
CAA
1,,, C GU
UUU

CGA
Arn
tl

A
vu\f

-l
tr ULI
i
i ^n^
vA\l lo,,.
AUU AGU- I U
ISer
AUc Ite AGCI
I -I
AUA I AGA A
lAro
AUG* Met AGG-]
GUU ITUU GGU U
GUC (,(/(,
Val Gll,
GUA GCA tr \rA A
uuu

*AUG in protein
r.r., as initiating
codon,besidescodingfor methionine
residue synthesis;
UAA,UAGandUGAcalledas nonsense
areresponsible
codons, of protein
fortermination synthesis,
 48
fMet
I codon (mRNA) is given
5' end
I 5, r\f\r.V\.r\ AUG /\;rl,r\/\1!
+ throughthe al terati ons
I Someof the mutationsare harmful.
Codon
Fig. 4.14 : Complementary binding of codon
(of nBNA) and antlcodon (of IRNA),
Godon.anticodon recognition
,l
The codon of the mRNA is recognizedby the
I
rl anticodonof IRNA (Fig.a,|a). They pair with each
I
ot he r i n a n ti p a ra l l edl i re c ti o n(5 ' + 3' of mR N A
)t
I
with 3' -+ 5' of IRNA). The usual conventional
I
c om p l e me n ta ry b a s ep a i ri n g(A = U , C = G) occurs
I betweenthe first two basesof codon and the last
two basesof anticodon.The third base of the
i codon is ratherlenientor flexiblewith regardto the
complementary base. fhe anticodon region of
IRNA consists of seven nucleotides and it
recognizes the three lettercodon in mRNA.
Wobble hypothesis
Wobble hypothesis,put forth by Crick, is the
phenomenonin which a singleIRNA can recognize
more than one codon. This is due to the fact that
the third base(3rbase)in the codon often fails to
recognizethe specificcomplementary base in the
anticodon(5'-base). Wobbling is attributedto the
differencein the spatialarrangementof the 5'-end
of the anticodon.The possiblepairing of 5'-end
B IOl E C H N OLO CY
base of anticodon (of IRNA) with the 3'-end base of
Anticodon Codon
(/-U
l ll basepairing
) Conventionat
v
il-G base
or A 1 Non-conventional
G - tl or C I pairing
(coloured)
Wobble hypothesisexplainsthe degeneracy of
the geneticcode, i.e. existence of multiplecodons
for a si ngl e ami no aci d. A l thoughthere are 61
codonsfor aminoacids,the numberof tRNAsis far
l ess(around40) w hi ch i s due to w obbl i ng.
Mutations and genetic code
Mutationsresult in the change of nucleotide
sequencesin the DNA, and consequentlyin the
RNA.The differenttypesof mutationsaredescribed
in Chapter3. The ultimateeffectof mutationsis on
3, mRNA
i n cod ons.
the transl ati on
The occurrenceof the diseasesickle-cellanemia
due to a single base alteration(CTC -+ CAC in
D N A , and C A G -+ C U C i n R N A ) i s a cl as sical
of mutations.The result
exampleof the seriousness
is that glutamateat the 6th positionof B-chainof
hemogl obi ni s repl acedby val i ne.Thi s happens
sincethe alteredcodon CUC of mRNA codesfor
valine insteadof glutamate(coded by CAC tn
normalpeopl e).
Frameshift mutationsare causedby deletionor
insertionof nucleotidesin the DNA that generates
alteredmRNAs.As the readingframe of mRNA is
continuous,the codons are read in continuation,
and amino acidsare added.This resultsin proteins
that may contain severalalteredamino acids,or
sometimes the proteinsynthesismay be terminated
prematurely.
The protein synthesis which involves the
translationof nucleotidebasesequenceof mRNA
into the languageof amino acid sequencemay be
divided into the following stages for the
convenienceof understanding.
of the comPonents
l. Requirement
l l . A cti vati onof ami no aci ds
 ]1AOtCT4 : TRANSCRIPTIONAND TRANSLATION 49

Beginningof Completely protein

synthesized
proteinsynthesis

Fig, 4,15 : A polyribosome in protein synthesis.

lll. Proteinsynthesisproper A site is for bindingof aminoacylIRNA and P sife

and proteinfolding
l\/. Chaperones is for bindingpeptidylIRNA, duringthe courseof
translation.Some authors consider A site as
modifications.
V. Post-translational
acceptorsite and P site as donor site. In case of
I . RE Q UI R EME N T O F T H E eukaryotes,there is another site called exist site
CO M P O N E N T S or E sife. Thus, eukaryotescontain three sites(A, P
and E) on the ribosomes.
The protein synthesismay be consideredas a
bioEhemical factoryoperatingon the ribosomes. As The ribosomesare located in the cytosomal
a factory is dependenton the supply of raw fractionof the cell. They are found in association
materialsto give a final product, the protein with rough endoplasmicreticulum(RER)to form
svnthesis also requiresmany components. clusters RER-ribosomes, where the protein
synthesisoccurs.The term polyribosome(polysome)
1. Amino acids : Proteinsare polymers of is used when several ribosomessimultaneously
amino acids. Of the 20 amino acids found in translateon a singlemRNA (Fig.a.l5).
protein structure, half of them (10) can be
svnthesizedbv man. About 10 essentialamino 3. MessengerRNA (mRNA) : The specific
acids haveto be providedthroughthe diet. Protein information requiredfor the synthesisof a given
c an o c c u ro n l y w h e n a l l th e a mi n oaci ds
s y nt hes is protein is present on the mRNA. The DNA has
neededfor a particularprotein are available.lf passedon the geneticinformationin the form of
there is a deficiencyin the dietarysupply of any codons to mRNA to translate into a orotein
one of the essentialamino acids, the translation seouence.
stops. lt is, therefore,necessarythat a regular 4. Transfer RNAs (tRNAs) : They carry the
dietarysupplyof essentialaminoacids,in sufficient amino acids,and hand them over to the growing
quantities,is maintained,as it is a prerequisite
for pepti dechai n.The ami noaci d i s coval entl ybound
proteinsynthesis. to IRNA at the 3'-end. Each IRNA has a three
As regards there is no requirement nucleotidebasesequence-Ihe anticodon,which is
prokaryotes,
of amino acids, since all the 20 are synthesized responsible to recognizethe codon(complementary
from the inorganiccomponents. bases) of mRNA for proteinsynthesis.
In man, there are about 50 differenttRNAs
2. Ribosomes: The functionallyactive ribo-
whereasin bacteriaaround 40 tRNAs are found.
somes are the cenfres or factories for protein
synthesis.Ribosomesmay also be consideredas S omeami noaci ds(parti cul arlthose
y w i th mul ti pl e
workbenchesof translation.Ribosomesare huge codons)have more than one IRNA.
complexstructures (70Sfor prokaryotes
and 80Sfor 5. Energy sources : Both AIP and GTP are
eukaryotes)of proteinsand ribosomalRNAs.Each required for the supply of energy in protein
ribosomeconsistsof two subunits-one big and synthesis.Some of the reactions involve the
one small.The functionalribosomehastwo sites- breakdown of ATPor CTP, respectively,to AMP
A siteand P site.Eachsitecoversboththe subunits. and GMP with the liberationof pyrophosphate.

Biotechnology
[4]
 50 B IOTECHNO LO CY

Amino acid

@-nrrrre-Aminoacid

ri
I

tRNA Aminoacyl
tRNA

Fig, 4.16 : Formation of aminocacy! iRNA (AA-Amino acid; E-Enzyme).

Each one of these reactionsconsumestwo hieh synthesis proceeds from N-terminal end to
energyphosphates(equivalentto 2 ATP). C-terminal end. Translationis directional ano
col l i nearw i th mR N A .
6. Proteinfactors : The processof translation
involvesa number of protein factors.These are The prokaryoticmRNAs arepolycistronic,since
neededfor initiation,elongationand terminationof a si ngl emR N Ahasmanycodi ngregi o nst hat code
protein synthesis.The protein factors are more for differentpolypeptides.In contrast,eukaryotic
complex in eukaryotes comparedto prokaryotes. mRNA is monocistronic, sinceit codesfor a single
polypeptide.
II. AC T IVA T IO N OF A M IN O A C ID S
In case of prokaryotes, translationcommences
Amino acidsareactivatedand attachedto tRNAs beforethe transcriptionof the gene is completed.
in a two stepreaction. A groupof enzymes-namely Thus,si mul taneous andtra nslat ion
transcri pti on ar e
aminoacylIRNA synthetases-are requiredfor this possible.This is not so in case of eukaryotic
process.Theseenzymesare highly specificfor the organi sms occursi n t he nucleus
si ncetranscri pti on
a mi n o a c i d a n d th e c o rre s pondi ng
IR N A . whereas translationtakes place in the cytosol.
The amino acid is first attachedto the enzyme Further, the primarytranscript(hnRNA)formedfrom
u ti l i z i n g A T P to fo rm e n z yme-A MP -ami noaci d DNA has to undergo several modificationsto
complex. The amino acid is then transferredto generate functi onalmR N A .
the 3' end of the IRNA to form aminoacvltRNA Proteinsynthesis is comparativelysimplein case
ffig. a.l6). of prokaryotescomparedto eukaryotes.Further,
many steps in eukaryotictranslationwere not
III. PR O T E IN SY N T H ES IS P R OP E R
understoodfor quite sometime.For thesereasons,
The protein or polypeptide synthesisoccurson majorityof the textbooksearlierused to describe
the ribosomes (ratherpolyribosomes). The mRNA is translationin prokaryotes in detail,and give most
read in the 5'-+3' direction and the polypeptide importantand relevantinformationfor eukaryotic
--i.ic'ji.-i 1R\\SCRIPTION AND TRANSLATION 5t

-:i--,:i,-,- ,'.:: t he adv anc esin m olec ularbio l o g y , thentransferredto 43Scomplex.Forthe appropriate
'rr tr-.,:-: :j c r ot ein bios v nt hes isin euk ar v o t e st s associ ati onof 43S prei ni ti ati oncompl ex w i th
Tr'lil"Pr - -'--E-S:OOd nOW. mRNA,energyhas to be suppliedby ATP. I

flzngJation in eukaryotes is briefly described Recognition of initiationcodon : The ribosomal !

Frr n : - i rr ith some relevant features of i ni ti ati on comol ex scans the mR N A for the i
iflir* .;-i :i : p rot ein bios y nt hes isTr
. ans lat ionpr o p e r i denti fi cati onof appropri atei ni ti ati on codon. !
5'-AUC is the initiationcodonand its recognitionis i
: r rer rto three stages-initiation, elongation
i'rj i:- ^ a tion ( as it is done f or t r ans c r ipt io n ) . facilitatedby a specificsequenceof nucleotides t
I
g
surroundi ngi t. Thi s marker sequencefor the tr
L
|flTrIATION OF TNANSLATTON identification of AUC is calledas Kozak consensus
sequences. In caseof prokaryotes the recognition
-.^ itjation of t r ans lat ion in euk ar y ote s i s
sequenceof initiationcodon is referredto as Shrne-
-: :\ ;nr,olving at least ten eukaryotic initiation
Dalgarno sequence.
tectors /elFs) Some of the elFs contain multiple
. -i ;-cu nits The pr oc es sof t r ans lat ion init ia t i o n
Formation of 8OS initiation complex
-.- := dirided into four steps (Fig. 4.171
- 4B S i ni ti ati oncomol exbi ndsto 605 ri bosomal
Rib osom aldis s oc iat ion.
subunito t formB 0Si ni ti ati oncompl ex.Thebi ndi ng
- Forma tion of 43S pr einit iat ion c om plex .
i nvol vesthe hydrol ysi sof C TP (boundto el F-2).
: Forma tion of 4BS init iat ion c om plex . This stepis facilitatedby the involvementof elF-5.
j Forma tion of B0S init iat ion c om plex .
A s the B 0S compl ex i s formed,the i ni ti ati on
factorsboundto 4BSinitiationcomplexarereleased,
Ribosomal dissociation
and recycled.Theactivationof elF-2requireselF-2B
805 ribosome dissociatesto form 40S and (al socal l edas guani nenucl eoti de factor)
exchange
-re
:'-S su bu nits. Two init iat ing f ac t or s nam ely e l F - 3 and CTP.The activatedelF-2 (i.e. bound to CTP)
.-: elF-1A bin d t o t he newly f or m ed 40S s ub u n i t , requireselF-2Cto form the ternarycomplex.
.'. th ere by bloc k it s r eas s oc iat ion wit h 6 0 5
=-run it. Fo r th is r eas onrs om e wor k er s nam e e l F - 3 Regulationo? initiation
,: anti-association factor. The elF-4F,a complexformed by the assembly
of three i ni ti ati onfactorscontrol si ni ti ati on,and
Formation of 43S preinitiation complex
process.
thusthe translation elF-4E,a componentof
\ ternary complex containing met-tRNAr and el F-4Fi s pri mari l yresponsi blfor e the recogni ti on
eiF-2 bound to CTP attaches to 40S ribosomar of mR N A cap. A nd thi s stepi s the rate-l i mi ti ng in
s;bu nit to fo r m 43S pr einit iat ion c om plex . T h e transl ati on.
3 resen ceof elF - 3 and elF- lA s t abiliz est his c om p l e x
el F-2w hi ch i s i nvol vedi n the formati onof 43S
,\ote : Me t-tRNA is s pec if ic allyinv olv ed in bin d i n g prei ni ti ati on compl ex al so control s protei n
:o the in itiat ion c ondon AUCs ; henc e t h e
biosynthesis to someextent.
=uperscrip/is used in met-tRNA/).

Initiation of translation in prokaryotes

Formation of 48S initiation complex
The formationof translationinitiationcomplex
Th e b ind ing of m RNA t o 43S pr einit i a t i o n
in prokaryotesis less complicatedcomparedto
co mple x re su lt s in t he f or m at ion of 4BS init ia t i o n
eukaryotes. The 30Sri bosomalsubuni ti s boundto
complex through the intermediate 43S initiation
initiation factor 3 (lF-3) and attachedto ternary
complex. This, however,involvescertain interactions
complex of lF-2, formyl met-tRNA and CTP.
between some of the elFs and activation of mRNA.
A nother i ni ti ati on factor namel y l F-l al so
elF-4F complex is formed by the associationof participates in the formation of preinitiation
elF-4C, elF-4A wit h elF- 4E.The s o f or m ed el F - 4 F compl ex.The recogni ti on of i ni ti ati oncodonA U C
(referredto as cap binding protein) binds to the cap is done through Shine-Dalgarno
of mRNA. Th en elF- 4A and elF- 48 bind t o m R N A ribosome unit is now
and reduce its complex structure. This mRNA is produce 70S initiation

-rt
a\*\._-*.--- tr .-1.
.;
il !
52 B IOTE C HNO LO CY

s',
cap

Met
@A I

nr o I
)
ADP + Pit'lY
Met

48S initiation comPlex

80S initiationcomPlex

Fig. 4.17 : A diagrammatic representation of initiation of protein biosynthesis (translation) in eukaryotic cells
(The eukaryotic initiation factors are represented by symbols n,A, andQ. By prefixing with elE
the fult names of the factors are obtained e.9. E represents elF-S)
 fnacner-l : TMNSCRIPTIONAND TRANSLATION 53

E,-OfIGATION OF TRANSLATION In caseof prokaryotes,

the elongationfactorsare
different,and they are EF-Tu,EF-Ts(in place of of
i. cosomeselongatethe polypeptidechain by a
EF-1a)and EF-C(insteado! EF-2).
.€olerrtialadditionof aminoacids.The aminoacid
:€rence is determinedby the order of the codons
lncorporation of amino acids
r re specificmRNA. Elongation, a cyclic process
-.,riring certainelongationfactors (EFs), may be lt is estimatedthat about six amino acids per
i', ded into three steps(Fig. 4,18). second are incorporatedduring the course of
elongationof translationin eukaryotes.In case of
i. B ind i n go f a m i n o a c yI-R
l N Ato A -s i te.
prokaryotes, as many as 20 amino acids can be
l. Peptidebond formation. incorporatedper second. Thus the processof
3. Translocation. protein/polypeptide synthesisin.translationoccurs
with great speed and accurary.
Binding of aminoacyl-tRNA to A-site
The B0Sinitiationcomplexcontainsmet-tRNAl TEBM'NATTON OF TBANSLATION
r the P-site, and the A-site is free. Another Termination is a simpleprocesswhen compared
arninoacyl-tRNAis placed in the A-site. This to initiationand elongation.After severalcyclesof
.equiresproper codon recognitionon the mRNA el ongati on,i ncorporati ngami no aci ds and the
.l
and the involvement of elongation factor q, formation of the specific protein/polypeptide
EF-la,)and supply of energy by CTP. As the molecule, one of the sfop or termination signals
s p l a c e di n th e A -s i te ,E F-1cr
am inoac y l -tR Ni A and (UAA, UAG and UCA) terminatesthe growing
CDP are recycled to bring another aminoacyl- polypeptide.The terminationcodonswhich act as
t RNA . stopsignalsdo not havespecifictRNAsto bind. As
the termination codon occupies the ribosomal
Peptide bond formation A-site,the releasefactornamelyeRFrecognizes the
The enzyme peptidyltransferase catalyses the stop signal.eRF-GTPcomplex,in association with
formationof peptide bond (Fig. 4.19).The activity the enzymepeptidyltransferase, cleavesthe peptide
of this enzymelies on 2BS RNA of 605 ribosomal bond between the polypeptide and the tRNA
subunit.lt is thereforethe rRNA (and not protein) occupyingP-site.In this reaction,a watermolecule,
referredto as ribozyme that catalysesthe peptide insteadof an amino acid is added.This hvdrolvsis
bond f or ma ti o n . A s th e a m i n o a c i d i n the releases the proteinand IRNA from the P-site.The
aminoacyl-tRNA is alreadyactivated,no additional B0S ribosomedissociatesto form 40S and 605
energyis requiredfor peptidebond formation. subuni tsw hi ch are recvcl ed.The mR N A i s arso
released.
The net resultof peptidebond formationis the
attachmentof the growing peptide chain to the ,NHIB'TOBS OF PNOTE"V SY'Y7HES'S
I RNA in t he A -s i te .
Translationis a complex processand it has
Translocation become a favourite target lor inhibition by
antibiotics.
Antibioticsarethe substances produced
As the peptide bond formation occurs, the by bacteriaor fungi which inhibit the growth of
ribosomemovesto the next codon of the mRNA otherorganisms. Majorityof the antibioticsinterfere
(towards3'-end).This processcalledtranslocation, with the bacterial protein synthesisand are
basically involves the movement of growing harmless to higherorganisms. This is due to the fact
peptidechain from A-site to P-site.Translocation that the processof translationsufficientlydiffers
requiresEF-2and GTP.CTP gets hydrolysedand betweenprokaryotes and eukaryotes. The actionof
suppliesenergy to move mRNA. EF-2 and CTP a few important antibiotics on translation is
complexrecyclesfor translocation. describedhere.
ln recent years, another site namely exist site Streptomycin: Initiationof proteinsynthesisis
(E-site)has been identified in eukaryotes.The inhibitedby streptomycin. lt causesmisreadingof
deacylatedIRNA movesinto the E-site,fiom where mRNA and interfereswith the normal pairing
it leavesthe ribosome. betweencodonsand anticodons.
54 B IOTE CHNO LO CY

80S initiation
complex

AAr
l-
t
AA,
r-..._A A n

Termination
codon

Met
I
AAr
I
AA2

GTP @Translocation t-
AAa
t-
AA"
G DP + Pi i

,-\ AAn
EF-2 ll..UAC/
5'M3',
mR N A Peptide
synthesized
Fig.4 .l a c ontd. ner t c ol um n

Fiq.4.18: Protein biosynthesis-Elongation and termination(fot initiationrefer Fi7.4.17). Met-Methionine;

P-site - Peptidyl IRNA binding site; A-site - Aminoacyl iRNA binding site. AA-Amino acid;
EF-Elongation factor; RF-Releasing factor.
 -+ : TRANSCRIPTION
--ra:i,i€. AND TRANSLATION 55
i
lA- II
ril
ili

HN:
li
R.-9-H i A-Site
ll
C=O i
Peptidyltransferase

I
I

3' m R N A 3'mRNA

Ribosome

Ftg- 4.19 : Formation of peptide bond in translation (P-site-Peptidyt IRNA s'tte;A-site-Aminoacvl IRNA sitd.

Tetra cyc line: lt inhibit st he binding of am i n o a c y l functionally active conformation e.g. danatured
:R\ I to the ribosomal complex. In fact, tetracycline pancreaticribonuclease.However, a vast majority of
-:n also bl oc k euk ar y ot ic pr ot ein s y nt hes i s .T h i s ,
proterns can attain correct conformation, only
^ r',\e ve r, d oes not happen s inc e euk ar y o t i c c e l l through the assistanceof certain proteins referredto
-e mbra ne is not per m eablet o t his dr ug. as chaperones. Chaperones are heat shock proteins
(originally discovered in response to heat shocr,t.
Puromycin : This has a structuralresemblanceto
They facilitate and favour the interactionson the
I RNA. Pur om y c in ent er s t he A s i t e a n d
"^ rin oa cyl polypeptide surfaces to finally give the specific
:ets incorporated into the growing peptide charn
ard causes its release. This antibiotic orevents conformation of a protein Chaperones can
crotein synthesisin 6oth prokaryotesand eukaryotes. reversibly bind to hydrophobic regions of unfolded
proteins and folding intermediates. They can
Chloramphenicol : lt acts as a competitlve stabilize intermediates, prevent formation
of
rhibitor of the enzyme peptidyltransferaseano incorrectintermediates,and also preventundesirable
:rus interfereswith elongation of peptide chain interactionswith other proteins. All these activitres
Erythro m y c in: lt inhibit s t r ans loc a t i o n D y of chaperones help the protein to attain compact
cin din g with 50S s ubunit of bac t er ial r ibos o m e . a n d biologicallyactive conformation.

Diphtheria toxin : lt prevents translocation rn Types of chaperones

e ukaryotic pr ot ein s y nt hes is by inac t i v a t i n g
e lon ga tion f ac t or eEFr . Chaperones are categorized into two major
groups
IV CI{APE RO NES AND PRO TEI N
1. Hsp70 system : This mainly consists of
FOLDIN G
Hsp70 (70-kDa heat shock protein) and Hsp40 (40-
The three dimensional conformation of oroteins kDa Hsp). These proteinscan bind individually to
is important for their biological functions. Some of the substrate (protein) and help in the correct
the proteins can spontaneouslygeneratethe correct formation of protein folding.
 56 B IOTE C HNO LO CY

Polypeptide

Folding
\---------
---t/Proteolytic Intein Chemical(covalent)
splicing modifications

Tertiarystructure

Fig. 4.20 : An outline of post-translational modifications of proteins,

2. Chaperonin system : This is a large the polypeptidesafter the initiation of their

oligomericassemblywhich forms a structureinto synthesisor, most frequently, after the protein
which the folded proteins are inserted. The synthesis is completed. These modifications
m a i n l yh a sH sp60andH spl0 i .e.
c h a p e ro n isny s te m include protein folding (described already),
6 0 k D a H s p a n d 1 0 k D a H sp. C haperoni ns are trimming by proteolytic degradation, intein
requiredat a laterpartof the proteinfoldingprocess, spl i ci ng and coval ent changes w hich ar e
and oftenwork in association with Hsp70system. collectively known as post-translational
modifications(Fig.a.2A.
Frotein misfolding and diseases
The failureof a proteinto fold properlygenerally Proteolytic degradation
leadsto its rapid degradation.Cysticfibrosis(CF)is
a commonautosomalrecessive disease. Somecases Many proteinsare synthesized as the precursors
il of CF with mutationsthat resultin alteredorotein w hi ch are much bi ggeri n si zethan the f unct ional
(cystic fibrosis transmembrane conductance proteins.Someportionsof precursormoleculesare
!i regulatoror in short CFTR)have been reported. removedby proteolysis to liberateactiveproteins.
Mutated CFTRcannot fold properly, besidesnot This process-commonly referredto as trimming-
li being able to get glycosylatedor transported. may occur in Colgi apparatus,secretoryvesicles
Therefore,CFTRgetsdegraded. and, sometimes, afterthe secretionof proteins.The
formati onof i nsul i nfrom preproi nsul i n ,
conver sion
Certainneurological diseases which are due to
of zymogens (inactive digestive enzymes e.g.
cellular accumulationof aggregates of misfolded
trypsinogen)to the active enzymes are some
proteinsor their partially degradedproducts have,
exampl esof tri mmi ng.
beenidentified.Thetermprions(prcteinousinfectious
agents)is usedto collectivelyrepresent them. The synthesisof the proteins as inactive
P ri o n se x h i b i t th e c h a ra c teri sti cs
of vi ral or orecursors and their later conversioninto active
microbialpathogensand have been implicatedrn form, may be, to protectthe functionalproteinunit
many diseases. e.g. mad cow disease,Creutzfeldt- from the envi ronmental i nsul ts.
Jacob disease,Alzheimer'sdisease,Huntington's
disease. Intei n spl i ci ng

V. POST.TRANSLATIONAL Inteins are intervening sequences in certain

M O D IF IC A T IO N S OF P R OTE IN S proteins. These are comparableto introns in
mRNAs. Inteins have to be removed, and exteins
The proteinssynthesizedin translationare, as Iigated in the appropriateorder for the protein to
such, not functional.Many changestake place in becomeactive.
 57

2. Hydroxylation : During the formation of

Tlur.'r4.2 Selectedexanplesof post- collagen, the amino acids proline and lysine are
kzrshtional modificationsof protelns respectively converted to hydroxyproline and
throughtheir aminoacids hydroxylysine. This hydroxylation occurs in the
e n d o p l a s m i c r e t i c u l u m a n d r e q u i r e sv i t a m i n C .
tnin o a cid Post-translational
modificationk) 3. Glycosylation : The attachment of carbo-
hydrate moiety is essential for some proteins to
ir- -c-:erminal acetylation,
Glycosylation,
perform their functions. The complex carbohydrate
j- 10 4cl0 formylation.
myristoylation,
moiety is attached to the amino acids, serine and
Saroxyterminal ADP-ribosylation
Methylation, t h r e o n i n e ( O - l i n k e d ) o r t o a s p a r a g i n e( N - l i n k e d ) ,
3r' no acid l e a d i n g t o t h e s y n t h e s i so f g l y c o p r o t e i n s
l' c , nine Methylation
Vitamin K dependent carboxylation of glutamic
lscarticacid hydroxylation
Phosphorylation,
acid residues in certain clotting factors is also a
irsieine(-SH) (-S-S-) formation,
Cystine o o s t - t r a n s l a t i o n am
l odification.
formation,
selenocysteine
glycosylation. ln the lable 4.2, selected examples of post-
13.rtamic y-carboxylation. t r a n s l a t i o n a lm o d i f i c a t i o n o f p r o t e i n s t h r o u g h t h e i r
acid Methylation,
amino acids are given
- strdine phosphorylation
Methylation,
_ysrne methylation,
Acetylation,
hydroxylation,
biotinylation.
Methionine formation,
Sulfoxide
Phenylalanine hydroxylation.
Glycosylation, The eukaryotic proteins (tens of thousands) are
Proltne glycosylation.
Hydroxylation, distributed between the cytosol, plasma membrane
Serine glycosylation. a n d a n u m b e r o f c e l l u l a r o r g a n e l l e s ( n u c l e u s ,
Phosphorylation,
Threonine methylation
Phosphorylation, mitochondria, endoplasmic reticulum etc.). At the
glycosylation. appropriate places, they perform their functions.
Tryptophan Hydroxylation. The proteins, synthesizedin translation, have to
Tyrosine phosphorylation, reach their destination to exhibit their biological
Hydroxylation,
iodination.
sulfonylation, activity. This is carried out by a process called
protein targeting or protein sorting or protein
Iocalization. The proteins move from one
Covalent modifications compartmentto another by multiple mechanisms.
T he pr ot e i n s s y n th e s i z e di n tra n s l a ti onare The protein transport from the endoplasmic
subjectedto many covalent changes.By these r e t i c u l u m t h r o u g h t h e C o l g i a p p a r a t u s ,a n d b e y o n d
m odif ic at ioni sn th e a m i n oa c i d s ,th e p ro te i n smay usescarrier vesicles.lt may be, however, noted that
be convertedto active form or inactive form. only the correctly folded proteins are recognized
Selectedexamplesof covalent modificationsare as the cargo for transport Protein targeting and
describedbelow. oost-translational modifications occur in a
1. Phosphorylation: The hydroxyl group coordinated manner.
containingan-rinoacidsof proteins,namelyserine,
Certain glycoproteins are targeted to reach
threonihe and tyrosine are subjected to
lysosomes,as the lysosomalproteins can recognize
n .h e p h o s p h o ry l a ti oma
phos phor y la ti o T n y e i ther
the glycosidic compounds e.g. N-acetyl-
increaseor decreasethe activity of the proteins
glucosaminephosphate.
A group of enzymes called protein kinases
c at aly s ephos p h o ry l a ti ownh i l e p ro te i np h o spha- For the transport of secretoryproteins, a special
tasesare responsible for dephosphorylation (removal mechanism is operative. A signal peptide
of phosphategroup).Many examplesof enzymes c o n t a i n i n g 1 5 - 3 5 a m i n o a c i d s , l o c a t e d a t t h e
that undergophosphorylation or dephosphorylationamino terminal end of the secretory proteins
are known in metabolisms. facilitates the transoort.
58 B IOTE CHNO LO CY

Protein targeting to mitochondria chondriaare derivativesof prokaryotes.mtDNA is

ci rcul ar i n nature and contai nsabout 16, 000
Most of the proteins of mitochondria are
nucleotidebases.
synthesizedin the cytosol,and their transportto
mitochondriais a complexprocess.Majorityof the A vast maj ori ty of structuraland f unct ional
proteinsare synthesized as largerpreproteins with proteinsof the mitochondriaare synthesized in the
N-terminalpresequences for the entry of these cytosol , under the i nfl uenceof nuclear DNA.
proteins into mitochondria. The transport of H ow ever,certai n protei ns(around13), m ost of
unfoldedproteinsis oftenfacilitatedby chaperones. them being the componentsof electrontransport
One protein namely mitochondrial matrix chai n, are synthesi zedi n the mi to chondr ia.
targetingsignal, involved in protein targetinghas Transcri pti ontakes pl ace i n the mi tochondr ia
been identified. This protein can recognize l eadi ngto the synthesi sof mR N A s,tRNAs and
mitochondrial receptor and transport certain rRNAs.Two types of rRNA and about 22 species
proteinsfrom cytosolto mitochondria.This is an of IR N A have been so far i denti fi ed.This is
energy-dependent process. fol l ow ed by transl ati on"resul ti ng i n pr ot ein
synthesi s.
Protein targeting to other organelles
The mitochondriaof the spermcell do not enter
Specificsignalsfor the transportof proteinsto the ovum duringfertilization,therefore,mtDNA is
organellessuch as nuclei and peroxisomeshave inherited from the mother. Mitochondrial DNA is
been identified. subjectedto high rateof mutations(aboutl0 times
T h e s ma l l e rp ro te i n sc a n easi l y passthrough more than nuclear DNA) that causes inherited
nuclearpores.However,for largerproteins,nuclear defects in oxidative phosphorylation.The best
Iocalization signals are needed to facilitate their known among them are certain mitochondrial
e n try i n to n u c l e u s . myopathies and Leber's hereditary optic
neuropathy. The latteris mostlyfound in malesand
is characterizedby blindnessdue to lossof central
vision as a result of nquroretinaldegeneration.
Leber's hereditary optic neuropathy is a
consequence of single base mutationin mtDNA.
The mitochondrialDNA (mtDNA)hasstructural D ue to thi s,the ami no aci d hi sti di ne,i n placeof
and functional resemblanceswith prokaryotic arginine,is incorporatedinto the enzyme NADH
DNA. This fact supports the view that mito- coenzymeQ reductase.
 r\ r r the c hem ic al v ehic le of her edity , i s 2. Negative regulation : A decreasein the gene
LJ - - -posed of functional unifs, namely genes. expression due to the presence of a regulatory
-: -.-T genome refers to the total genetic infor- element (negative regulator) is referred to as
-.- - con tain ed in a c ell. The bac t er ium Es ch e r i - negative regulation.
- ,: - r'r contains about 4,400 Benes present on a
-.: It may be noted here that double negativeeffect
clro mos om e. The genom e of hum an s i s
on gene regulationresultsin a positivephenomenon.
'=
= ::oommoleplex x . ,w 233 p
h 2
witi th pair so
a i rsoff (d i p l o i d )c hr
( diploid) h romo-
omo-
-- corn nnta
t:i n i nining
o A billion
6 hillinn ( 6 xx 10e)
t6 hr cc e pair
1O9t bas nair c s o
n{f
:, * ith an estimated 30,000-40,OOO genes. At Gonstitutive and inducible genes

= ren time, only a f r ac t ion of t he genom e i s The genes are generally considered under two
: ' : -a :S€d categones.
--e living cells pos s es sa r em ar k ablepr oper t yt o 1 . Constitutive genes : The products (proteins)
::::r to cha ng es in t he env ir onm ent by r egula t i n g of these genes are required all the time in a cell.
'- sen e e xp r es s ion. For ins t anc e, ins ulin i s Therefore, the constitutive genes (or housekeeping
-
,"^ :^ esize d b y s pec ializ ed c ells of panc r eas a n d genes) are expressedat more or less constant rate in
^ -: ov ce lls of ot her or gans ( s ay k idney , li v e r ) , almostall the cells and, further,they are not subjected
.:-,ruBh th e n uc lei of all t he c ells of t he b o d y to regulatione.g. the enzymes of citric acid cycle
-:a :n th e in s ulin Benes . ' M olec ular r egul a t o r y
2. Inducible genes : The concentration of the
-=cra nisms fa c ilit at et he ex pr es s ionof ins ulin g e n e
p r o t e i n ss y n t h e s i z e db y i n d u c i b l e g e n e si s r e g u l a t e d
- ca ncrea s, w hile pr ev ent ing it s ex pr es s io n i n
b y v a r i o u s m o l e c u l a r s i g n a l s .A n i n d u c e r i n c r e a s e s
.-er cells.
the expression of these genes while a repressor
decreases, e.g. tryptophan pyrrolase of liver is
G ENE REGU LATI O N_G ENERAL
induced by tryptophan.
The regulation of the expression of genes is
acsolutely essential for the growth, development, One cistroh.on€ subunit concept
difierentiation and the very existence of an
T h e c h e m i c a l p r o d u c t o f a g e n e e x p r e s s i o ni s a
lq an ism. Th er e ar e t wo t y pes of gene r egula t i o n -
protein which may be an enzyme. lt was originally
losrtive an d negat iv e.
believed that each gene codes for a specific
1 Positive regulation : The gene regulation is e n z y m e , l e a d i n g t o t h e p o p u l a r c o n c e p t , o n e g e n e -
said to be positive when its expressionis increased o n e e n z y m e . T h i s h o w e v e r , i s n o t n e c e s s a r i l yv a l i d
lr a regulatory element (positive regulator). due to the fact that several enzymes (or proteins)

59
60 B IOTE CHNO LO CY

are comoosedof two or more nonidentical subunits Repression of lac operon

(p o l y p e p ti dceh a i n s ).
The regul atorygene (l ) i s consti tut ive.lt is
The cistron is the smallest unit of genetic expressed at a constantrateleadingto the synthesis
expression. lt is the fragmentof DNA codingfor the of lac repressor. Lac repressoris a tetrameric(4
s u b u n io
t fa p ro te i nm o l e c u l e.
The ori gi nal
concept subunits) regulatory protein(totalmol. wt. 150,000)
of one gene-one enzyme is replaced by one w hi ch speci fi cal lbi y ndsto the operatorgene( O ) .
cistron-onesubunit. Thi s preventsthe bi ndi ng of the enzym e RNA
polymeraseto the promoter site (P), thereby
Models for the study of blockingthe transcription of structuralgenes(2, Y
gene expression and A ). Thi s i s w hat happensi n the a bsenceof
lactosein E. coli.The repressormolecule actsas V
of geneexpressi on negative regulator of gene expression.
El u c i d a ti oonf th e re g u l a t i on
in prokaryotes has largelyhelpedto understand the
frinciplesof the flow of informationfrom genesto Derepression of lac operon
mRNA to synthesizespecific proteins. Some
importantfeaturesof prokaryoticgene expression In the presenceof lactose (inducer) in the
are describedfirst. This is followed by a brief medium,a smallamountof it can enterthe E. coli
accountof eukaryoticgeneexpression. cells.The repressor moleculeshave a high affinity
for lactose.Thp lactosemoleculesbind and induce
a conformational changein the repressor. The result
is that the repressor getsinactivatedand,therefore,
cannot bind to the operatorgene (O). The RNA
polymeraseattachesto the DNA at the promoter
The operon is the coordinatedunit of genetic site and transcriptionproceeds,leading to the
expression in bacteria.The conceptof operonwas formationof polycistronicmRNA (for genesZ, Y
i n tro d u c e db y J a c o ba n d M o nod i n 1961 (N obel and A) and, finally,the 3 enzymes.Thus, lactose
Prize 1965), basedon their observations on the i nduces the synthesi sof the three enzym es
regulationof lactosemetabolismin E. coli. This is B-galactosidase,galactoside permease and
popularlyknown as lac operon. galactosideacetylase.Lactoseacts by inactivating
the repressor molecules, hence this process is
LACTOSE (LACI OPERON known as derepression of lac operon.

Structure of lac operon Gratuitousinducers: Therearecertainstructural

anal ogs w hi ch can i nducethe lac oper on
of l actose
The lac operon(Fig.5.1\ consistsof a regulatory
but are not the substratesfor the enzyme p-
gene (l; I for inhibition),operatorgene (O) ano
galactosidase.Such substancesare known as
threestructuralgenes(2, Y,A). Besides thesegenes, gratuitousi nducers.lsopropylth iogalactoside (lPTC)
there is a promotersite (P), next to the operator
is a gratuitousinducer,extensivelyused for the
gene, where the enzyme RNA polymerasebinds.
studyof lac operon.
The structuralgenes7, Y and A respectively, code
for the enzymes p-galactosidase,galactoside The catabolitegeneactivatorprotein: The cells
permeaseand galactosideacetylase.p-Calacto- of E. coli utilize glucosein preferenceto lactose;
sidasehydrolyses lactose(p-galactoside)to galactose when both of them are oresentin the medium.
and glucosewhile permeaseis responsible for the After the depletion of glucose in the medium,
transportof lactoseinto the cell. The functionof utilization of lactose starts.This indicatesthat
acetylase(codedby A gene)remainsa mystery. glucosesomehowinterfereswith the inductionof
l ac operon.Thi s i s expl ai nedas fol l ows.
The structuralgenesZ, Y and A transcribeinto
a s i n g l el a rg emR N Aw i th 3 i n dependent
transl ati on The attachmentof RNA polymeraseto the
units for the synthesisof 3 distinctenzymes.An promotersite requiresthe presenceof a catabolite
mRNA codingfor more than one proteinis known gene activator protein (CAP)bound to cyclic AMP
as polycistronic mRNA. Prokaryotic organisms (Fig. 5.2). The presenceof glucose lowers the
containa largenumberof polycistronicmRNAs. of cA MP by inact ivat ing
concentrati on
i ntracel l ul ar
 61
N F C E N EEX PIE 9 !! 9N
" ! : - = ' 5 : RE C U L A T IO O

Regulatory Promoter OPerator Structuralgenes

gene site gene

Ar P U z A

P z A

rTl
|_-1-L
lll
Repressor
tetramer

CAP-cAMP

of lac operon (B) Repression of lac operon

Fig. 5,1 : Model of lactose operon in E.coli (A) Structure
(C) Derepressionof lac operon' (CAP-cAMP---catabolite gene activator
protein bound to cAMP; RNAP-RNA polymerase)'

of l ac operonby depl eti ngcA MP l evel s'

tfre enzyme adenylyl cyclaseresponsiblefor the expressi on to initiate
of exogenouscAMP is found
s int hes isof c A M P.D u e to th e d i m i n i s h e dl e v e l sof Addition
formation of CAP-cAMP is low' the transcription of many inducible operons/
..lVR the
the bindingof RNA polymerase to DNA i ncl udi ng l ac oP eron.
Therefore,
due to the absence of CAP-cAMP)and the It is now clear that the presenceof CAP-cAMP
p re s e of
nce is essential of structuralgenes
for the transcription
is a l mo s tn e g l i g i b l ei n th e
t r ans c r ipt ion
qluc os e. T hu s , g l u c o s e i n te rfe re s w i th the of lac operon. Thus, CAP-cAMP acts as a positive
62 B IOTE C HNO LO CY

Glucose corepressor. Thus lactose operon (described

already) is inducible, whereas tryptophan operon is
repressible The tryptophan operon is said to be
d e p r e s s e dw h e n i t i s a c t i v e l y t r a n s c r i b e d

Glucose
Tryptophan operon regulatlon
J by a repressor
o
Tryptophan acts as a corepressorto shut down
the synthesisof enzymes from tryptophan operon.
T h i s i s b r o u g h t o u t i n a s s o c i a t i o nw i t h a sp e ci fi c
protein, namely tryptophan repressor. Tryptophan
r e p r e s s o r ,a h o m o d i m e r ( c o n t a i n s t w o i d e n ti ca l
s u b u n i t s )b i n d s w i t h t w o m o l e c u l e s o f t r yp to p h a n ,
and then binds to the trp operator to turn off the
transcription.lt is of interestto note that tryptophan
n r e p r e s s o r a l s o r e g u l a t e s t h e t r a n s c r i p t i on o f th e
ffi
lacoperon gene (frpR) responsiblefor its own synthesis.
I Two polycistronic mRNAs are produced from
Polycistronic
mRNA tryptophan operon-one derived from all the frve
structural genes, and the other obtained from the
Fig. 5,2 : Control of lac operon by catabolite gene last three genes.
activator pratein (CAP) and the role of glucose.
Besides acting as a corepressor to regulate
t r y p t o p h a n o p e r o n , t r y p t o p h a n c a n i n h i b i t th e
a c t i v i t y o f t h e e n z y m e a n t h r a n i l a t es y n t h eta seTh . is
regulator for the gene expression. lt is, therefore,
i s r e f e r r e dt o a s f e e d b a c k i n h i b i t i o n , a n d i s b r o u g h t
evident that lac operon is subjectedto both positrve
o u t b y b i n d i n g o f t r y p t o p h a n a t a n a l l o s t e r i csi te o n
(by repressor, described above) and negative
anthrani late synthetase.
r egulat ion.

Attenuator as the second control s;te

for tryptophan operon

Attenuator gene (rrpa)of tryptophan operon lies

T ry p to p h a ni s a n a ro ma ti cami no aci d, and i s
upstream of trpE gene Attenuation is the second
requiredfor the synthesis of all proteinsthat contain
level of regulation of tryptophan operon. The
tryptophan.lf tryptophan is not present in the a t t e n u a t o rr e g i o n p r o v i d e s R N A p o l y m e r asew h r ch
me d i u mi n a d e q u a teq u a n ti tyth
, e bacteri alcel l has
r e g u l a t e s t r a n s c r i p t i o n . I n t h e p r e se n ce o f
to make it, as it is requiredfor the growth of the t r y p t o p h a n , t r a n s c r i p t i o ni s p r e m a t u r e l yt e r m i n a te d
bacteria. at the end of attenuator region However, in the
The tryptophanoperonof E. coli is depictedrn absenceof tryptophan, the attenuatorregion has no
Fig. 5.3. This operoncontainsfive structuralgenes effect on transcription.Therefore,the polycistronic
(trpE, trpD, trpc, trpB, trpA), and the regulatory m R N A o f t h e f i v e s t r u c t u r a l q e n e s ca n D e
elements-primarypromoter(trp P1,operator(frpO), s y n t h e s i z e d .
attenuator (trpa), secondary internal promoter
(TrpP2),
and terminator(trpt).
The five structuralgenesof tryptophanoperon
code for threeenzymes(two enzymescontaintwo
d i ffe re n ts u b u n i ts )re q u i re dfor the synthesi sof
tryptophanfrom chlorismate.
E a c h c e l l o f t h e h i g h e r o r g a n i s m c o n ta i n s th e
The tryptophanrepressoris alwaysturned on, entire genome. As in prokaryotes,gene expression
u n l e s si t i s re p re s s ebdy a s p e ci fi cmol ecul ecal l ed in eukaryotes is regulated to provide the
 N F C EN EEX PR ES S ION
Chaot er5 : R EC U L A T IO O 63

STRUCTURAL
GE N E S

REGULATORY
ITpTELEMENTS

II __l_r-_T mRNAs

J tl
jiJ
Anthranilate
synthase(CoII) Phosphoribosyl Tryptophan Tryptophan
Phosphoribosyl POLYPEPTIDES
anthranilate synthetaseB synthetasea
anthranilate rsomerase,
transferase Indoleglycerol
phosphate
synthetase

Anthranilatesynthetase Tryptophan
synthetase ENZYME
(Col + CoII) COMPLEXES
(rlz9z\

CATALYSED
REACTIONS
Glutamine L-Serine

Fig. 5.3 : Tryptophan operon in E.coli [regulatory elements are promoter (trpP), operator (trpo),
attenuator (trpa), secondary internal promoter (trpPr) and terminator (trpt); Col, Coll -Component land
component Il; PRPP-5-Phosphoribosyl 1-pyrophosphate;CdRP-Carboxyl-phenylamino 1 deoxyribulose
S-phosphate; lnGP - Indole S-glycerol phosphatel.

appropriate responseto biological needs. This may CHROMATIN SRUCTURE

o ccur in the following way s AND GENE EXPRESSION

. Expressionof certain genes (housekeepinggenes) The DNA in higher organisms is extensively

in mo st o f t he c ells .
o Activation of selected genes upon demand.
. Perma ne ntinac t iv at ionof s ev er aleenes in a l l b u t
a few types.
In case of prokaryotic cells, most of the DNA is
o rga nized into genes whic h c an be t r ans c r ib e d .I n
contrast, in mammals, very little of the total DNA
is organized into genes and their associated
regulatory sequences.The function of the bulk of
the extra DNA is not known.
Eukaryotic gene expression and its regulation
are highly complex. Some of the important aspects
a re b iiefly d es c r ibed.
folded and packed to form protein-DNA complex
called chromatin. The structural organization of
DNA in the form of chromatin plays an important
role in eukaryotic Bene expression. In fact,
chromatin structure provides an additional level of
control of gene expression.
A selected list of genes (represented by the
products) along with the respective chromosomes
on which they are located is given in Table 5.1 .
In general, the genes that are transcribedwithin
a particular cell are less condensedand more open
in structure. This is in contrast to genes that are
not transcribed which form highly condensed
chromatin.
64 B IOTE CHNO LO CY

Methylation of DNA
Irru 5.1 A selectcdllst of genes{representedlo}r and inactivation of genes
the products)alongwlth rcspective chlonosones
Cytosine in the sequenceCC of DNA gets
Genes Chromosome methylatedto form 5'-methylcytosine. A major
number portion of CC sequences(about 20%) in human
DNA exists in methylated form. In general,
phosphatase
Alkaline 1 methylation leadsto lossof transcriptionalactivity,
Apolipoprotein
B 2 and thus inactivation of genes.This occurs due to
Transfenin bi ndi ng of methyl cytosi nebi ndi ng pr ot eins t o
methylatedDNA. As a result,methylatedDNA is
Alcohol
dehydrogenase ^
not exposedand boundto transcription factors.lt is
HMG CoAreductase interestingto note that methylation of DNA
21-hydroxylase
Steroid correlateswith deacetylationof histones This
Arginase 7 providesa double meansfor repression of genes.
Carbonic
anhydrase I of genes,
The activationand normalexpression
Interferon and gene inactivationby DNA methylationare
I
depicted in Fig. 5.4.
Parathyroid
hormone 11
Glyceraldehyde
3-phosphate
dehydrogenase12
Adenosinedeaminase 13
o,,-Antitrypsin 1n

CytochromePouo 15
Hemoglobina-chain lo

Growlhhormone 17
Prealbumin 18
phosphokinase
Creatine (Mchain) IJ

Adenosinedeaminase 20
dismutase
Superoxide 21
(1.chain)
lmmunoglobulin zz

Glucose6-phosphatedehydrogenase X
Steroid
sulfatase oruRmetnvtation
f

Histone acetylation and deacetylation

EukaryoticDNA segmentsare wrappedaround
histoneproteinsto form nucleosomeAcetylation
I u"tnyt.yto.in"
proteins
or deacetylationof histonesis an important factor IbindinO
in determining the gene expression.In general,
acetylationof histonesleadsto activationof gene
expressionwhile deacetylation reverses
the effect.
Acetylationpredominantly occurson the lysine Y

re s i d u e si n th e a mi n o te rm i nalends of hi stones. Gene inactivation and no erpression

This modificationin histonesreducesthe positive Fig. 5.4 : Methylation of DNA and inactivation of
chargesof terminalends(tails),and decreases their genes (A) Gene activation in the absence of DNA
binding affinity to negatively charged DNA. methylation (B) Gene inactivation due to methylation
Consequently, nucleosome structureis disruptedto
(fi represent CG sequences).
a l l o w tra n s c ri o ti o n .
 OF C EN EE XP R E S S ION
: - , i. t er 5 : R E C U L AT ION 65

ET{HANCERS AND TI SSUE. SPECI FI C

GE}'E EXPRESSI O N

'i-
i-^ancers (or activators)are DNA elementsthat
:aie or enhance gefie expression. The
I
Gene activated
:--:rcers p rov ide binding s it esf or s pec if ic pr o t e i n s
:-= :e gu late tr ans c r ipt ion.They f ac ilit at e bin d i n g
--r t1e transcription complex to promoter
-?- 3ns. Enhancers differ from promoters in two I
I
: ;: 'lct lvays +
Gene activated
.: Enhancersmay be located thousandsof base
:,. 's away from the start of transcription site tz l

:'cmoters are close to the site of transcription).

2 They can work in either orientation i.e.

:-iancers can worK upstream (5') or downstream
j' irom the oromoter.
Several eukaryotic genes containing enhancer
: ements at various locations relative to their
,oding regions have been identified.
Some of the enhancers possessthe ability to
cromote transcription in a tissue-specificmanner.
For instan ce ,g ene ex pr es s ionin ly m phoid c el l s f o r
the pro du ctio n im m unoglobulins ( lg) is pr om o t e d
b,v the enhancer associatedwith lg genes between
J a nd C re gio ns .
Transgenicanimals are frequently used for the
study of tissue-specificexpression. The available
evidence from various studies indicates that the
tissue-specific gene expression is largely mediated
through the involvement of enhancers.
COMBINATION O F DNA ELEM ENTS
AND PROTEI NS I N G ENE EXPRESSI O N
Cen e e xp res s ionin m am m als is a c om plica t e d
oro ce ss with s ev er al env ir onm ent al s t im uli o n 'a
single gene. The ultimate response of the gene
which may be positive or negative is brought out
by the associationof DNA elements and proteins.
In th e illust r at iongiv en in t he Fig. 5. 5, gen e I i s
activatedby a combination of activators1, 2 and 3.
Cene ll is more effectively activated by the
combined action of 1, 3 and 4. Activator 4 is not
in direct contact with DNA, but it forms a bridge
between activators 1 and 3, and activatesgene ll.
As regards gene lll, it gets inactivated by a
co mbin atio n o f 1, 5 and 3. ln t his c as e, pr ot e i n 5
interfereswith the binding of protein 2 with the
DNA and inactivatesthe gene.
Gene inactivated
Fig. 5.5 : A diagrammatic representation of the
association of DNA elements and proteins in gene
regulation. A, B and C represent genes l, ll and lll
(1...5 representprateins).
MOTIFS IN P R OTE IN S
A N D GE N E E X P R E S S ION
A moti f l i teral l ymeansa domi nantel ement.
Certainmotifs in proteinsmediatethe binding of
regulatoryproteins(transcription factors)to DNA.
The specificcontrol of transcription occursby the
bindingof regulatoryproteinswith high affinityto
the correctregionsof DNA.
A great majority of specific protein-DNA
interactionsare broughtout by four uniquemotifs.
. H el i x-turn-hel (H
i x TH
r Zi nc fi nger
r Leuci nezi pper
o H el i x-l oop-hel i(H
x LH ).
The above listed amino acid motifs bind with
high affinityto the specificsite and low affinityto
otherpartsof DNA. The motif-DNAinteractions are
maintainedby hydrogenbondsand van der Waals
forces.
Helix.turn.helix motif
The helix-turn-helix(HI$ motif is about 20
amino acids which represents a small part of a
largeprotein.HTSis the domainpart of the protein

Biotechnology [5]
66 B IOTE C HNO LO CY

w hic h s pec if ic ally int er ac t s w i t h t h e D N A

( Fig. 5. 6A) . Ex am ples of heli x - t u r n - h e l i x m o t i f
, nd cyclic AMP
pr ot eins inc lude lac t os e r epr es s o r a
catabolite activator protein (CAP) of E. coli, and
s ev er al dev elopm ent ally im po r t a n t t r a n s c r i p t i o n
f ac t or s in m am m als , c ollec t iv e l y r e f e r r e d t o a s
homeodomain proteins. The term homeodomain
refers to the portion of the protein of tne
t r ans c r ipt ion f ac t or s t hat r e c o g n i z e s D N A (A) Helix-turn-helix
Hom eodom ain pr ot eins play a k e y r o l e i n t h e
dev eloom ent of m am m als

Zinc finger motif

/Z\

Sometime ago, it was recognized that the ,z Ct--r-------C\

t (Zn) )
t r ans c r ipt ion f ac t or TFI I I A r eq u i r e s z i n c f o r i t s \ 6'lv---g/
ac t iv it y O n analy s is , it was r e v e a l e d t h a t e a c h
/\
TFI I I A c ont ains z inc ions a s a r e p e a t i n g ,/\
c oor dinat ed c om plex . This c om p l e x i s f o r m e d b y (B) Zinc finger (Cys-Cys) Zinc finger (Cys-His)
t he c los ely s pac ed am ino ac i d s c y s t e i n e a n d
c y s t eine,f ollowed by a his t idin e - h i s t i d i n e p a i r . I n
s om e ins t anc es ,His - His is r ep l a c e d b y a s e c o n d
Cys-Cys pair rFig. 5.68t.
The z inc f inger s bind t o t h e m a j o r g r o o v e o f
DNA, and I ie on t he f ac e of t he D N A T h i s b i n d i n g
makes a contact with 5 bp of DNA. fhe steroid
hormone receptor transcription factors use zinc
f inger m ot if s t o bind t o DNA.
The oc c ur r enc e of a m ut a t i o n r e s u l t i n g i n a
s ingle am ino ac id c hange o{ z i n c f i n g e r m a y l e a c l
to resistanceto the action of certain hormones on
gene ex pr es s ion.A m ut at ed z in c f i n g e r r e s i s t a n t o
t he ac t ion of c alc it r iol ( ac t iv e f o r m o f v i t a m i n D ) (C) Leucinezipper
has been ident if ied. This m av u l t i m a t e l v r e s u l t i n
r ic k et s ( v it am in D def ic ienc y ) .

Leucine zipper motif

The basic regions of leucine zipper (bZlP)

pr ot eins ar e r ic h is t he am ino a c i d l e u c i n e T h e r e
oc c ur s a per iodic r epeat of l e u c i n e r e s i d u e s a t
every seventh position. This type of repeat structure
allows t wo ident ic al m onom er s o r h e t e r o d i m e r st o
zip together and form a dimeric complex This
protein-protein complex associates and interacts
wit h DNA ( Fig. 5. 6a Cood e x a m p l e s o f I e u c i n e
z ipper pr ot eins ar e t he enhanc e r b i n d i n g p r o t e i n s
( EBP) - f os and jun.

Helix - loop- helix m ot if (D) Helix-loop-helix

Two am phipat hic ( lit er ally m e a n s a f e e l i n g o f

Fig, 5.6 : Diagrammatic rcpresentatian of common
closeness)a-helical segmentsof proteins can form matifs in p.rateins interacting with DNA.
helix - looo- helix m ot if and b i n d t o D N A . T h e
 OF CENEEXPRESSION
lln"acnei : REGULATION 67

---_---I
D:n c-,: -:- of t he pr ot ein ac t ually binds t o D N A
fA. t-6D

------I
----=I
-______I
--_-__-I
--€ important features of eukaryotic Sene --_____I
___---I
,r'i[N-=*s on along with the regulatoryaspectsare --------r
:rs:-:ed in the preceeding pages. Besides
-,-,-'iption, eukaryoticcells also employ variety Fig. 5.7 : Diagrammatic representation of
,i -i^ei mechanisms to regulategene expression. gene amplification (the genes are depicted in
- . - E : 1os t i mp o rta n to n e s a re l i s te d b e l o w , and colour shade and colout).
:r =tr describednext.
: Cen e a m p l i fi c a ti o n
now explainedon the basisof generearranSement
L Cene rearrangement or transpositionof genesor somatic recombination
3. P r oce s s i nogf R N A of DNA.
The structure of a typi cali mmunogl obul imoln e-
- 1.A lt e rn a tem R N A s p l i c i n g cule consistsof two light (L) and two heavy (H)
5. Transport of mRNAfrom nucleusto cytoplasm chai ns.E achone of thesechai ns(L or H ) contai ns
an N-terminalvariable(V) and C-terminalconstant
6. Degradation of mRNA. (C ) regi ons.The V regi onsof i mmunogl obul i ns are
responsiblefor the recognitionof antigens.The
Gene amplification phenomenon of gene rearrangementcan be
In this mechanism,the expression of a gene is understood from the mechanismof the synthesis of
rncreased l i ght chai nsof i mmunogl obul i (Fi
ns g.5.8).
severalfold. This is commonlyobserved
during the developmentalstagesof eukaryotic Each light chain can be synthesizedby three
organisms. For instance, in fruit fly (Drosophila), distinct DNA segments,namelythe variable(Vr),
the amplificationof genes coding for egg shell the joining (11)and the constant(CL).The mamma-
proteinsis observedduringthe courseof oogenesls. lian genomecontainsabout 500 V, segments, 6 Jt
T he am pli fi c a ti o no f th e g e n e (D N A) c an be se8ments and 20 C, segments. Duringthe courseof
observedunderelectronmicroscope(Fig.5.V. differentiation of B-lymphocytes, one Vr segment
(out of the 500) is broughtcloserto J1 and C,
The occurrenceof gene amplificationhas also
segments. This occurson the same chromosome.
been reported in humans. Methotrexateis an 100thV L,3tdJ, and 1Oth
Forthe sakeof i l l ustrati on,
ant ic anc e r d ru g w h i c h i n h i b i ts th e e nzyme
C,_ segmentsare rearrangedin Fig. 5.8. The
dihydrofolate reductase. The maliSnant cells
rearrangedDNA (with V1, J,_and C, fragments)is
developdrug resistance to long term administration
then transcribed to producea singlemRNA for the
of methotrexate by amplifyingthe genescodingfor
synthesis of a specificlight chain of the antibody.
dihydrofolatereductase.
B y i nnumerabl ecombi nati onsof V r, l , and C t
Gene rearrangment segments, the body'simmunesystemcan generate
mi l l i ons of anti gen speci fi c i mmunogl obul i n
The body possesses an enormouscapacityto mol ecul es.
synthesizea wide range of antibodies. lt is
estimatedthat the humanbody can produceabout The formationof heavy(H) chainsof immuno-
e apti gen gl obul i nsal sooccursby.rearrangement
10 billion ( 1 0 1 0a) n ti b o d i e isn re s p o n sto of 4 di sti nct
s t im ulat io n sT. h e m o l e c u l a rm e c h a n i s mof thi s genes-variable (Vn), diversity(D), joining (ls) ano
antibody diversity was not understood for long. lt is constant (Cn).
68 B IOTE CHNO LO CY

v (500) c (20)
,, I
-)) 4..#)) O1ginatDNA

r-t-r-l- * RearrangedDNA
Vr oo

Primarytranscript

mRNA
-|-|_

Protein
(lightchainof lg)

Fig. 5.8 : Diagrammaticrepresentationof gene rearrangementfor the synthesis of light chain of immunoglobulin.

Processing of RNA It appearsthat the ends of mR N A m olecules

The RNA synthesized in transcription undergoes determi ne the stabi l i ty of mR N A . A t ypical
m o d i fi c a ti o nres s u l ti n gi n a f uncti onalR N A . The eukaryoti cmR N A has 5' -non-codi ngsequences
(5' -N C S ),
a codi ng regi onand a 3' -N CS.All t he
c h a n g e si n c l u d ei n tro n -e x ospln i ci ng,pol yadenyl a-
tion etc. (Chapter 4). mRNAs are capped at the 5' end, and mostof them
have a polyadenylatesequenceat the 3' end
Alternate mRNA splicing (Fig.5.9).The 5' cap and poly (A) tail protectthe
mRNA againstthe attackby exonuclease. Further,
E u k a ry o ti cc e l l s a re c a p a bl eof carryi ngout
stem-loopstructuresin NCS regions,and AU rich
alternate mRNA processing to control gene
regionsin the 3' NCSalsoprovidestabilityto mRNA.
expression. DifferentmRNAscan be producedby
a l te rn a tes p l i c i n gw h i c h c o d e for di fferentprotei ns
(for more details,ReferChapter4).

Degradation of mRNA
The expression of genesis indirectlyinfluenced
by the stability of mRNA. Certain hormones
regulatethe synthesisand degradationof some
mRNAs.For instance,estradiolprolongsthe half-
life of vitellogeninmRNA from a few hours to
a b o u t2 0 0 h o u rs .
C e n e e x p r e s s i o no r g e n e r e g u l a t i o n i s u su a l l y
s t u d i e d a t t h e t r a n s c r i p t i o n a ll e v e l , i . e . p r o d u cti o n
of mRNA from the gene. The methods to elucidate
gene expressionare designedto provide information
on one or more of the followine
. Sequence of the gene
a Size of the transcript(mRNA)
a S t a r t i n ga n d f i n i s h i n g p o i n t s o f g e n e s to p r o d u ce
the transcript.

Stem loop a N u m b e r a n d p o s i t i o n o f i n t r o n s o n t he g e n e s.
AU structure a The activity of the promoter.
structure richregion
Some of the importantand general methods
Fig. 5.9 : A diagrammatic representation of a typical
employedto study the gene regulationare briefly
eukaryotic m BNA. (N0S-Non-coding seguences)
descri bed.
 - ' " ac r - e' i : R EC U L A T IO OF
N C E N EE XP RE S S ION 69

Sorrthern blot Exon Intron Exon lntron Exon

DNA
i:,uthern blot is a novel techniqueto detecta
*"r:'*n tragmentof DNA in the DNA preparation mRNA
of
a- ; " = anis mT. h i ste c h n i q u ei s p a rti c u l a rluysefulto
rs€r: ihe presenceof a foreign DNA in the
{l:-et callv modified organismsor to identify the
:r=€nce and copy number of genes in an
:r.-aeism'sgenome.The detailson this technique
i- : gi\ en els e w h e re (C h a p te7r ).

lforthern blot
\orthern blot specificallydetectsthe size and
Huenc e of th e mR N A . T h e to ta l m R N A i s DNA with exons
:r'i'acted from a cell or tissue suspension,ano mRNA
=carated by agarosegel electrophoresis and then
:etectedby hybridization(ReferChapter7).
Fig. 5,10 : A diagrammatic representation of
Huc leas e S l m a p p i n g mapping of introns by nuclease Sl digestion.
\uclease Sl is an enzymethat can specifically
degradesingle-stranded nucleicacids.NucleaseSl
r.apping is used to determine the number of f t t t 'rt
introns presentin a gene (Fig. 5.10\. LabeledDNA
The mature mRNA is hybridized with its (mRNA)
Testtranscript
c or r es pondi n gg e n e (i .e . g e n o mi c D N A). The
Annealing
portion of the intron on the gene which is not
transcribedis looped out. This looped-outintron (A)
can be specificallydigestedby nucleaseSl, which
degradesthe single-stranded DNA. The number
t t
and presenceof introns can be identified by
analysingthe fragmentedDNAs.
NucleaseSl
Nuclease protection assay
In nucleaseprotectionassay,the test transcript ir 1'
tmRNA)is hybridizedwith excessquantitiesof in
(B) '!, t
vltro synthesizedand radioactivelylabeled DNA
molecules(usuallyobtainedfrom cloned genes). NucleaseDrotected
fragment
T he anneale d h y b ri d s w h i c h a re l a b e l e d, are
subjected to digestion by nuclease Sl which A and B subiectedto
degrades single-strandednucleic acids. The electroporesisand
autoradiography
nuclease-treated and untreated hybridized
moleculesare separated by agargel electrophoresis
and identified(Fig.s.ll).
Nuclease protection assay is a variant of
nuclease5l mappingand providesinformationas
regardsthe presenceof introns, transcriptional
termini and the test transcriptproper.

Primer extension
Fig. 5.11 : A diagrammatic representation of nuclease
Primerextensionmethodis a reliabletechniqueto protection assay (A) Hybridized DNA not treated with
determinethe 5' end of the transcripts.For this nuClease Sl (B) Nuclease Sl trcated hybridized DNA.
purpose,
a synthetic5'-labeled
oligonucleotideprimer
70 B IOTE C HNO LO CY

Promoter TranscriptionTermination

I
stad site site
J 5,DNA
Cene tagging broadly involves the insertion of a
3 'm R N A
I recognizable DNA fragment within a gene so that
primer
Oligonucleotide t h e f u n c t i o n o f a g e n e i s d i s r u p t e d ,a n d t h e g e n e l s
I Reversetranscriptase identified by virtue of the inserted DNA fragment
I
J T-DNA (transferredDNA) is the part of tumor-
3 'm R N A i n d u c i n g p l a s m i d ( T i p l a s m i d )D N A f o u n d i n th e so i l
H bacterium Agrobacterium tumefaciens (For detaiis,
cDNA Refer Chapter 49) Transposons or transposable
e l e m e n t sa r e m o b i l e g e n e t i c e l e m e n t s( D N A p i e ce s)
that can move from one place to another in a DNA
Sequenceanalysisof cDNA
molecule (ReferChapter 3). Transposonsor T-DNA
Fig. 5,12 : A diagrammatic of primer
representation c a n b e u s e d i n g e n e t a g g i n g a n d g e n e a n a l ysi s.
extensiontechniqueto detectthe transcription The transposontagging of a gene is depicted in
starl site. F i g . 5 . 1 3 . Wh e n a t r a n s p o s o n i n a p l a sm i d i s
introduced into a cell, it gets incorporated into
DNA, and the gene gets disrupted' Consequently,
containing complementarybase sequenceto a small transposon insertion produces a mutant (A-). This
porlion of the testtranscriptis used Both are allowed
to hybridize,and the enzyme reversetranscriptasets
used to extend the primer till it reachesthe 5' end of
the mRNA (Fig. 5.12). This resultsin the synthesisof
complementary DNA (cDNA) representing the
distancebetween S'-end of the primer and s'-end of
the mRNA. The cDNA can be separated by
electroohoresisand detected.

Rapid amplification of cDNA ends

(RACEI
The 5' - and 3' - ends of c om p l e m e n t a r v D N A Transposon
( c DNA) c an be m apped by us e o f p o l y m e r a s ec h a i n
r eac t ion.This m et hod is eit her 5 '- R A C Eo r 3 '- R A C E
depending on t he end t o be m ap p e d . F o r d e t a i l so n t-
RACE. refer Chapter B rransposrllon
J
Reporter assays
Reporter genes are the genes that form protein
products which can be detected without destoying
. eluc idat e a S e n e e x p r e s s i o n ,r t s
t he t is s ues / c ellsTo
promoter is fused with a reporter gene, and then
(A-)
Mutant
int r oduc ed int o t he c ells .
I
The specific products (e.g. Iuciferase,B-galacto-
sidase, chloramphenicol acetyltransferase)of the {{-Gene a tagged
reporter genes can be identified. The activity of the ldentificationof gene
reporter gene reflects the activity of the promoter a taggedtransPoson
gene, and c ons equent lyt he ge n e e x p r e s s i o n .
Fig. 5.13 : Transposon tagging of a gene in
Reporter assaysare very useful for the study of gene analvsis.
gene expression in vivo in the tissues/cells.
 = ' r : RE C U L A T IO O
N F C E N EEX PR ES S ION 71

- -'.:- ca n b e ident if ied by it s phenot y pe and a (A)

-:-: :-arr'. Fur t her ,t he m ut ant c an be s c r een e o VeciorDNA t- O"nu for phage
" --= cresence of transposon. By identifying the
,Tetion oi insertion of transposon, the location of
-he specific gene can be identified.

Fusedframeof DNA

--e
operation of the genome can be evaluated +
I
^ e stu dy of pr ot eom e. Thus , by s t udy ing th e Transformation
of
E. coli
--: io ns of p rot eins , it is pos s ible t o under s t a n d
- - .,, t1e genome operatesand how a dysfunctional
::-cme activitv c en r es gll in dis eas es t at ess uc h a s
t_
tsxpfe$srcn
:: l C e f
I

P.oteomics broadly involves the methodology

D
Protein
-:' ch ara cte rizi ngt he pr ot ein c ont ent of t he c e l l .
--is can be do ne by pr ot ein elec t r ophor es is m I Proten displayed
, ass I
,::ctrometry etc. J
lde ntificatio nof pr ot ein- pr ot ein int er ac t ion is a /n
p\ )l
-:cent approach to study proteome. fhe protein
interaction maps can be constructed to understand \r'
F) i cn l a r r nhana
lre rela tion be t ween t he pr ot eom e and c ellu l a r
biochemistry. Phage display and yeast two-hybrid
svstem are commonly used to study protein-
protein interactions

PHAGE DISPL AY
Phagedisplaylibrary
Phage display is a novel technique to evaluate
Eenome activity with particular reference to identify
proteins that interact with one another. lt basically
involves insertion of a foreign DNA into phage
genome, and its expressionas fusion product with a
phage coat protein (Fig. 5.1 A) This is followed by
screening of test protein by phage display library
lFig. 5.148).The technique is briefly describedbelow.
A spe cia l ty pe of c loning v ec t or s uc h as a
ba cte riop ha geor f ilam ent ous bac t er iophage ( e . g .
M13 ) are u se d f or phage dis play . A f r agm ent o f
DNA coding for the test protein is inserted into the
vector DNA (adjacentto phage coat protein gene).
After transformation of E. coli, this recombinant
gene (fused frame of DNA) results in the synthesis
of hybrid protein. The new protein is made up of
the test protein fused with the phage coat protein.
The phage particles produced in the transformed
E. coli display the test protein in their coats. Fig. 5.14 : Elucidation of protein-protein interaction by
phage display (A) Production of fusion protein
The test protein interaction can be identified by
displayed on phage (B) Screening of test protein by
u sin g a ph ag e d is play libr ar y For t his pur pos e,t h e
phage display library.
test pro tein is im m obiliz ed wit hin a well of a
72 B IOTECHNO LO CY

*- Transcriotional
activationdomain
Aarnal Bindingpartner
DNAbinding Recombinantgenes
domain protein encodingbait and prey PREY
BAIT introducedinto veast cell

interactionby yeast two-hybridsystem(RNAP'RNApolymeruse)

Fig. 5.15 : Elucidationof protein-protein

microtitertray,and the phagedisplaylibraryadded. (i.e.the proteinsinvolvedin promotingtranscription

After severalwashes,the phagesthat are retained of a gene) contain two distinctdomains- DNA
in the well are those displayinga protein that bi ndi ng domai n and transcri pt ionalact ivat ion
interactswith the test protein. domai n.W hen thesetw o domainsar e phyically
separated, the protein losesits activity.However,
Phage-displaying peptidescan be isolated,based
the same protein can be reactivatedwhen the
properti es,
o n th e i ra n ti b o d y -b i n di ng by empl oyi ng
domainsare broughttogether.Theseproteinscan
affinity chomatography. Severalroundsof affinity
bind to DNA and activatetranscription.
chromatographyand phage propagationcan be
usedto enrich phageswith desiredproteins. The target protein is fused to a DNA-binding
domain to form a bait. When this targetprotein
Phagernid display binds to another specificallydesigned protein
namelytheprey in the nucleus,they interact,which
Ph a g e mi d i n p l a c eo f pl asmi d,can al sobe used of the reporter
in turn switcheson the expression
for the displayof proteins.In fact, specialtypesof gene(Fig.5.f5). The reporter
Senescan be detected
phagemiddisplayvectorshavebeendevelopedfor
by growingthe yeaston a selectivemedium.
th i s p u rp o s e .
the bait and preyfusion
It is possibleto generate
P h a g ea n d p h a g e mi ddi spl aycan be successful l y proteinsby standardrecombinantDNA techniques.
used for selectingand engineeringpolypeptides
A singlebaid proteinis frequentlyusedto fish out
with novel functions.
interacting partners among the collection of
prey proteins.A largenumberof prey proteinscan
YEAST TWO.HYBRID SYSTEM
be produced by ligating DNA encoding the
When two proteinsinteractwith each other, activation domain of a transcriptionalactivator
their corresponding genesare known as interacting to a mistureof DNA-fragments from a cDNA library.
genes.The yeasttwo-hybridsystemusesa reporter
geneto detectthe physicalinteractionof a pair of Yeast three'hyhrid system
proteinsinsidea yeastnucleus.
The interactionsbetween protein and RNA
The two-hybrid method is based on the molecules can be investigatedby usinga technique
observation that mostof the transcriptional proteins known as yeastthree-hybridsystem.
 ilI I
Introducti onto C eneti c
E ngi neeri ng 75
B asi c Techni ques i n Geneti c
E ngi neeri ng 92
P ol ymeraseC hai n R eacti on
(D N A A mpl i fi cati on) 112
9 C ene Li brari es 120
10 S i te-di rectedMutagenesi sand
P rotei nE ngi neeri ng -129
-t'l Mani pul ati on of Gene E xpressi on
i n H ost C el l s " 137
12 HumanGenomeProject 148

,i t,
I,j

''

73 73
f enet ic e n g i n e e ri n gp ri ma ri l y i n v o l v e s the p S C 1 0 1 D N A . F r o g D N A f r a g m e n t s a n d p l a s m i d
\) manipulation of genetic material (DNA) to DNA fragmentswere mixed, and pairing occurred
ac hiev et he de s i re dg o a l i n a p re -d e te rmi n eway.d between the complementary base pairs. By the
Some ofher terms are also in common use to addition of the enzyme DNA ligase, a recombined
describegeneticengineering. p l a s m i d D N A w a s d e v e l o p e d .T h e s e n e w p l a s m i d s ,
rvhen introduced into E.coli, and grown on a
. Cene manipulation
nutrient medium resulted in the production of an
. Recombinant DNA (rDNA) technology extra protein (i.e. the frog protein). Thus, the genes
. Gene cloning (molecular cloning) of a frog could be successfullytransplanted, and
. Genetic modifications expressedin E.coli. This made the real beginning of
modern rDNA technology and laid foundations for
. New genetics.
the present day molecular biotechnology.

B RI E F HI S TOR Y OF R EC O MB IN A N T Some biotechnologists who admire Boyer-

DNA TECHNOLOGY Cohen experiments divide the subject into two
chronological categories.
The presentday DNA technologyhasits rootsin .l
the experimentsperformedby Boyer and Cohen in . BBC-biotechnology Before Boyer and Cohen.
1973. ln their exp-eriments, they successfully 2. ABC-biotechnology A[ter Boyer and Cohen.
r ec om bined t w o p l a s m i d s(p S C1 0 1 a n d p S C 102)
More information on the historicaldevelopments
and c lonedt he n e w p l a s mi di n E .c o l i P . l a s m i dpS C
o f g e n e t i c e n g i n e e r i n ga n d b i o t e c h n o l o g y i s g i v e n
101 pos s es s e sa g e n e re s i s ta n tto a n ti b i oti c
under the scope of biotechnology (Chapter1).
wh i l e p l a s m i dp S C1 0 2c o n ta i n sa g ene
t et r ac y c line
r es is t antt o a n o th e r a n ti b i o ti c k a n a m y c i n .The
An outline of recombinant DNA
newly dev el o p e d re c o mb i n e d p l a s m i d when
technology
incorooratedinto the bacteriaexhibitedresistance
to both the antibiotics-tetracvcline and kanamvcin. T h e r e a r e m a n y d i v e r s ea n d c o m p l e x t e c h n i q u e s
involved in gene manipulation. However, the basic
The second set of experimentsof Boyer and
principles of recombinant DNA technology are
Cohen were more organized.A gene encodinga
reasonably simple, and broadly involve the
protein(requiredto form rRNA)was isolatedfrom
following stages(Fig. 6.1).
the cells of African clawed ftog Xenophslaevis,by
use of a restrictionendonuclease enzyme(ECoRIl. 1 . Ceneration of DNA fragments and selection
The same enzymewas usedto cut open plasmid of the desired piece of DNA (e.9. a human gene).

75
 76 B IOTECHNO LO G Y

R ecombi nantD N A technol ogywit h special

Qn n
bL/()
referenceto the following aspectsis describedin
this chapter.

\-/ 1. Mol ecul artool sof geneti cengi n eer ing.

\
) PlasmidDNA 2. Host cells-thefactoriesof cloning.
DonorDNA I
r I Bestriction cl oni ngvehi cl es.
3. V ectors-the
I lendonuclease
I Restriction J 4. Methodsof genetransfer.
endonusease
I 5. C ene cl oni ngstrategi es.
+l gui del i nes.
6. C eneti cengi neeri ng
o".,offi',"."
I
-
\-/ 7. fhe futureof geneticengineering.
I Cut plasmidDNA
L -Tl
Y
I Lrgase

tf An engineeris a personwho designs,constructs

\-/ (e.g. bri dges,canal s,rai l w ays)and manipulat es
DNA
Recombinant accordingto a set plan. The term geneticengineer
-\
may be appropri atefor an i ndi vi d ualwho is
I Introduce
into
hostcells involved in genetic manipulations.The genetic
J
engineer'stoolkit or molecular tools namely the

o@ enzymes most commonly used in recombinant

DNA experiments are brieflydescribed.

t Multiplication
I Selectionof clones
Ftg. 6.1 : The basic principle of recombinant DNA
technology.
2. Insertion of the selected DNA into a cloning
vector (e.g. a plasmid) to create a recomhinant
DNA or chimeric DNA (Chimera is a monster in
Creek mythology that has a lion's head, a goat's
body and a serpent'stail. This may be comparable
to Narasimha in Indian mythology).
3. Introduction of the recombinant vectors inro
host cells (e.9. bacteria).
4. Multiplication and selection of
c ont aining t he r ec om binant m o l e c u l e s .
5. Expressionof the gene to produce the desired
oroduct.
R E S TR IC TION E N D ON U C LE A S ES_
D N A C U TTIN G E N ZY ME S
Restrictionendonucleases are one of the most
importantgroupsof enzymesfor the manipulation
of DNA. Theseare the bacterialenzymesthat can
cut/splitDNA (from any source)at specificsites.
They were first discoveredin E.colirestrictingthe
replicationof bacteriophages, by cuttingthe viral
DNA (The host E.coli DNA is protected from
cleavageby additionof methyl groups).Thus,the
enzymesthat restrictthe viral replicationare known
as restrictionenzymesor restrictionendonucleases.
Hundreds of restrictionendonucleaseshave
been isolatedfrom bacteria,and some of them are
commerciallyavailable.The progressand growth
of bi otechnol ogyi s uni magi nabl ewit hout t he
availabilityof restrictionenzymes.
N omencl ature
clones Restrictionendonucleasesare named by a
standardprocedure, with particularreference
to the
bacteriafrom which they are isolated.The first
letter(in italics)of the enzymesindicatesthe genus
 T O C EN ET ICEN CIN E E R IN C
J - ac t er 6 : I NT R OD U C T IO N 77

-,:-e. followed by the first two letters(also in

:: cs of the species,then comesthe strainof the Tlru 6.1 Characterlsticsof dlfferent types
and f in a l l ya R o ma nn u m e ra li n d i c a ti ng
:, ' E anis m of restrlction endonucleases
-r order of discovery.A couple of examplesare
Type Salient features
: . en below.
A singleenzyme with3 subunits
for
EcoRl is from Escherichia(D coli (co), strain recognition,
cleavage andmethylation.
i, I3 (R)and firstendonuclease (0 to be discovered. It cancleaveupto 1000bpfrom
Hindlll is from Haemophilus(f0 influenzae(in), recognition
site.
;r'ain Rd (d) and the third endonucleases (lll) to be
ll Twoditlerentenzymeseither to
: scovered.
cleave
or modifytherecognilion
sequence.Cleavagesiteis lhesame
Types of endonucleases orcloselo recognition
site.
At least 4 d ifferent types of restriction ill A singleenzymewith2 subunits
for
endonucleases are known-type1 (e.gEcokl2), type recognition
andcleavage.
Cleavage
ll e.g. EcoRf, type lll (e.9.EcoPI)and type lls.Their siteis 24-26bpfromrecognition
site.
characteristicfeatures are given in Table 6.1. lls Twodifierent
enzymes,
cleavage
site
{mong these,type ll restrictionendonucleases are
is upto 20bpfromrecognition
site.
most commonlyused in genecloning.

Taslr 5.2 Some restrictlon enzynes with sources, recognltlon sequencesand the products fonned

Enzyme (source) Recognition sequence Products

EcoRl . 3'
s' ..ch A-T-T-C,. A-A-T-T-C
(Escherichia
colll . 5',
3' C-T-T-A-Aip. G
G
C-T-T-A-A

BamHl 5',. Gh-A-T-C-C. G-A-T-C-C

(Bacill
us anyIolique faciensl 3',.....C-C-T-A-GilG n
G
c o-T-A-G
Haelll .G-GTC-C
s',.... ....3', xc4
(Haemophilus aegyptius) 3', C-CIG-G.s', tJ \l
*^^
' \f -r.,

Hlndlll 5'. .AffA-G-GT_T.. 3' A-G GT-T

(Haenophilusinfluenzae) 3'... .T-T-rc-AJ(A...5' A
.A
..T-T-C-G-A

Notl 5',..c-cffc-c-c-c-c-c
3' u-u-u-u-u-u .
(Nocardia
otitidis) 3'. ..c-G-c-c-G-Gtc-G5' ii
v-\:...
..t -u
.c-G-c-GG-G,
(l{ole: Sabsorsindicate * Theproducts
thesitesol cleavage. arewithbluntendswhilefot thercst,theWoducts
arewithstbkyendsl.
 B IOTE CHNO LO CY
7a

Recognition sequences

Recognition sequence is the site where the

DNA is cut by a restriction endonuclease'
Res t r ic t ion endonuc leas es c a n specifically
recognize DNA with a particular sequence of 4-B
nuc leot idesand c leav e Eac h r e c o g n i t i o ns e q u e n c e
has two fold rotational symmetry i'e the same
nucleotide sequence occurs on both strands of
DNA which run in opposite direction (Table 6'2)'
Suc h s equenc es ar e r ef er r ed t o a s p a l i n d r o m e s '
s inc e t hey r ead s im ilar in bot h d i r e c t i o n s ( f o r w a r d s
and backwards).

Gleavage Patterns
Majority of restriction endonucleases
r]
( par t ic ular lyt y pe ll) c ut DNA a t d e f i n e d s i t e sw i t h i n ol-l I
I
recognition sequence. A selected list of enzymes,
O=P-O-
recognition sequences,and their products formed I I
I
I
is given in Table 6.2.
I
I
The cut DNA fragments by restriction I

endonucleases may have mostly sticky ends

(cohesiveends) or blunt ends,as given in Table 6 2'
DNA fragments with sticky ends are particularly
us ef ul f or r ec om binant DNA e x p e r i m e n t s 'T h i s i s o
because the single-strandedsticky DNA ends can I
eas ilv pair wit h any ot her D N A f r a g m e n t h a v i n g I
comPlementarYstickY ends' I
o
I
DNA LI G ASES _ DNA J O I N I N G ENZYMES I

The c ut DNA f r agm ent s a r e c o v a l e n t l y j o i n e d

together by DNA ligases. These enzymes were
or iginally is olat edf r om v ir us e s .T h e y a l s o o c c u r i n
E.coli and eukaryotic cells' DNA ligases actively
participated in cellular DNA repair process'
The ac t ion of DNA ligas esi s a b s o l u t e l yr e q u i r e d
t o per m anent ly hold DNA pi e c e s T h i s i s s o s i n c e
the hydrogen bonds formed between the
complementary bases (of DNA strands) are not
strong enough to hold the strands together. DNA
ligas ejoins ( s eals )t he DNA f r a g m e n t sb y f o r m i n g a
phosphodiesterbond between the phosphateSroup
of S'-carbon of one deoxyribose with the hydroxyl
group 3'-carbon of another deoxyribose (Fig. 6.2).
PhageT4 DNA ligase requiresATP as a cofactor
while E.coli DNA ligase is dependent on NAD+' ln
each case, the cofactor (ATP or NAD+) is split to
form an enzyme-AMP complex that brings about
the formation of phosphodiesterbond. The action
of DNA ligase is the ultimate step in the formation
of a r ec om binant DNA m ole c u l e .
Fig. 6.2 : Action of DNA ligase in the formation of
PhosPhodiesterbond (B-base)'
Homopolymer tailing
The compl ementary D N A strandscan be joined
together by annealing. This principle is utilized in
homopol ymertai l i ng.The techni quei nvolvest he
addi ti on of ol i go (dA ) to 3' -end s of som e
D N A mol ecul esand the addi ti on of oligo ( dT)
to 3' -endsof other mol ecul es'The hom opolym er
extensi ons (by addi ng 10-40 residues)can
be synthesi zed by usi ng termi nal deoxy-
nucl eoti dyl transferase(of cal f t hym us) '
H omopol ymertai l i ng, achi evedby a nnealingis
illustratedin Fig. 6.3.
Linkers and adaPtors
Linkersand adaptors arechemically synthesized,
short, double'stranded DNA molecules. Linkers
 T O C E N E T ICE N C IN E E R IN G
: ' r aot er6 : IN T R OD U C T IO N 79

:o:sessrestriction enzymecleavagesites.They can 5' _ 3'

ligat edt o b l u n te n d so f a n y D N A m o l e c u l eand J-c
-:-i rvith specificrestrictionenzymesto produce
)\A fragmentswith stickyends (Fig.6.4. Terminal
oeoxy-
.\daptorscontain preformedsticky or cohesive nucleotidyl-
transferase
.nds. They are useful to be ligated to DNA
:'agmentswith blunt ends. The DNA fragments A)nA 3'
:eld to linkersor adaptorsare finally ligatedto 3' A(A).A- 5'
rector DNA molecules(Fig 6.a).

ALKALINE PHOSPHATASE
Mix and anneal
Alkalinephosphatase is an enzymeinvolvedin

.4,-_>>
the removalof phosphategroups.This enzyme is
usefulto preventthe unwantedligation of DNA
molecules which is a frequentproblemencountered
in c loningex p e ri me n ts .
When the linearvectorplasmidDNA is treated
rvith alkaline phosphatase, the 5'-terminal
phosphateis removed(Fig 6.fl. This preventsboth
and plasmidDNA dimerformation.
recircularization
It is now possibleto insertthe foreignDNA through Fig. 6.3 : Homopolymer tailing.
the participation
of DNA ligase.
(A) (B) Til
DNA MODIFYING ENZYMES r-l
Someauthorspreferto usethe broadterm DNA
ForeignDNA t]
modifyingenzymesto all the enzymesinvolvedin
recombinant DNA technology.These enzymes
'epresentthe cutting andjoining functionsin DNA
ranipulation. They are broadly categorizedas
rucleases,polymerasesand enzymes modifying
ends of DNA molecules,and briefly described
below,and illustratedin Fig. 6.6. f-T------r-t
I
Nucleases lecoar
Nucleasesare the enzvmes that break the
phosphodiesterbonds (that hold nucleotides
rc
ForeignDNA with
together)of DNA. Endonucleases
act on the internal cohesiveends
phosphodiesterbondswhile exonucleases degrade
DNA from the terminalends(Fig6.64). Restriction
endonucleases,described already, are good
examplesof endonucleases.Someother examples
of endo- and exonucleases
are listed.
Endonucleases
. NucleaseS, specificallyacts on single-stranded
VectorDNA Recombinant
DNA
DNA or RN A m o l e c u l e s .
Fig. 6.4 : Use of linkers (A) and adaptors (B) in the
| (DNasel) cuts eithersingle
. Deoxyribonuclease
formation of recombinant DNA (Note that the linkers
DNA moleculesat random
or double-stranded have blunt ends while adaptors have sticky ends).
sites.
 B IOTECHNO LO CY
80

(A) Nucleases

t*"h DNA

lnternalcuts Removalof nucleotides

Plasmidcut oPen Recircularization from the ends
(linearDNA) of plasmid

(B) DNA PolYmerases

Alkaline DNAtemPlate RNA temPlate

2P\ phosphatase
I orun-d*p"no"nt I mln'a"p"ro"ot
DNAfolVmerase DNAOotYmerase
I J
DN{A
ligase
DNA copies DNA copies
---)(-+ No reaction
(A)'
Fig. 6.6 : An outline of the activities of nucleases
and DNA PolYmerases (B)'

HO - P
P - HO Exonucleases
ForeignDNA
fragment r E xonucl easel l l cuts D N A a nd Sener at es
mol ecul esw i th protrudi ng5' -ends'
. N ucl easeB al 31 i s a fastacti ng3'- exonuclease'
Its acti on i s usual l ycoupl edw it h slow act ing
endonucl eases.
A diagrammaticrepresentation of the action of
endo-and exonucleasesis given in Fig' 6'7'

Besidesthe DNA cutting enzymes,there are

prevenl which are referredto as
Fig. 6.5 : Action of alkaline phosphatase to RNA specificnucleases,
lhe recircularization (religation) of vector plasmid' (RNases).
rihonucleases

51 .
Nuclease
Sinqle-stranded
DNAoTRNA

DNaseI

DNA
Double-stranded
ffl u
Exonuclease .,

DNA
Double-stranded
NucleaseBal31 .

DNA
Double-stranded

Fig.6.7:Modeofactionofselectedendo_andexonucleases(DNase|_Deoxyribonucleasel)'
 o : INTRODUCTIONTO CENETICENCINEERINC
il'h"ir*-=r 8t

Iru 6.3 The most conilonly used enlymes In recombinant DNA technology/genetlc englneerlng

Enzrme Use/reaction
,rlair€ phosphatase Removes phosphate groupsfromS'-ends DNA(orRNA).
of double/single-stranded
h l' 'tuclease Fortheprogressive
shorteningof DNA.
l t,a Ease JoinsDNAmolecules bylorming phosphodiester linkages
between
DNAsegments.
I
lirr oolymerase DNAcomplementary
Synthesizes to a DNAtemplate.
:hes€ | Produces nicksin DNA.
single-stranded
lr:rrdease lll Removes nucleotidesfrom3'-end of DNA.
;r :rSnuclease Removes nucleotidesfromS'-end o1DNA.
br-ucleotidekinase phosphate
Transfers fromATPto 5'-0Hendsof DNAor RNA.
:esrction enzymes Cutdouble-strandedDNAwitha specilic recognition
site.
=euersetranscriptase DNAfromRNA.
Synthesizes
Ei\laseA Cleavesanddigests RNA(andnotDNA).
=ltase H Cleavesanddigests theRNAstrand of RNA-DNA heteroduplex.
-r DNApolymerase Usedin polymerasechainreaction
S ruclease Degrades DNAandRNA.
single-stranded
transferase
-erminal Addsnucleotides
to the3'-ends of DNAor RNA.Useful in homopolymer
tailing.

Polymerases . Terminal transferase (also called termina,

deoxynucleotidyltransferase)repeatedlyadds
Thegroupof enzymesthat catalysethe synthesis
nucleotides
to any available3'-terminalends,the
:' rucleic acid moleculesare collectivelyreferred
most suitablebeing the protruding3'-ends.This
:c as polymerases.lt is customaryto usethe name
enzyme is particularly useful to add
: j the nucleic acid template on which the
homopolymertails prior to the constructionof
:or\merase acts (Fig. 6.6A. The three important
recombi nantD N A mol ecul es.
:oirmerasesare given below.
The mostcommonlyusedenzymesin recombinant
. DNA-dependent DNA polymerasethat copies '
DNA technology/genetic
engineering
are listedin
D\A from DNA.
Table6.3.
. RNA-dependent DNA polymerase (reverse
t'anscriptase) DNA from RNA.
that synthesizes
. DNA-dependent RNA polymerase that produces
R)lA from DNA.
Fog more details on the functions of these The hostsare the living systemsor cellsin which
rz\ mes.referChaoter4. the carrierof recombinantDNA moleculeor veclor
can be propagated.There are differenttypes of host
Enzymes modifying the ends of DNA cells-prokaryotic (bacteria)and eukaryotic(fungi,
animalsand plants).Someexamplesof host cells
There are certain enzvmes that act on the usedin geneticengineering given
are in Table6.4.
:='rrinalendsof DNA and modifythesemolecules.
-"e imoortantones are listed. Host cells,besideseffectivelyincorporating the
vector's genetic material, must be conveniently
. Alkaline phosphatasethat removesthe terminal cultivatedin the laboratory
to collectthe products.
chosphategroup (seeFig.6.5). ln general, microorganisms are preferred as host
. Polynucleotidekinaseinvolvedin the additionof cells, since they multiply faster comparedto cells
chosphategroups. of hi gherorgani sm(pl antsor ani mal s).

3ctechnology[6]
a2 B IOTECHNO LO G Y

PROKARYOTIC HOSTS
Tlcrr 6.4 Some examples of host cells used
Escherichia coli in genetic engineering
The bacterium,Escherichiacoli was the first
Group Examples
organismusedin the DNA technologyexperiments
and continuesto be the host of choice by many Prokaryotic
workers.Undoubtedly,E.coli, the simplestCram Bacteria Escherichia
coli
negativebacterium(a commonbacteriumof human Bacillus
subtilis
),a spl ayeda key rol e i n the
a n d a n i ma li n te s ti n e h
Streptomyces
sp
developmentof presentday biotechnology. Under
suitableenvironment,E.colican double in number Eurkaryotic
e v e ry 2 0 mi n u te s T . h u s ,a s the bacteri amul ti pl y, Fungi Saccharomycescerevisiae
th e i r p l a s mi d s(a l o n g w i th forei gn D N A ) al so Aspergillus
nidulans
multiplyto producemillionsof copies,referredto Animals Insect
cells
as colonv or in short clone. The term clone is Oocytes
broadlyusedto a massof cells,organisms or genes
Mammalian cells
th a ta re p ro d u c e db y mu l tipl i cati on
of a si ngl ecel l ,
or 8ene.
Wholeorganisms
o rg a n rs m
Plants Protoplasts
l i m i ta ti o n s o f f. c o l i : There are certai n Intact
cells
i s a host.Thesei ncl ude-
l i m i ta ti o n isn u s i n gE .c o l a Wholeplants
causationof diarrheaby some strains,formation
of endotoxinsthat are toxic, and a low export
ability of proteinsfrom the cell. Another mayor cannotbe synthesized by bacteriacan be produced
drawback is that E.coli (or even other prokaryotic by mammal i an cel l s e.g. ti ssue plasm inogen
organisms) cannot perform post-translational acti vator.Thi s i s mai nl y becauseth e m am m alian
modifications. cells possessthe machinery to modify the protein
to its final form (post-translational modifications).
Bacillus subtilis
It may be notedherethatthe genemanipulation
Bacillussubtilisis a rod shapednon-pathogenic experi ments i n hi gherani mal sand pl a nt sar eusually
bacterium.lt hasbeenusedas a hostin industryfor carried out to alter the genetic make up of the
the productionof enzymes,antibiotics,insecticides organismto createtransgenic animals(Chapter+1;
etc. Some workers consider B.subtilis as an and transgenicplants(Chapter49), ratherthan to
alternative to E.coli. isolategenesfor producingspecificproteins.

EUKARYOTIC HOSTS
Eukaryoticorganismsare preferredto produce
human proteinssince these hosts with complex
structure(withdistinctorganelles) are moresuitable
to synthesize complex proteins. The most
commonly used eukaryoticorganismis the yeasf,
Saccharomycescerevisiae.lt is a non-pathogenic
o rg a n i s mro u ti n e l yu s e d in brew i ng and baki ng
industry.Certainfungi havealsobeenusedin gene
c l o n i n ge x p e ri m e n ts .
Mammalian cells
Despitethe practicaldifficultiesto work with
and high cost factor, mammaliancells (such as
mouse cells) are also employed as hosts. The
advantageis that certaincomplex proteinswhich
Vectorsare the DNA molecules,which can carry
a foreign DNA fragment to be cloned.They are self-
replicatingin an appropriatehost cell. The most
important vectors are plasmids,bacteriophages,
cosmidsand phasmids.
Characteristics of an ideal vectol
A n i dealvectorshoul dbe smal li n size,wit h a
si ngl e restri cti onendonucl ease si te , an or igin of
replicationand 1-2 genetic markers(to identify
reci pi entcel l scarryi ngvectors).
N atu r allyoccur r ing
plasmidsrarelypossess all thesecharacteristics.
a' r aot er6 : I N T R OD U C T IO N
T O C EN ET ICEN C IN E E R IN C 83

P LA S M I DS
Hind lll
P las m ids a re e x tra c h ro mo s o m a l d , o u bl e-
circular,self-replicating DNA molecules.
'ri.anded,
\ lm os t all t he b a c te ri ah a v ep l a s m i d sc o n ta i n i nga
o\ \ ' c opy nu m b e r (1 -4 p e r c e l l ) o r a hi gh
c opy num ber (1 0 -1 0 0 p e r c e l l ). T h e s i z e of
: . e plas m idsv a ri e sfro m 1 to 5 0 0 k b . U s u al l y, PstI
ciasmidscontributeto about 0.5 to 5.0% of the
:otal DNA of bacteria (Note : A few bacteria
contain linear plasmids e.g. Streptomycessp,
Borella burgdorferi).

Types of plasrnids
T her e ar e m a n y w a y s o f g ro u p i n gp l a s mi ds.
They are categorizedas conjugativeif they carry a
;et of transfer genes (fra genes) that facilitates
bacterialconjugation,and non-conjugative,if they Fig. 6.8 : Genetic map of plasmid cloning
do not possess such genes. vector pBR322

Another classificationis based on the copy

number.Stringentplasmidsare presentin a limited tetracycline(Telr)that serve as markersfor the
number(1-2 per cell)while relaxedplasmidsoccur i denti fi cati onof cl ones carryi ng pl asmi ds.The
in lar genum be ri n e a c hc e l l . plasmidhas uniquerecognitionsitesfor the action
F-plasmids possess genesfor their own transfer of restrictionendonucleases suchas EcoRl,Hindlll,
from one cell to another,while R-plasmidscarry BamHl, Sa// and Pstll (Fig. 6.8.
genesresistance to antibiotics.
Other plasmid cloning vectors
In general,the conjugativeplasmidsare large,
show stringentcontrolof DNA replication,and are The otherplasmidsemployedas cloningvectors
pr es entin low n u m b e rsOn . th e o th e r h a n d ,n on- i ncl ude pU C 19 (2,686 bp, w i th ampi ci l l i n
c onjugat iv epl a s m i d sa re s m a l l , s h o w re l a xed resistancegene), and derivativesof pBR322-
controlof DNA replication,and are presentin high pB R 325,pB R 328and pB R 329.
num oer s .
BACTERIOPHAGES
Nomenclature of plasmids Bacteriophages or simply phagesare the viruses
thaLreplicatewithin the bacteria.ln caseof certain
It is a commonpracticeto designateplasmidby
phages, their DNA gets incorporatedinto the
a lower case p, followed by the first letter(s)of
bacterial chromosome and remains there
researcher(s)namesandthe numericalnumbergiven
permanently.Phage vectors can accept short
by the workers. Thus, pBR322 is a plasmid
fragmentsof foreignDNA into their genomes.The
discovered by Eolivar and frodriguez who
advantagewith phages is that they can take up
designated it as 322. Some plasmids are
Iarger DNA segmentsthan plasmids.Hence phage
givennamesof the placeswheretheyarediscovered
vectorsare preferredfor working with genomesof
e g. pUC is plasmidfrom Universityof California.
humancel l s.
pBR322-the most common
Bacteriophage )"
plasmid vector
Bacteriophage lambda (or simply phage L), a
pBR322of E.coliis the most popular and widely
virus of E.coli, has been most thoroughly studied
usedplasmidvector,and is appropriately regarded
as
and develooedas a vector.ln order to understand
:he parentor grand parentof severalother vectors. how bacteriophagefunctions
as a vector, it
cBR322has a DNA sequenceof 4,361 bp. lt is desirableto know its structureand life cycle
e r a mp i c i l l i n(Amp r)and (Fig.6.9). Phagel, consistsof a head and a tail
: : - $ genesr e s i s ta n cfo
 a4 B IO TECHNO LO CY

( bot h being pr ot eins )and i t s s h a p e i s c o m p a r a b l et o (about 25%) has to be deleted from the vector to
a m iniat ur e hy poder m ic sy r i n g e .T h e D N A , l o c a t e d make space for the foreign DNA (about l Bkb).
in t he head, is a linear m o l e c u l e o f a b o u t 5 0 k b . A t
Replacementvectors : These vectors have a pair
each end of the DNA, there are single-stranded
of restrictionsitesto remove the non-essentialDNA
ex t ens ions of I 2 bas e le n g t h e a c h , w h i c h h a v e
(stuffer DNA) that will be replaced by a foreign
c ohes iv e ( c os ) ends .
DNA. Reolacement vectors can accommodale
On attachmentwith tail to E.coli, phage L injects up to 24kb, and propagate them.
it s DNA int o t he c ell I ns i d e E . c o l i ,t h e p h a g e l i n e a r
Many phage vector derivations (insertion/
DNA cyclizes and gets ligated through cos ends to
replacement) have been produced by researchers
f or m a c ir c ular DNA. T h e p h a g e D N A h a s t w o
f o r u s e i n r e c o m b i n a n t D N A t e c h n o l o g y.
fates-lytic cycle and lysogenic cycle. I
The main advantage of using phage vectors is
Ly t ic c y c le : The c ir c u l a r D N A r e p l i c a t e s a n d
that the foreign DNA can be packed into the phage
it als o dir ec t s t he s y nth e s i s o f m a n y p r o t e i n s
(in vitro packaging), the latter in turn can be
nec es s ar yf or t he head, t a i l e t c , o f t h e p h a g e . T h e
injected into the host cell very effectively (Note :
c ir c ular DNA is t hen c le a v e d ( t o f o r m c o s e n d s )
N o t r a n s f o r m a t i o ni s r e q u i r e d ) .
and pac k ed int o t he he a d o f t h e p h a g e . A b o u t
.l
00 phage par t ic les ar e p r o d u c e d w i t h i n 2 0
Phage Mr" vectors
m inut es af t er t he ent r y o f p h a g e i n t o E . c o l i . T h e
hos t c ell is t hen s ubjec t e d t o l y s i s a n d t h e p h a g e s P h a g e M , , ( b a c t e r i o p h a g e M 1 3 ) i s a si n g l e -
ar e r eleas ed. Eac h pr og e n y p h a g e p a r t i c l e c a n stranded DNA phage of E.coli. Inside the host cell,
inf ec t a bac t er ial c ell. a n d o r o d u c e s e v e r a l M,, synthesizesthe complementary strand to form
hundr eds of phages . l t i s e s t i m a t e d t h a t b y a double-strandedDNA (replicativeform DNA; RF
r epeat ing t he I y t ic c y c l e f o u r t i m e s , a s i n g l e DNA). For use as a vector, RF DNA is isolated and
phage c an c aus e t he d e a t h o f m o r e t h a n o n e a foreign DNA can be inserted on it. This is then
billion bacterial cells. lf a foreign DNA is spliced r e t u r n e d t o t h e h o s t c e l l a s a p la sm i d . Si n g l e -
into phage DNA, without causing harm to phage stranded DNAs are recovered from the phage
genes, the phage will reproduce (replicate the p a r t i c l e s .P h a g e M , ', i s u s e f u l f o r s e q u e n ci n gD N A
foreign DNA) when it infects bacterial cell. This through Sanger'smethod (Refer Chapter 7).
has been ex ploit ed in p h a g e v e c t o r e m p l o y e d
c loning t ec hniques . cosMtDs
Lysogenic cycle : In this case, the phage DNA Cosmids are the vectors possessingthe
I ( ins t ead of independen t l y r e p l i c a t i n g ) b e c o m e s of both plasmidand bacteriophage 1".
I characteristics
integrated into the E.coli chromosome and Cosmidscan be constructedby adding a fragment
r eplic at esalong wit h t he h o s t g e n o m e . N o p h a g e of phagel" DNA includingcos site,to plasmids.A
particles are synthesizedin this pathway foreign DNA (about40 kb) can be insertedinto
cosmidDNA. The recombinant DNA so formedcan
Use of phage )" as a vector be packed as phages and injected into E.coli
Only about 5O"h of phage l, DNA is necessary (Fig. 6.10). Once inside the host cell, cosmids
f or it s m ult iplic at ion and o t h e r f u n c t i o n s . T h u s , a s behave j ust l i ke pl asmi dsand r eplicat e.The
m uc h as 50% ( i. e. up t o 2 5 k b ) o f t h e p h a g e D N A advantagewith cosmidsis thatthey can carrylarger
c an be r eplac edby a dono r D N A f o r u s e i n c l o n i n g fragments of foreignDNA comparedto plasmids.
experiments. However, several restriction sites are
presenton phage )" which is not by itself a suitable Phasid vectors
vector.The l"-basedphage vectorsare modifications
P hasi dsare the combi nati onof plasm id and
of t he nat ur al phage wit h m u c h r e d u c e d n u m b e r o f
phageandcanfuncti onasei therone ( i. easplasm id
r es t r ic t ion s it es . Som e o f t h e m a r e d i s c u s s e o
or phage).P hasi dspossessfunctionalor iginsof
nereunoer. plasmid phage 1., and
replicationof both and
lnsertion vectors : They have just one unique thereforecan be propagated (asplasmidor phage)
cleavage site, which can be cleaved, and a foreign in appropriateE.coli.The vectorsphasidsmay be
DNA lieat ed in. lt is es s e n t i a lt h a t s u f f i c i e n t D N A usedi n many w ays i n cl oni ngexp er im ent s.
. ] \ T R O D U C T IO NT O C EN ET ICEN C IN E E R IN C
o
86 B IO TECHNO LO CY

acceptlargefragments of foreignDNA (particularly

human D N A ). l t i s thus possi blet o clone lar ge
DNA pieces by using YAC. YACs are the most
sophisticatedyeast vectors, and representthe
largest capacity vectors available. They possess
centromericand telomericregions,and therefore
the recombi nantD N A can be m aint ainedlike a
yeastchromosome.

Bacterial artificial chromosomes (BACSl

cos
-+-----.{ 37.5 kb cos The constructionof BACs is based on one
F-pl asmi dw hi ch i s l argerthan the ot her plasm ids
IlPackaoinorn yftro used as cloning vectors.BACs can accept DNA
J insertsof around 3-00 kb. The advantagewith
bacterialartificialchromosome is thatthe instability
problemsof YACscan be avoided.In fact, a major
part of rhe sequencingof human Senome has been
accompl i shed by usi ng a l ibr ar y of BAC
recombinant.

51{UTTLE VECTORS
The plasmid vectors that are specifically
I designedro replicate in two different hosfs (say in
llnfection E.coli and Streptomyces sp)are referredto as shuttle
J vectors.The originsof replicationfor two hostsare
/-rG\.,
( combined in one plasmid.Therefore,any foreign
/ . *Plasmidform
'i ' (orcosmid) DNA fragmentintroducedinto the vector can be
( J
\Y/ expressed shuttlevectorscan
in eitherhost.Further,
\Qplical/ be grown in one host and then shiftedto another
E. coli host (hencethe name shuttle).A good numberof
eukaryoticvectorsare shuttlevectors.
Fig. 6.10 : Cosmids as vectots.
OH OIC E OF A V E C TOR
Amongthe severalfactors,the sizeof the foreign
ART l F I c I Jii'- CF;sli,,Fi.
Jc :.,i{:;
l}1€ V ECTO RS DNA is very importantin the choiceof vectors.The
Hunralr artiflciat cl;rl -ll;rsonne (HAG) efficiency of this process in often crucial for
determiningthe successof cloning. The size of
D e v e l o p e di n 1 9 9 7 (by H . W i l l ard); human DNA insert that can be accepted by different
artificial chromosomeis a syntheticallyproduced vectors is shown in Table 5.5.
vector DNA, possessing the characteristics of
human chromosome.HAC may be consideredas a
self-replicatingmicrochromosomewith a size
ranging from 1/1Oth to 1/5th of a human
chromosome.The advantagewith HAC is that it Introducinga foreign DNA (i.e. the Sene)into
can carry human genesthat are too long. Further, the cells is an importanttask in biotechnology. The
HAC can carry genesto be introducedinto the efficiency of this process is often crucial for
c e l l s i n g e n eth e ra p y . .determi ni ngthe successof cl oning. The m ost
commonly employed gene transfer methods,
Yeast crrlifi*ial ehrn;no;omes (YACs)
namely transform6tion, conjugation, electro-
lntroducedin 1987(by M. Olson),yeastartificial poration and lipofection,and direct transferof
chromosome(YAC)is a syntheticDNA that can DNA are brieflydescribed.
lt @E - : : I \ T R OD U C T IO NT O C EN ET ICEN C IN E E R IN C 87

usediethylaminoethyldextran(DEAE-dextran)for
hr 5"5 Tle different clonlng veetors with DNA transfer.
h cnresponding hosts and the slzes
of foreign insert DNAs coNJucTroh!
Host Foreign insert Conjugation is a natural microbial
DN,4,size recombination process.During conjugation,two
l i ve bacteri a(a donor and a reci pi ent)come
er@g;_ E. coli 5-25kb together,join by cytoplasmicbridgesand transfer
l:rrc i- E. coli 35+5 kb single-stranded DNA (from donor to recipient).
ra-c artifical E. coli 100-300kb Insi de the reci pi entcel l , the new D N A may
integrate with the chromosome(ratherrare)or may
r'rircsome (PAC)
remai nfree (as i s the casew i th pl asmi ds).
b.eral artificial E. coli 100-300
kb
C onj ugati oncan occur among the cel l s from
rnnosome (BAC) different Beneraof bacteria (e.g Salmonella and
r=as1chromosome S. cerevisiae 200-2000
kb Shigellacells).This is in contrastto transformation
w hi ch takespl ace amongthe cel l s of a bacteri al
genus.Thusby conjugation,transferof genesfrom
TRA NS F O RM AT ION two differentand unrelatedbacteriais possible
-'ansformationis the method of introducing The natural phenomenonof conjugation is
r:re:gn DNA into bacterialcells (e.g. E.colD.fhe exploitedfor gene transfer.This is achievedby
- m ak eof plas m i dD N A b y E.c o l ii s c a rri e do u t i n pl asmi d-i nsert
transferri ng D N A from one cel l to
:e-cold CaCl, (0-5"C),and a subsequentheat another.In general,the plasmidslack conjugative
;-.:ck (3745"C for about 90 sec). By this functions and therefore,they are not as such
::r:rnique, the transformation frequency, which capableof transferring DNA to the recipientcells.
-:fers to the fraction of cell population that can be However, some plasmids with conjugative
tansferred,is reasonably good e.g. approximately propertiescan be preparedand used.
c r e c ell f or 100 0 (1 0 -3 )c e l l s .
Transformation efficiency : lt refers to the E LE C TR OP OR f,Ti f
-umber of transformants per microgramof added Electroporation is basedon the principle that
D\A. For E.coli, transformation by plasmid,the hi ghvol tageel ectri cpul sescan i nducecel l pl asma
:.ansformation efficiencyis about 107ro 108 cells membranesto fuse. Thu_s,electroporationis a
per microgram of intact plasmid DNA. The technique involving electric field-mediated
bacterialcellsthat can takeup DNA areconsidered membrane permeabilization.Electric shocks can
as competent.The cornpetencecan be enhanced al so i nduce cel l ul ar uptakeof exogenousD N A
bv alteringgrowth conditions. (believedto be via the pores formed by electric
The mechanismof the transformation processis pul ses) from the suspendi ng sol uti on.
Electroporation is a simple and rapidtechniquefor
not fully understood.lt is believedthat the CaCl,
i ntroduci nggenes i nto the cel l s from vari ous
affectsthe cell wall, breaksat localizedregions,
organi (mi croorgani sms,
sms pl antsand ani mal s).
e r b i n d i n go f D N A to cel l
a nd is als or es p o n s i b lfo
surface.A briefheatshock(i.e.the suddenincrease The basic technique of electroporationfor
in temperature from 5'C to 40"C) stimulatesDNA transferri ng genesi nto mammal i an cel l si s depi cted
uptake. In general, large-sizedDNAs are less i n Fi g, 6.11. The cel l s are pl aced i n a sol uti on
efficientin transforming. containingDNA and subjectedto electricalshocks
to causeholesin the membranes. The foreignDNA
Other chemical methods for fragments enter through the holes into the
transformation cytoplasmand then to nucleus.
Calc ium ph o s p h a te(i n p l a c e o f C a C l r) i s is an effectiveway to transform
Electroporation
oreferredfor the transferof DNA into cultured E .col icel l s contai ni ngpl asmi dsw i th i nsertD N A s
m a y re s u l ti n longerthan 100 kb. The transformation
c ells .S om et im e sc,a l c i u mp h o s p h a te efficiencyis
precipitateand toxicityto the cells.Someworkers around 109 transformants per microgram of DNA
88 B IOTE CHNO LO CY

fusewith themto transferDNA fragments.Thus,the

DNA entersthe cell and then to the nucleus,The
positively
chargedliposomes complex
veryefficiently
with DNA, bind to cellsand transferDNA rapidly.

Cells DNA fragments Lipofectionis a very efficienttechniqueand is

usedfor the transferof genesto bacterial,animal
and ol antcel l s.
l_ _J
The differentmethodsof genetransferare briefly
I Mixed describedabove. More details are given in the
together
I respectivechaptersapplying these techniquesto
+
achievegenetransfer.

TR A N S D U C TION
*K Sometimes,the foreign DNA can be packed
i nsi deani malvi ruses.Thesevi rusesca n nat ur ally
Special DNA
cuvetle fragment

a').
,)
a ( o^h^
a DNA enterscell
throughpores

withDNA
Liposome
( @)
DNAenters
nucleus

Fig. 6.II : Gene transler by electroporation.

(Note : Magnification depicted on right side)

for small plasmids(about3kb) and about 106 for

l a rg ep l a s mi d s(a b o u t1 3 0 k b ).

L IPOS O ME .M ED IAT E D G E N E TR A N S FE R
L i p o s o m e as re c i rc u l a rl i pi d mol ecul es,w hi ch Fusionbetween
plasmamembrane
have an aqueousinterior that can carry nucleic andliposome
acids.Severaltechniqueshave been developedto
encapsulateDNA in liposomes.The liposome-
DNA
mediatedgenetransfer,referredto as lipofection, is nucleus
depictedin Fig. 5.12.
On treatmentof DNA fragmentwith liposomes,
Flg. 6.12 : Liposome-mediated gene transfer
the DNA piecesget encapsulated
insideliposomes.
(Note : For clarity, the native cell DNAis not shown).
Theseliposomescan adherto cell membranes and
Chapt er6 : I N T R O D U C T IO N
T O C E N E T ICE N C IN E E R IN C 89

infect the cells and introducethe DNA into host

cells. The transferof DNA by this approach is GENERATION
OF DNA FRAGMENTS
referredto as transduction.

DIRECT TRANSFER OF DNA

It is possibleto directlytransferthe DNA into INSERTIONINTOA CLONINGVECTOR
t he c ell nuc l e u s . Mi c ro i n j e c ti o na n d p a r ti cl e
| (Ligationof bluntends or cohesiveends,
bombardmentare the two techniquescommonly homopolymer tailing,linkermolecules)
I
usedfor this purpose. I
J
Microinjection INTRODUCTION INTOHOSTCELLS
DNA transferby microinjectionis generallyused | (Transformation,
transfection,transduction)
I
for the culturedcells.This techniqueis also useful
to introduceDNA into largecells such as oocytes, +
I
eggsand the cells of early embryos. SELECTION OR SCREENING
(Hybridization,
PCR,immonochemical
Some more details on gene transfermethods
methods,protein-proteininteractions,
inc luding par ti c l e b o mb a rd me n ta re g i v e n i n functionalcomplementation)
Chapter49.
The term transfection is used for the transfer
Fig. 6.13 : An overview of cloning strategies in
DNA into eukaryotic cells,by various physicalor
rccombinant DNA Iechnology.
c hem ic alm ea n s .

CLONING FROM GENOMIC DNA

OR MRNA?

A clone refersto a group of organisms,cells, DNA representsthe complete genetic material

moleculesor other objects, arising from a single of an organism which is referred to as genome.
individual. Clone and colony are almost Theoreticallyspeaking,cloning from genomic DNA
synonymous. i s s u p p o s e dt o b e i d e a l . B u t t h e D N A c o n t a i n s n o n -
coding sequences (introns), control regions and
Cene c lo n i n g s tra te g i e s i n re l a ti o n to repetitive sequences.This complicates the cloning
recombinantDNA technologybroadlyinvolvethe strategies,hence DNA as a source material is not
followingaspects(Fig.6.13). preferred, by many workers. However, if the
objective of cloning is to elucidate the control o{
o Cenerationof desiredDNA fragments.
gene expression, then genomic DNA has to be
o Insertionof thesefragmentsinto a cloning vector. invariably used in cloning.
o lntroductionof the vectorsinto host cells. The use of mRNA in cloning is preferredfor the
r Selectionor screeningof the recipientcells for following reasons.
the recombinantDNA molecules. . mRNA representsthe actual genetic information
(or being expressed.
It is obviousthat for a good understanding
account)of gene cloningstrategies,
thischapter has . Selection and isolation mRNA is easy.
to be learntin detail.In addition,the readermust o As introns are removed during processing,mRNA
invariably refer gene libraries (Chapter 9) for reflects the coding sequence of the gene.
cloning of genesand screening strategies,and the
. The synthesisof recombinant protein is easy with
polymerasechain reaction (Chapter Bl for in vitro
mRNA cloning.
generation of largequantitiesof DNA. Further,gene
cloningalsoinvolvesthe expression of geneswhich Besides the direct use of genomic DNA or
is described under manipulation of gene expression mRNA, it is possible to synthesize DNA in the
in host cells(Chapter11). laboratory (Chapter 7), and use it in cloning
 90 B IOTECHNO LO CY

experiments. This approach is useful if the gene l n 1974, a groupof ten sci enti stled
s by Paul
sequence is short and the complete sequence of Bergwrote a letterthat simultaneously appearedin
am ino ac ids is k nown. the presti geousj ournal s-N ature,Science and
Proceedings of the NationalAcademyof Sciences.
The different strategies for the cloning of
The dangersof DNA technologywere printedout
genom ic DNA and m RNA a r e d e s c r i b e d u n d e r
in that letter(highlightsgiven below) :
gene libr ar ies ( Chapt er 9) .
"Recent advances in techniques for isolation
and rejoining of segmentsof DNA now permit
construction of biologically active recombinant
DNA molecules in vitro. Although such
experimentsare likely to facilitate the solution of
For addit ional inf or m a t i o n o n t h e g e n e t i c
important theoretical and practical biological
problems, they would also result in creation of
engineeringof plants,the reader must refer Chapter
49. Some details on the following aspectsare given
novel types of DNA elements whose biological
in that chapter
propertiescannot be completely predicted. Thereis
a serious concern that some of these DNA
. Cene transfer methods (vector-mediated gene molecules could prove biologically hazardous".
t r ans f er - T DNA, plan t v i r u s e s ; d i r e c t D N A
transfer- physical and chemical methods). The letteralso appealedto molecularbiologists
w orl dw i de for a moratori umon many kinds of
. Marker genes for plant transformation.
recombinant DNA research,particularly those
o Promoters and terminators. i nvol vi ngpal hogeni organi
c sms.
o Transgene stability, expression and gene
s ilenc ing. A S I LOMA R R E C OMME N D A TIO NS
o Chloroplast transformation. ln February1975,a groupof 139 scientists from
17 countriesheld a conferenceat Asilomar,a
conferencecenterin California.USA.Thevassured
the uneasypubl i cthat the mi croorg anismused
s in
DNA experiments were specificallybredand could
not surviveoutsidethe laboratory. Thesescientists
With the success of Boyer-Cohen experiments formulated guidelines and recommendations for
(in 1973), it was realised that recombinant DNA conducting experiments in genetic engineering.
technology could be used to create organismswith
novel genes. This created worldwide commotion N IH GU ID E I.IN E S
(among scientists,public and government officials)
N ati onal l nsti tute of H eal th (NlH) , USA
about the safety, ethics and unforeseen
constituted the Recombinant DNA Advisory
c ons equenc esof genet ic m a n i p u l a t i o n s . S o m e o f
Committee (RAC) which issueda set of stringent
the phrases quoted in media in those days are
guidelinesto conductresearchon DNA. RAC was
8r v en. in fact overseeingthe researchprojectsinvolving
o Manipulation of life. genespl i ci ngand recombi nant
DNA.
. Play ing Cod. S ome of the i mportant original NI H
. M an m ade ev olut ion. recommendations on recombinantDNA researcn
relateto the followingaspects.
o The most threatening scientific research.
. Physical (laboratory)containment levels for
It was feared that some new organisms,created
conductingexpermiments.
inadvertently or deliberately for warfare, would
cause epidemics and environmental catastrophes. . Biological containment-thehost into which
Due to the fears of the dangerous consequences,a foreign DNA is insertedshould not proliferate
cautious approach on recombinant DNA outsidethe laboratoryor transferits DNA into
experiments was suggested. otherorqani sms.
: iapt er 6 : I NT R OD U C T IO N
T O C EN ET ICE NC IN E E R IN C 9l

r For research on pathogenicorganisms, elaborate, Genetically engineered organisms

controlled and self-contained rooms were {GEOs)
recommended.
RecombinantDNA researchhas resultedin the
. For researchon lessdangerousorganisms,units creationof manygeneticallyengineered organisms.
equippedwith high qualityfilter systemsshould Thesei ncl udemi croorgani sms, ani mal sand pl ants.
be used. The latter two respectivelyresult in transgenic
animalsand transgenicplants.The safetyaspects
" \o deliberatereleaseof any organismcontaining
r ec om binanDt N A i n to th e e n v i ro n me n t. and other relatedmattersof CEOsare discussed at
appropriate places in text, and some major
It may be noted here that althoughthe NIH hi ghl i ghts are gi ven i n C hapter61.
euidelinesdid not have the legal status,most
rnstitutions, companiesand scientistsvoluntarily
c om olied.

Relaxation of NlFl guidelines

l IH g u i d e l i n ew
I t was in 198 0 ,th e o ri g i n a N
considerablyrelaxedby NIH-RAC,basedon the
experienceand experimentaldata obtainedfrom
the NIH-sponsored sludieson recombinantDNA
research.lt was almost agreedthat the original
apprehensions on recombinantDNA research
unfounded.
It is a fact that the geneticengineeringresearch
flourishedand progressed rapidlyafterrelaxationof
NIH guidelines.lt may howeverbe notedthat NIH-
RAC continuesto be a watchdogover the DNA industryand environment.The major oblectiveof
technologyexperiments.
Pharmaceutical products of
recombinant DNA
As the recombinant DNA technology
progressed, many pharmaceuticalcompoundsof
human health care are being producedthrough
geneticmanipulations. Mostcountriesconsiderthat
the existing regulations for approval of
pharmaceuticals of commercialuseare adequateto
ensure safety since the process by which the
product manufacturedis irrelevant. Thus, the
recombinantDNA product(protein,vaccine,drug)
is evaluatedfor its safetyand efficacylike any other
pharmaceutical product.
s ere
DNA technologyhas largelyhelpedscientists to
understand the structure,functionand regulationof
genes. The development of neVmodern
biotechnologyis primarilybasedon the successof
DNA technology.Thus, the presentbiotechnology
were
(more approp riately mol ecular biotechnology) has
its main roots in molecular biology.
Biotechnologyis an interdisciplinary
approach
for applicationsto human health, agriculture,
biotechnologyis to solveproblemsassociated with
human health,food production,energyproduction
and environmentalcontror.
The major contributionsof geneticengineering
throughthe new disciplinebiotechnology aregiven
i n thi s book.
It is an acceptedfact that the recombinantDNA
technologyhasenteredthe main streamof human
life and has becomeone of the most significant
applicationsof scientificresearch.Biotechnologyis
regarded as more an art than a science.
After the successfulsequencing of human
genome/many breakthroughsin biotechnology
are
exoectedin future.
 her e ar e s ev er al t ec h n i q u e s u s e d i n
r ec om binant DNA t ec h n o l o g y o r gene
m anipulat ion. The m os t f r equ e n t l y u s e d m e t h o d s
ar e lis t ed.
. Agarose gel electrophoresrs.
o I s olat ion and pur if ic at ion of n u c l e i c a c i d s .
r ls olat ion of c hr om os om es .
. Nuc leic ac id blot t ing t ec hni q u e s .
r DNA s equenc ing.
o Alt er nat e m et hods of DNA s e q u e n c i n g .
. Chem ic al s y nt hes isof DNA.
o Methods of gene transfer (Chapters6 and 49).
. Poly m er as ec hain r eac t ion ( C h a p t e r B ) .
r Pr oduc t ionof m onoc lonalan t i b o d i e s( C h a p t e r1 7 ) .
o Cons t r uc t ionof gene libr ar y ( C h a p t e r 9 ) .
. Radiolabelingof nuc leic ac i d s ( C h a p t e r 9 )
Som e det ails on t he f ir s t se v e n t e c h n i q u e s a r e
des c r ibed in t his c hapt er while o t h e r t e c h n i q u e a r e
discussed elsewhere (as referred in the
correspondingchapters).lt may however, be noted
t hat s om e s pec if ic applic at ion so f t h e s e t e c h n i q u e s
are frequently referred to as when needed in the
c or r es pondi ng c hapt er s .
AGAROSE GEL ELEGTBOPHOREEIS
Electrophoresis refers to the movement of
charged molecules in an electric field. The
negatively charged molecules move towards the
p o s i t i v e e l e c t r o d e w h i l e t h e p o s i t i v e l y ch a r g e d
molecules migrate towards the negative electrode
C e l e l e c t r o p h o r e s i si s a r o u t i n e l y u s e d a n a l yti ca l
technique for the separation/purificationof specific
D N A f r a g m e n t s . T h e g e l i s c o m p o s e d o f e i th e r
polyacrylamide or agarose. Polyacrylamide gel
electrophoresis(PACE)is used for the separationof
s m a l l e r D N A f r a g m e n t s w h i l e a g a r o se e l e ctr o -
p h o r e s i s i s c o n v e n i e n t f o r t h e s e p a r a t i ono f D N A
f r a g m e n t sr a n g i n g i n s i z e f r o m 1 0 0 b a s e p a i r s to 2 0
k b p a i r s . C e l e l e c t r o p h o r e s i sc a n a l s o b e u se d fo r
t h e s e p a r a t i o no f R N A m o l e c u l e s .
Agarose is a polysaccharide derived from
s e a w e e d s .l t f o r m s a s o l i d g e l w h e n d i sso l ve d r n
aqueous solution at concentrations between 0.5
and 2.0% (wlv) lt may be noted here that the
agarose used for electrophoresisis more purified
form of agar when compared to that used for
culture purposes.
A d i a g r a m m a t i c v i e w o f t h e a g a r o se g e l
e l e c t r o p h o r e s i su n i t i s s h o w n i n F i g . 7 '1 .
T h e D N A s a m p l e sa r e p l a c e d i n t h e w e l l s o f th e
g e l s u r f a c ea n d t h e p o w e r s u p p l y i s s w i t ch e d o n . As
t h e D N A i s n e g a t i v e l y c h a r g e d , D N A fr a g m e n ts
 7 : B A S ICT E C H N IQU ES
. - - ^apt er l N C EN ET ICE N C E N E E R IN C 93

fi el dori entati on
changes,
the D N A mol ecul es
al i gn
themselves and migratein new directions.
limitation of PFGE: The maior limitationof
pul sed-fi elgel
d el ectrophoresi i ssthat the sampl es
Cover do not mi gratei n strai ghtl i nes.Thi s makesthe
furtheranal ysi sof D N A ratherdi ffi cul t.
Gel
CONTOUR.CLATUIPEDHOMOGENEOUS
+ve
electrode E LE C TR IC A L-FIE LD E LE C TR OP I{ OR E S IS
-ve . . . . . . . .. . . ' - . . I
:lectrocle Migrationof Buffer {cH E Fl
DNA
C H E F i s an i mproved method to appl y
Fig. 7.1 : A diagrammatic representation of agarose alternating electrical fields to separate DNA
gel electtophoresis system. mol ecul es
i n el ectrophoresiIns.thi s approach,
the
reorientationangle of electricalfield is fixed at
120'C (or even at 90') and electrophoresis carried
nove through the gel towards the positive out. By usingCHEF,largeDNA fragments (200-300
e lec t r ode.T he ra te o f mi g ra ti o n o f D N A i s kb) can be routinelyseparated in a matterof hours.
clependent on th e s i z e a n d s h a p e . In g e n e ral ,
..mallerlinearfragmentsmovefasterthan the larger POLYACRYTAMIDE GEL
ones. Hence, gel electrophoresiscan be E LE C TR OP H OR E S IS (P A GE I
conveniently
usedfor the separation
of a mixtureof gel i s composedof chai nsof
P ol yacryl ami de
DNA fragments,basedon their size. acryl ami de monomers cross-l i nked w i th
Agaroseformsgelswith pore sizesrangingfrom methylenebisacrylamide units.The pore sizeof the
100 t o 300 nm i n d i a m e te r.T h e a c tu a lp o re s i ze gel is dependent on the total concentrationof the
dependson the concentration of the agarose.The monomers and the cross l i nks.
size of the pores determinesthe range of DNA Polyacrylamidegel electrophoresis (PACE)is
fragments that can be separated on electrophoresis.used for the separation of single-stranded DNA
For instance,a 0.3'/' agaroseis used for the molecuels that differ in length by just one
separation of DNA fragments between5 and 50 kb, nucleotide. Agarosegels cannot be used for this
rvhile a 5% agarosecan separate100-500 bp purpose.This is becausepolyacrylamide gels have
m olec ules .
The migrationof DNA fragmentsduring the
course of electrophoresis can be monitored by Wellsfor
sampres
usingdyeswith known migrationrates.Thesedyes
Agarosegel
are addedto the DNA samplesbeforeloading.The
bandsof the DNA can be detectedby soakingthe UV transparenl
plasticsupport
gel in ethidium bromide solution.When activated
b y ult r av iolet ra d i a ti o n , D N A b a s e p a i rs i n
wit h e th i d i u m b ro mi d e e mi t o ra n ge
as s oc iat ion | = ,n' o,rrbromi de
fluorescence. And in this way the DNA fragments solution(soaking)
separated in agarose electrophoresiscan be J
identified(Fig.7.2).

P ULS E D" F I E L D GE L EL E C T R O P H OR ES IS
(PFGEI
T he lar ges iz e dD N A mo l e c u l e s(- 1 0 Mb ) c an lTt
UV light
in a g a ro s g
be s epar at ed e e l sb y u s i n gP F G ET. h i si s
madepossibleby periodicallyalteringthe direction Fig. 7.2 : ldentification of DNA bands separated
of DNA migrationby changingthe orientationof by agarose gel electrophoresis.
electricfield with respectto the gel. As the electric
 94 B IOTECHNO LO CY

s m a l l e rp o re s i z e sth a n agarosegel s and al l ow P U R IFIC A TION OF C E LLU LA R DNA

precise separation of DNA molecules from
The firststepfor DNA purificationis to openthe
10 -15 0 0 b p .
cel l s and rel easeD N A . The method should oe
Po l y a c ry l a m i d eg e l el ectrophoresi si s a gentle to preserve the native DNA. Due to
wonderfultechniquefor the fine separation of DNA variabilityin cell structure,
the approachesto break
moleculesthat differ from each other even by 1-3 the cells are also different.
n u c l e o ti d e sP.AC Ei s u s e di n D N A sequenci ng,
and
for the identification of amplifiedproductsof DNA l-ysis of cells
b y p o l y m e ra sceh a i n re a cti on(P C R ).
Bacterialcells : The bacterialcells(e.g.E. coli)
OT H ER U SE S OF GE L can be lysedby a combinationof enzymaticand
EL E C T R O P H OR ES IS chemical treatments.The enzyme lysozymeand
the chemicalethylenediamine tetraacetate (EDTA)
Besidesthe separationof DNA fragments,gel are usedfor this purpose.This is followed by the
electrophoresis is also usefulfor understanding the addition of a detergentsuch as sodium dodecyl
m o l e c u l a rc o n fi g u ra ti o n
o f D N A mol ecul es,and sul fate(S D S ).
orotein-nucleic acid interactions. The latteris baseo
o n th e p ri n c i p l eth a t b i n d i ngof a protei nto D N A A ni malcel l s: A ni malcel l s,parti c ular ly
cult ur ed
usuallyresultsin the reductionof electrophoretic ani mal cel l s, can be easi l y opened by dir ect
mobility. treatmentof cells with detergents (SDS).

The electrophoretictechnique for protein- P l ant cel l s : P l antcel l s w i th stro ngcell walls
n u c l e i ca c i d i n te ra c ti o n
i n vol vesthe addi ti onof a requireharshtreatmentto breakopen.The cellsare
desiredproteinto double-stranded DNA fragments, frozenand then groundin a morterand pestle.This
separationof this complex and naked DNA by is an effectiveway of breakingthe cellulosewalls.
electrophoresis. The separatedpatternscan be
visualizedto understandthe orotein-nucleicacid Methods to purify DNA
interactions.
There are two differentapproachesto purify
D N A from the cel l ul arextracts.
1. Purificationof DNA by removing cellular
components: Thi s i nvol vesthe de gr adat ionor
I
compl eteremovalof al l the cel l ul a rcom ponent s
I
Al m o s t a l l th e e x p e ri ments deal i ngw i th gene other than D N A . Thi s approachi s' s uit ableif t he
m a n i p u l a ti o nre s q u i rep u reformsof ei therD N A or cel l sdo not contai nl argequanti ti e of s lipidsand
RNA, or sometimeseven both. Hence there is a carbohydrates.
n e e dfo r th e re l i a b l ei s o l a t i onof nucl ei caci dsfrom The cellularextractis centrifuged at a low speed
the cells.The purificationof nucleicacidsbroadly to removethe debris(e.g.piecesof cell wall) that
involvesthree stages. forms a pellet at the bottom of the tube. The
1 . Bre a k i n go r o p e n i n gof the cel l s to expose supernatant is collectedand treatedwith phenolto
n u c l e i ca c i d s . precipitateproteinsat the interfacebetween the
organic and aqueouslayers.The aqueouslayer,
2 . S e p a ra ti o no f n u c lei c aci ds from other
nucl ei caci ds,is collect ed
contai ni ngthe di ssol ved
c e l l u l a rc o mp o n e n ts . (RNase).
and treatedwith the enzymeribonuclease
3 . R e c o v e ry o f n u c l e i caci dsi n a pure form. The R N A i s degradedw hi l e the DNA r em ains
Analyticalproceduresinvolvinga few stepsto i ntact.Thi s D N A can be preci pi ta t ed by adding
severalsteps are in use for the purificationof ethanol and isolated after centrifugation,and
nucleic acids.ln fact, commercialkits are readily suspendedin an appropriatebuffer.
availablethesedaysto enablepurificationof either 2. Direct purification of DNA : In this
DNA or RNA from differentsources. approach,the DNA itself is selectivelyremoved
The basicprinciplesand procedures for nucleic from the cellular extractand isolated.There are
acid purificationare brieflydescribed. two ways for direct purificationof DNA.
 - - apt er 7 : B A S ICfEC H N IQ U ESl N C E N E T ICE N C E N E E R IN C 95

ir one method, the addition of a detergent

cettltrimethyl ammonium (CTAB) results in the
Upper band with
' - -rra tion of a n ins oluble c om plex wit h nuc le i c
chromosomalDNA
=:rd s This comp lex , in t he f or m of a pr ec ipit at ei s and open plasmidDNA
:: lected after centrifugation and suspended in a
- rh-salt solu tion t o r eleas e nuc leic ac ids . B y
:'ea imen t with RN as e,RNA is degr aded.Pur e DN A Lower band with
: :1 b e isola ted by et hanol pr ec ipit at ion. closedcircular
plasmidDNA
The se co nd te c hnique, is bas ed on t he pr inc ip l e
: i tig ht bin din g b et ween DNA and s ilic a par t ic le s
- the presence of a denaturing agent such as
.ran idin ium th iocy anat e.The is olat ionof DNA c a n Fig. 7.3 : Purification of plasmid DNA by isopycnic
re a ch ieved b y th e dir ec t addit ion of s ilic a par t ic le s centrifugation in CsCl-EtBr gradient.
;'rd gu an idin ium t hioc y anat et o t he c ellular ex t r ac t ,
-cllowed by centrifugation. Alternately, a column
:hro mato gra ph yc ont aining s ilic a c an be us ed, an d thereis a narrowrangeof pH (12.0-12.5)at which
.rrou gh this the ex t r ac t and guanidiniu m denaturation of linear DNA and not closedcircular
:niocyanate are passed. The DNA binds to the DNA occurs:
s ilica p articles i n t he c olum n whic h c an b e
When the clearedlysateis subjectedto carefully
recovered.
moni toredal kal i nepH (12.0to 12.5),the pl asmi d
D N A mol ecul esremai ni n sol uti onw hi l e al l other
PURIFICATION O F PLASM I D DNA
DNA moleculesget denaturedand precipitate. This
Pure forms of plasmid DNA are often required in precipitatecan be removedby centrifugation. The
qenetic engineering experiments. The prerequisite pl asmi d D N A i n the supernatantcan oe
for a successful isolation of olasmid DNA is the concentrated by ethanolprecipitation.Sometimes,
appropriateand gentle lysis of the host cells, so that further puri fi cati onof pl asmi d D N A may be
th e pla smid DNA is r eleas ed wit hout t oo m uc h necessary which can be achievedby gel filtration.
conta mina tion o f c hr om os om al DNA. The hig h
m o lecula r weig ht hos t c ell c hr om os om al DNA c a n Factors affecting plasmid DNA
be re moved a lon g wit h t he c ell debr is by high - purification
speed centrifugation. This results in the formation
Several factors influence the purification of
of a cleared lysate from which pure plasmid DNA
plasmidDNA. The most importantonesare briefly
can be isolated. Of the several methods in use ro
described.
isolate plasmid DNA, two are briefly described.
1. Plasmid copy number : The number of
lsop ycnic ce ntr if ugat ion m et hod plasmidspresentin the host cells at the time of
harvestis important.The copy number in turn rs
The cleared lysate (referred above) is treated
influencedby growth medium, genotypeof host
with a solu tion o f c es ium c hlor ide ( Cs Cl)c ont ainin g
cell and the stageof growth.In general,the higher
ethidium bromide (EtBr). EtBr can bind with linear
the copy number, the better is the yield of plasmid
DNA molecules (chromosomal DNA fragments and
DNA on purification.
open pla smid DNA) and not wit h c ir c ular plas m i d
DNA. Th e d en sity of DNA- Et Br c om plex is m uch 2. Preparation of cleared lysate : The
lower than the plasmid DNA. The circular plasmid methodologyadoptedand the care taken for the
DNA can be separatedby isopycnic centrifugation lysisof the host cells to prepareclearedlysateis
in a CsCl-EtBrgradient (Fig. 7,3). imoortant.
3. Host cells with end A gene : The wild-type
B irnb oim an d Doly m et hod
host cells possesendA gene that encodes the
Th e te ch niq ue dev eloped by Bir nboim and Dol y enzyme endonucl easel . The functi onsof thi s
(197 9) is wide ly us ed f or t he pur if ic at ionof plas m i d enzyme are not clearly known. lt is however,
DNA. This me tho d is bas ed on t he pr inc iple t ha t observed .that cell strains bearing endA
 96 B IOTECHNO LO CY

Afterthe cellsare disruptedon lysisby different

{- Cellulosebead techni ques(S ee p.99), the cel l ul ar ext r act is
with oligo (dT)
T deproteinised by treatmentwith phenolor phenol/
T chl oroformmi xtures. t he nucleic
On centri fugati on,
T
T acidsget concentrated in the upperaqueousphase
T w hi ch may then be preci pi tatedby using
T
i sopropanol or ethanol .
The puri fi cati onof mR N A can be achievedby
affi ni tychromatography usi ngol i go (dT) - cellulose
(Fig.7.9. This is basedon the principlethat oligo
Columnwith
oligo(dT)-cellulose (dT)-cel l ul ose y nd to the poly ( A)
can speci fi cal lbi
tai l sof eukaryoti cmR N A .Thus,by this appr oach,
I i t i s possi bl to
e i sol atemR N Afrom D N A, r RNAand
J tR N A .
A s the nucl ei caci d sol uti oni s passedt hr ough
an affinitychromotographic column, the oligo(dT)
bi nds to pol y(A )tai l s of mR N A . B y washingt he
columnwith high-saltbuffer,DNA, rRNAand IRNA
can be el uted,w hi l e the mR N A i s ti ght lybound.
Thi s mR N A can be then el utedby washingwit h
low-salt buffer. The mRNA is precipitatedwith
ethanoland collectedby centrifugation (Fig.7.4).

High-saltwash

i Separation of largechromosomes of eukaryotes

is not possibleby conventionalelectrophoresis.
The individualchromosomes of eukaryotes can be
separated l:y fluorescence-activated cell sorting
(FACS), also known as flow cytometry or flow
I karyotyping.
Ethanol
supernatanl FLUORESCENCE-AGTIVATED
C E LL S OR TIN G
To carry out FA C S ,the di vi di ng cells ( wit h
condensedchromosomes)are carefully broken
open, and a mixture of intact chromosomesis
Low-saltwash mRNA
prepared.Thesechromosomes arethenstainedwith
dye.The quantityof the dye that binds
a fluorescent
Fig. 7.4 : Purification of nRNA by affinity to a chromosomedependson its size.Thus,larger
chromatography with oligo(dT)-cellulose. chromosomes (with more DNA) bind moredye and
fluorescemore brightlythan the smallerones.
The dye-mi xedchromosomes are dilut ed and
mutationsimprovethe stabilityand yieldof plasmid passedthrougha fine aperturethat resultsin the
D N A. formation of a streamof droplets.Each droplet
containsa singlechromosome. The fluorescence of
PU R IF IC AT ION O F m R N A the chromosomes is detectedby a laser.When the
Among the RNAs,mRNA is frequentlyrequired fluorescence indicates that the chromosome
in a pure form for geneticexperiments. i l l umi natedby the l aser i s the on e desir ed,
 Chapt er7 : B AS ICT E C H N IQU ES
l N C EN ET ICE N C E N E E R IN G 97

that do not containthe desiredchromosomepass

througha wastecollectionvessel.

C OLLE C TION OF C H R OMOS OME S

Mixtureof W ITH ID E N TIC A L S IZE
chromosomes
(withdye) The directapplicationof FACS(described
above)
is not suitablefor the separationof chromosomes
with identicalsizes.e.g. chromosomes 21 and 22
i n humans.C ol l ecti onof such chromosomes
can
be achievedby use of specialdyes (e.g.Hoechst
33258 and chromomycin Ar) which bind to
AT-rich DNA or CC-rich DNA. The rest of tne
procedureis the samethat has been described.
It is convenient to separatetwo or more
chromosomes with identicalsizesby differentdyes.
A This is possiblesince no two chromosomesare
t/)
likely to containidenticalGCIATcontents.

O
/-------\- | Siqnal I
Charger+( e )-l ampliiicationI
--t-----/ | an0 SeleCIlOn i
<)
o Blotting techniques are very widely used
analytical tools for the specific identification of
desired DNA or RNA fragmenfsfrom thousandsof
molecules. Blotting refers to the prccess of
immobilization of sample nucleic acids or solid
support (nitrocellulose or nylon membranes). The
blottednucleicacidsare then usedas targetsin the
hybridization experiments for their specific
detection.An outline of the nucleic acid blotting
techniqueis depictedin Fig, 7.6,

Types of blotting techniques

The mostcomonly usedblottingtechniquesare
listedbelow
. Southernblotting(for DNA)
. Northernblotting(for RNA)
Desired All other
chromosome chromosomes r Dot blottinS(DNA/RNA)
. Colony and plaque hybridization(cells from
Fig. 7.5 : Separationof chromosomesby
colony).
cell softing (FACS|
fluorescence-activated
The Southernblottingis namedafterthe scientist
Ed Southern(1975) who developedit. The other
electricalcharge is specificallyapplied to these names Northern blotting and Westernblotting are
droplets(and no others)which get charged.This laboratory jargons which are now accepted.
resultsin the deflectionof the dropletswith the Western blotting involves the transfer of protein
desiredchromosomewhich can be separated from blots and their identification by using specific
the restand collected(Fig.7.5).Unchargeddroplets antibodies.

Biotechnologyftl
98 B IOTECHNO LO CY

The genomicDNA isolatedfrom cells/tissues is

digestedwith one or more restrictionenzymes.
Southernblot (DNA) This mixtureis loadedinto a well in an agaroseor
Northernblot (BNA) polyacrylamide gel and then subjected to
Dot-blot(DNA/RNA)
Colonyand plaqueblot
(Cellsfrom colony)

GenomicDNA

, lResl errao+rrpfe
,Y
\-
\- t'
,' ,t ,l
/ \r

\- \ t /
\\ "'

DNA fragments

Fig, 7.6 : An outline of the nucleic acid

blotting techniques.

A diagrammaticrepresentationof a typical
blottingapparatusis depictedin Fig. 7.7.

SOUTHERN BLOTTING
t
' S o u th e rnb l o tti n gte c h n iquei s the fi rst nucl ei c
acid blotting proceduredeveloped in 1975 by
Southern. lt is depicted in Fig. 7.8, and brietly
described.

{_weight

Nitrocellulose
(or nylon membrane)
or.rn
nrooe
{-Membrane
_______t(_ J
r___________ Gel
Plastic

Filterpaperbutfer
wick
Autoradiograph
Fig.7.7 : Diagrammatic representation
of a typical
blottingapparatus. Fig. 7.8 : Southern blotting lechnique.
C hapt er7 : B A S ICT EC H N IQ U ES
l N C E N E T ICE N C E N E E R IN C 99

DNA, being negativelycharged

electrophoresis.
migratestowards the anode (positivelycharged
electrode);
the smallerDNA fragmentsmovefaster.
The separated DNA moleculesare denaturedby
ex pos ur et o a m i l d a l k a l i a n d tra n s fe rre dto
nit r oc ellulosor
e n y l o n p a p e r.T h i s re s u l tsi n an
exact replicaof the patternof DNA fragmentson
the gel.T he DNA c a n b e a n n e a l e d to th e p a p e ro n
ex pos urteo heat(8 0 " C ).T h en i tro c e l l u l o soer n y l o n
paper is then exposedto labeledcDNA probes. rRNAbands
Theseprobeshybridizewith complementary DNA
molec ules on t he p a p e r.
I atottino
The paperafterthoroughwashingis exposedto I Hybriciza-tion
j Autoradiography
X-rayfilm to developautoradiograph.This reveals
specificbandscorrespondingto the DNA fragments
recognizedby cDNA probe.
DNAprobe
Factors affecting Southern blotting hybridizesto RNA
1. Stringentconditions(stringencycontrol) :
Stringencyrefersto the specificitywith which a
particularDNA targetsequenceis detectedby a Fig. 7.9 : An outline ot Norlhern blotting.
pr obe. T hus , w i th h i g h s tri n g e n t c o n d i ti ons
(elevatedtemperatureand low salt concentration)
o nly c om plet elyc o m p l e me n ta ry D N A s e q u e n ces . Forensi cal lappl
y i edto detectmi nutequanti ti es
w ill bind and hy b ri d i z e .H o w e v e r,l o w s tri n g ent of DNA (to identifyparenthood,thieves,rapists
condit ionswill a l l o w h y b ri d i z a ti o no f p a rti a l l y etc.).
matchedsequences. Therefore,stringencycontrol . H i ghl yusefulfor the determi nati on
of restri cti on
is very essentialfor specific detectionof DNA fragmentlengthpolymorphism(RFLP) associated
. th g o o d c o n trol
m olec ulesin S ou th e rnb l o tti n gWi w i th pathol ogi cal
condi ti ons.
of stringency,it is now possibleto detecta DNA
m olec ulejus t wit h a s i n g l eb a s ep a i r d i ffe re n c e . . D N A pi ecesfrom one speci es(e.g.human)can
be used to detect DNA moleculesfrom related
2. Membranesfor blot transfer : In the early
speci es(e.g.chi mpanze, cow ).Thi stechni quei s
year snit
, r oc ellu l o swea s u s e dfo r i m m o b i l i z a ti oof
n
referred to as Zoo-blotting.
DNA moleculesduringblot transfer. The drawbact<
s th a t i t i s fra g i l ea n d h a sto be N OR TH E R N
wit h nit r oc ellulo si e
B LOTTIN G
carefullyhandled.In recentyears,mostlaboratorres
use nylon membranesas they posseshigh tensile N orthern bl otti ng i s the techni que for the
strength,and better binding capacityfor nucleic specific identification of RNA molecules. The
a c ids . procedure adopted i s al most si mi l ar to that
describedfor Southernblottingand is depictedrn
Applications of Southern blotting Fig. 7.9. RNA molecules are subjected to
electrophoresis, followed by blot transfer,
Southernblottingtechniqueis extremelyspecific
hybridizationand autoradiography.
alt h o u g hi t i s a s i m p l ete c h n i q ue.
a nd s ens it iv e,
Someof the applicationsare listed. R N A mol ecul es do not easi l y bi nd to
. lt is an inv alu a b l eme th o di n g e n ea n a l y s i s . ni trocel paper or nyl on membranes.B l ot-
l ul ose
transferof RNA moleculesis carriedout by usinga
. lm por t antf or t h e c o n fi rma ti o no f D N A c l o n i ng chemically reactive paper prepared by
results. diazotizationof aminobenzyloxymethyl to create
. Useful for mapping restrictionsites around a diazobenzyloxymethyl (DBM)paper.The RNA can
singlecopy gene sequence. covalentlybind to DBM paper.
ro o B IOTE CHNO LO CY

Some workers have later developed appropriate c o n v e r t e d t o m e t a l l i c s i l v e r a t o m s . T h i s r e su l ts r n

c ondit ions t o blot RNA ban d s o n n i t r o c e l l u l o s e t h e d e v e l o p m e n t o f a v i s i b l e i m a g e w h i ch ca n b e
paper , and m odif ied ny lon m e m b r a n e s .T h e s e 'a r e easily detected.
now widely us ed in RNA blot t i n g . T h e u s e o f D B M
paper s is alm os t dis c ont inue d . Direct autoradiography

The blot + r ans f er r edRNA m o l e c u l e s h y b r i d i z e Direct autoradiography is ideally suited for the
with DNA probes which can be detected by detection of weak to medium strength p-emitting
autoradiography. r a d i o n u c l i d e s e H , 1 4 C , 3 s S ) .I n t h i s t e c h n i q u e , th e
s a m o l e i s p l a c e d i n d i r e c t c o n t a c t w i t h th e fi l m .
Northern blotting is theoretically, a good The radioactiveemissionsresult in the developmenr
t ec hnique f or det er m ining t h e n u m b e r o f g e n e s of black areas.
( t hr ough m RNA) pr es enton a g i v e n D N A B u t t h i s
is not r eally pr ac t ic able s inc e e a c h g e n e m a y g l v e Indirect autoradiography
r is e t o t wo or m or e RNA t r a n s c r i D t s .A n o t h e r
dr awbac k is t he pr es enc eof e x o n s a n d i n t r o n s . For the detection of highly energetic B-particles
''zs},
(e g. 32P, direct autoradiography is not
DCT-BI-OTTING s u i t a b l e . T h i s i s b e c a u s e t h e s e e m i ssi o n s p a ss
through and beyond the film, and a major part of
Dot-blotting is a modification of Southern and the energy gets wasted.
Nor t her n blot t ing t ec hniques d e s c r i b e d a b o v e . I n
this approach, the nucleic acids (DNA or RNA) are I n d i r e c t a u t o r a d i o g r a p h y i s u s e f ul fo r th e
directly spotted onto the filters, and not subjected d e t e c t i o n o f h i g h l y e n e r g e t i c B - p a r t i cl e s. In th i s
t o elec t r ophor es is The
. hy br id i z a t i o n p r o b e d u r e i s technique, the p-particleenergy is first converted to
t he s am e as in or iginal blot t i n g t e c h n i q u e s l i g h t b y a s c i n t i l l a t o r ,w h i c h t h e n e m i t s ph o to n s o n
e x p o s u r e t o p h o t o g r a p h i ce m u l s i o n .
Dot - blot t ing t ec hnique is p a r t i c u l a r l y u s e f u l i n
obt aining quant it at iv e dat a f o r t h e e v a l u a t i o n o f Applications of autoradiography
gene ex pr es s r on.
A s a l r e a d y d e s c r i b e d ,a u t o r a d i o g r a p h yi s cl o se l y
WESTERN associated with blotting techniques for the
BLOTTING
detection of DNA, RNA and proteins.
Western blotting involves the identification of
proteins. lt is to very useful to understand the COLONY AND PLAQUE BLOTTING
nuc leic ac id f unc t ions , par t i c u l a r l y d u r i n g t h e
c our s e of gene m anipulat ions . Colony and plaque blotting is a process of
hybridization for the specific identification and
The technique of Western brlottinginvolves the purification of colonies (e.9. bacterial clones). This
transfer of electrophoresed protein bands from technique is depicted in Fig.7.10, and briefly
poly ac r y lam ide gel t o ny lo n o r n i t r o c e l l u l o s e d e s c r i b e d h e r e u n d e r .
membrane. These proteins can be detected oy
The desired bacteria are grown as colonies on
s pec if ic pr ot ein- ligand int er a c t i o n s .A n t i b o d i e s o r
lec t ins ar e c om m only us ed f o r t h i s p u r p o s e . a n a g a r p l a t e . Wh e n a n i t r o c e l l u l o s ef i l te r p a p e r i s
overlaid on the agar plate, the colonies get
transferred.They are permanentlyfixed to the paper
AUTOAAD'OGBAPHY
b y h e a t . O n t r e a t m e n tw i t h a l k a l i ( N a O H ) , th e ce l l s
Autoradiography is the process of localization lyse and the DNA gets denatured When these
and recording of a radiolabel within a solid DNA prints are exposed to specific probes
specimen, with the production of an image in a ( r a d i o l a b e l ) , h y b r i d i z a t i o n o c c u r s . T h e h yb r i d
photographic emulsion. These emulsions are complex can be localized and detected by
c om pos ed of s ilv er halide c r y s t a l s s u s p e n d e d r n autoradiography.
gelatin
A similar strategy(describedabove for bacteria;
When a B-particle ot a y-tay from a radiolabet can be used for the identification of phages and
pas s es t hr ough t he em uls io n s , s i l v e r i o n s a r e f r a g m e n t so f p h a g e l i b r a r i e s .
Chapt er7 : B A S ICT E C H N IQU ES
l N C EN ET ICEN GE N E E R IN C l Oi

Double-stranded
DNA
I
Bacterialcolonieson l-

agar plates Single-stranded

DNA(labeled)

Distributedinto 4 tubes

..t / \\

A +G
(Specificbasesdestroyedand fragmentsformed)

I rrugr"nt.separated
byelectrophoresis
I
+
A +G T +C C
Longer
I

A
g

Specificcolony T
detected

Fig. 7.10 : Colony and plaque blotting technique. A

T
Shorter A
Bandson autoradiograph

ATACTGCGACT Seouencedstrand
TATGACGCTGA Complementary
strand
Determination of nucleotide sequencein a DNA
m o lecule is the b as ic and f undam ent al r equir em e n t
in b iote ch no log y .DNA s equenc ing is im por t ant t o Fig. 7.11 : Maxam and Gilbert method for DNA
r-inderstandthe functions of genes, and basis of sequenctng.
inh erite d disord er s .Fur t her ,DNA c loning and gen e
m a nip ula tion in v ar iably r equir e k nowledge o f
accurate nucleotide sequence. C i l bert (1977).Thi s method i s bri efl y descri bed
bel ow (Fi g.7.11).
MAXAM AND G I LBERT TECHNI Q UE
A strandof sourceDNA is labeledat one end
The first DNA sequencing technique, using with 32P. The two strands of DNA are then
chemical reagents, was developed by Maxam and The labeledDNA is distributedinto four
seoai'ated.
1|J2 B IOTECHNO LO CY

s am ples ( in s epar at e t ub e s ) . E a c h s a m p l e i s
subjected to treatment with a chemical that
(n) @o-9Hz Base
(A,C, G,T)
specifically destroys one (C, C) or two bases
( A + C, T + C) in t he DNA. T h u s , t h e D N A s t r a n d s
ar e par t ially diges t ed in f ou r s a m p l e s a t s i t e s C ,
A + C, T + C and C. This r e s u l t s i n t h e f o r m a t i o n
of a seriesof labeled fragmentsof varying lengths.
The actual length of the fragment depends on the
(a) @-cHz Base
site at which the base is destroyedfrom the labeled (A,C, G,T)
end. Thus for instance, if there are C residues at
pos it ions4, 7, and 10 away f r o m t h e l a b e l e d e n d ,
then the treatmentof DNA that specificallydestroys
C will giv e lebeled piec es o f l e n g t h 3 , 6 a n d 9 OH H
bases.The labeled DNA fragmentsobtained in the
four tubes are subjected to electrophoresisside by
side and they are detected by autoradiograph.The Fig. 7.12 : Structure of (A) dideoxynucleotide
triphosphate and (B) deoxynucleotide triphosphate
s eouenc e of t he bas es in t h e D N A c a n D e
(Note the difference at s'-cafuon).
constructedfrom the bands on the electroohoresis.

DIDEOXYNUCLEOTIDE METI.IOD
single-stranded DNA to be sequencedis chosenas
Currently, the preferred technique for a template.lt is attachedto a primer(a shortlength
det er m ining nuc leot ide s eq u e n c e i n D N A i s t h e compl ement art oy a sm all
of D N A ol i gonucl eoti de)
one developed by Sanger (1980). This is an sectionof the template.The 3'-hydroxylgroup of
enzymatic procedure commonly referred to as the the pri meri ni ti atesthe new D N A syn t hesis.
dideoxynucleotide method or chain termination
DNA synthesisis carried out in four reaction
method (Note : Fredrick Sanger won Nobel prrze
tubes.Eachtube containsthe primedDNA, Klenow
twice, once for determining the structureof protein,
subunit(the largerfragmentof DNA polymerase of
ins ulin; t he s ec ond t im e f o r s e q u e n c i n g t h e (ddATP,
E. coli), four dideoxyribonucleotides
nuc leot ides in an RNA v ir u s ) .
ddCTB ddCTP or ddTTP). lt is necessaryto
324 the pri mer or one of t he
A dideox y nuc leot ide i s a l a b o r a t o r y - m a d e radi ol abel(w i th
c hem ic al m olec ule t hat lack s a h y d r o x y l g r o u p a t deoxyri bonucleotides.
both the 2' and 3'carbons of the sugar (Fig. 7.12). As the new DNA synthesisis completed,each
This is in contrast to the natural deoxyribo- one of the tubes containsfragmentsof DNA of
nucleotide that possessesat 3' hydroxyl group on varyinglengthboundto primer.Letus considerthe
the sugar. first reactiontube with dideoxyadenosine (ddATP).
In this tube, DNA synthesisterminates whenever
Termination role of dideoxynucleotide the growing chain incorporated ddA
I n t he nor m al pr oc es s o f D N A r e p l i c a t i o n , a n (complementary to dT on the template strand).
inc om ing nuc leos idet r iphos p h a t ei s a t t a c h e db y i t s Therefore, this tube will contain a seriesof different
5'-phosphategroup to the 3'-hydroxyl group of the length DNA fragments, each ending with ddA. In a
last nucleotide of the growing chain (Refer Chapter smilar fashion, for the other 3 reaction tubes,DNA
3) when a dideoxynucleotide is incorporatedto the synthesis stopsasthe respective dideoxynucleotides
gr owing c hain, no f ur t her r e p l i c a t i o no c c u r s . T h i s i s are incorporated.
bec aus e dideox y nuc leo t i d e , lacking a of new DNA fragmentsin the four
The synthesis
3'-hydroxyl group, cannot form a phosphodiester tubes is depictedin Fig. 7.13.
bond and t hus t he DNA s y n t h e s i st e r m i n a t e s .
The DNA pieces are denaturedto yield free
strandswith radiolabel.The samplesfrom eacn
Sequencing method
tube are sepaiated by polyacrylamide gel
The pr oc es s of sequencing DNA by electrophoresis. techniqueresolves
This separation
dideoxynucleotide method is briefly described. A DNA pieces,differentin size even by a single
 Chapt er7 : B A S T C
T E C H N Ie U ES
tN C E N E T| CE N C E N E E R TN G 103

Template
G C A TC GA A T5'
5',+
\JN Newlysynthesized
DNA*
Fnmer

Reaction tube Primerwith nucleotide Primer with sequence of

with dldeoxynucleotide extended nucleotides extended
ddATP Primer+ 4 Primer-CGTddA

Primer+ 9 Primer-CGTAGCTTddA

ddCTP Primer+ 1 Primer-ddC

Primer+ 6 Primer-CGTAGddC

ddGTP Primer + 2 Primer-CddG

Primer+ 5 Primer-CGTAddG

rHd,'---1
i Primer+ 3 Primer-CGddT
Primer+ 7 Primer-CGTAGCddT
Primer+ 8 Primer-CGTAGCTddT

Fis'7'13: svnthesis
of r*tn" sizeofthe
;";.?-,: J:,',#:";;"':::;"::i;::l:i:",::#::1,:"o|ll"'

nucleotide.The shortestDNA will be the fastest Limitations of dideoxynucleotide

moving on the electrophoresis. method
The sequenceof basesin a DNA fragmentts There are mai nl y tw o l i mi tati ons i n thi s
determined by identifying the electrophoretic techni ques. The need for a si ngl e-stranded DNA
(radiolabeled)bands by autoradiography. In the templateand the use of a primer to an unknown
Fig, 7,14, the sequenceof the newly synthesized seguences. Theseproblemscan be overcomeby
DNA fragrnent that is complementary to the original usi ngbacteri ophage M,,.
DNA piec eis s h o w n .l t' i s c o n v e n ti o n atol re a dthe
bandsfrom bottomto top in 5' to 3' direction'By BACTEBIOPHAGE Mr3 AS A CLONING
noting the order of the bandsfirst C, secondC, AND DNA SEQUENCING VECTOR
third T and so on, the sequenceof the DNA can be
determined accurately.As many as 350 base ' The l i fe cycl e of phage M* i s depi ctedrn
sequencesof a DNA fragment can be clearly Fig. 7.15. This virus contains a single-stranded
ident if iedby us i n ga u to ra d i o g ra p h s . circularDNA. When it infectsE. coli, the double-
strandedD N A i s synthesi zed w hi ch i s a repl i cati ve
Modifications of dideoxynucleotide form (R F).R F D N A repl i catesunti l 50-.100 copi es
method are produced. Now the mode of replicationis
Replacementof 32P-radiolabel by 33p or. 3s5 changed to synthesi ze si ngl e-strandedD N A
improvesthe sharpness of autoradiographic images' (ssD N A )mol ecul es.E achof the ssD N Aof phage
parti cl es
DNA poly m era s e o f th e th e rmo p h i l i cb a c te ri um, M,, i s coatedw i th a protei n.The phage
(in placeof Klenowfragmentof then freely pass through the membrane to be
Thermusaquaticus
released out. Thus, the E. coli cells infected with
E. coli DNA polymerasel) or a modifiedform of
(sequenase) improves M,, can conti nuousl secrete
y i nfecti veparti cl es,
phageT7 DNA polymerase
t he t ec hnique . w i thout undergoi l
ng ysi s.
t04 B IO TECHNO LO CY

ddATP ddCTP ddGTP ddTTP Sequence

3'
Largest
A
-
T
-
rIIt I

-
u
-
E A

T
-
u
-

Smallesl

Fig. 7.14 : Sequence ot the newly synthesized DNA fragment (complementary to original strand).

Bac t er iophageM . ,, is e m p l o y e d a s a c l o n i n g fragments (frequently overlapping), the final

and DNA s equenc e de t e r m i n i n g v e c t o r . T h i s i s seouencecan be bui l t.
possible since the double-strandedreplicative form
c an be is olat ed and us e d a s a p l a s m i d , w h i l e t h e D N A S E QU E N C IN G B Y P R IMER
s ingle- s t r andedphage DN A c a n b e e m p l o y e d a s a W A LK IN G
t em plat e f or DNA s equ e n c i n g . U s u a l l y , a t a r g e t Primerwalkingor primer extensiontechniqueis
DNA wit h les s t han 500 b p c a n b e c l o n e d a n d employed for sequencedeterminationof long
s equenc ed in phage M t . . pi ecesof D N A (> 5,000 bp). I n t his m et hod,
This double- s t r andedR F f o r m s o f p h a g e M ', a r e pl asmi d cl oned D N A contai ni n gt ar get DNA is
isolated and the DNA to be cloned is inserted This anneal ed w i th a pri mer(synthetiocligonucleot ide) .
phage whic h is c or npar a b l et o a p l a s m i d i s r e t u r n e d The ori mer bi nds w i th the D N A close t o t he
to E coli. (The uptake of phageswith insertscan be inserted DNA. Now the tar8et DNA (250-350
screened by using lac Z marker). As the phage nucl eoti des)i s sequenced,most f r equent lyby
undergoes its life cycle (Fig. 7.15), single-stranded dideoxynucleotidemethod (describedalready).
DNA molecules of the insertedDNA are recovered. From the base sequenceof a fragmentof the
Sequenc e det er m inat io n o f t h e s i n g l e - s t r a n d e d target DNA (determinedin the first round), the
DNA can be done by dideoxynucleotide method secondpri meri s chosen.l t i s an ol igonucleot ide to
( Seep. 102) . The pr im er u s e d i s c o m p l e m e n t a r yt o bind to a region approximately300 nucleotides
a par t of phage M , , DN A w h i c h i s c l o s e t o t h e away from first primer binding.The next fragment
point of ins er t ion.Ther efo r e ,a s i n g l e p r i m e r c a n b e of target DNA can be sequencedby another
used for different DNA molecules inserted. 250-300bases(secondround).Thissequence will
For det er m ining t he s e q u e n c e o f a l a r g e r D N A be usefulto choosethe thi rdpri me rand det er m ine
(< 2,000 bp), it is necessary to clone different the next 250-350 bases (thi rd r ound) . I n t his
pieces of the DNA. From the sequences of these fashi on,the pri merw al ki ngi s con t inuedunt il t he
 l N C EN ET ICE N C E N E E R IN C
Chaot er7 : B A S ICT EC H N IO U ES l O5

-l
lol
-(_

f 11 I Phage
L_"J
Phage E. coli

lnfe

Single-
strandedDNA
oooo
o o o oo
oo

o oo
oo o
oo o f-\-

Single-
strandedDNA 50-1 00 copies
(double-stranded
DNA)

Fig. 7,15 : The life cycle of bacteriophage Mts.

complete target DNA is sequenced. Three rounds o f t h e m a r k e r .T h i s i n i t i a l p r o b e w i l l h y b r i d i z e o n , l y

of prime r wa lkin g t ec hnique f or DNA s equenc i n g with the clones containing fragment A. This
are depicted in Fig. 7.16. fragment A can be isolated, cloned and used as a
probe to detect fragment B. This procedure of
CHROMOSOME WALKING IN DNA cloning and probing with fragments is repeated
SEQUENCING again and again until fragment D hybridizeswith
fragment E.
Chro mosomewalk ing is a DNA bas es equenc i n g
me tho d in wh ic h t he c hr om os om e is analy s ed b y As is evident from the above description,
extending one tip to reach the other. The technique chromosome walking uses probes derived from the
basically involves systematicallymoving along the ends of overlapping clones to facilitate a walk
chromosome from a known location to an along with the DNA sequence. ln the processof
unknown location, and thus determining the DNA this walk, the sequence of the desired target gene
sequence. The method adopted in chromosome can be identified.
walking is depicted in Fig. 7.17, and briefly
described below. Ghromosome iunnping

A DNA fragment with approximately 40,000 An improved strategyof chromosome walking is

base pairs can be sequenced.To start with a marker
is used to identify the appropriateSene probe (from
the DNA probe library). This gene probe must have
a section of the base pair identical to the base pairs
chromosome iumping. Many regionsof DNA that
are difficult to be cloned by walking can be
j u m p e d . T h e p r o c e d u r e i n v o l v e st h e c i r c u l a r i z a t i o n
of large genbmic fragments generated by the
106 B IOTECHNO LO CY

P1

(A) l-T-lt ,
Plasmid ClonedDNA
DNA

P2

Fig. 7.16 : Primer walking technique for DNA sequencing (A) First round (B) Second round (C) Third
round (P1, Pr, Pr-Primers 1, 2, 3).

digestionof endonucleases. This is followed by (A)

cloning of the region loweringthe closureof the Linked Linked
marker2
fragment.By this approach,the DNA sequences marker1
Iocated at far off places can be brought together I I
+
and cloned. A chromosomejumping can be
constructedin this manner and used for long Targetgene
distancechromosomewalks. Intact DNA fragment

Applications of chromosome walking

and jumping
The gene responsiblefor the diseasecystic
fibrosis (CF) has been identified by chromosome
w a l k i n g a n d j u m p i n g . The gene of protei n
re s p o n s i b l efo r C F i s cal l ed cysti c fi brosi s ourlrm
transmembrane conductanceregulator(CFTR)and
is locatedon the long arm of chromosome7.

AI' T O MA T ED D N A S EQU E N C IN G
DNA sequencingin the recentyearsis carried
out by an automated DNA sequencer.In this Fig. 7.17 : (A) Chromosome walking
technique,flourescenttags are attachedto chain- (B) Chromosome iumping.
terminatingnucleotides(dideoxynucleotides).This
 l N C EN ETICE N C E N E E R IN C
Chapt er7 : BA SICT EC H N IQ U ES lo7

tag gets incorporated into the DNA molecules, {- DNAtemplate

wh ile te rminat ing new s t r and s y nt hes is . F o u r
differentfluorescentdyes are used to identify charn-
termin atin g r eac t ions in a s equenc ing gel . T h e
DNA bands are separated by electrophoresisand Nucleotidase
detected by their fluorescence.Recently,four dyes dATP Degraded
tha t exhib it st r ong abs or pt ion in las erar e in u s e f o r +
automated sequencing. Nucleotidase
dGTP---------------- Deqraded
Advantages of automated sequencing : lt is a
ra pid a nd ac c ur at e t ec hnique. Aut om at ed D N A
seq ue ncer can ac c ur at ely s equenc e up t o 1 0 0 , 0 0
nucleotides per day. The cost works out to be not
more than $0.2 per nucleotide. Automated DNA
sequencing has been successfully used in the
Nucleotidase
human genome project, dTTP ----------+ Degraded

Some groups of research workers have

developedalternatemethodsto Sangermethodfor
sequencingof DNA. Unfortunately,despite the
initial excitement,most of these methods have
disappeared from the scene.Thereare at leasttwo Fig. 7.18 : The technique of pyrosequencing.
m et hodswit h s o mep ro m i s efo r D N A s e q u enci ng-
pyrosequencing,and genechips (microarrays).
nucleotide is incorooratedinto the new DNA
P Y RO S E Q U EN C IN G strand,a moleculeof pyrophosphate is released.
This can be convertedby the enzymesulfurylase
P y r os eq u e n c i ni sg a D N A s e q u e n c i n gm ethod
i nto a fl ashof l i ght (chemi l umi nescence).B y the
that is basedon the principleof determiningwhich additionof
eachdNTPseparately, one afteranother,
one of the four bases(A, G, C, T) is incorporated the order of the nucleotidesaddedto the growing
at each step while'a DNA template is copied. ln DNA strandcan followed,and the
be sequence can
, e te m p l a tec o p i e s i n a strai ght be identified.
t his t ec hniq u e th
forward manner whithout added dideoxy-
nucleotides (ddNTPs).As the new strand is Detectionof the molecule pyrophosphateis the
synthesized,the order in which the deoxy- basis of DNA sequencing, hence the name
nucleotides(dNTPs)are incorporatedis detected, pyrosequencing.Since pyrosequencingdoes not
and the seouencecan be read as the reaction requireelectrophoresis or any other DNA fragment
proceeds(Fig. 7.18). The identificationof base separationtechnique,it is more rapid than chain
additionbecomesoossiblesincethe additionof a termi nati on sequenci ng.
nucleotideis accompaniedby the releaseof a
The major limitation of pyrosequencingis that it
moleculeof pyrophosphate. This can be detected
is suitable to detect the seguenceof ahout 200
by c hem ilumi n e s c e nte c ec h n i q u e .
nucleotides.This is much less than the Sanger
In the procedureof pyrosequencing, eachdNTP method. Many improvementsare being made
al ong in pyrosequencingso that much longer DNA
is addedind i v i d u a l l y(n o t a l l fo u r to g e th e r ),
with a nucleotidasb enzyme.Thisenzymedegrades moleculescan be sequenced. In fact,an automated
the dNTP if it is not incorporatedinto the newly system for pyrosequencingis also available
synthesizedDNA strand. Once the appropriate now.
r08 B IOTE CHNO LO CY

DNA CH|PS (MTCROARRAYSI

D N A c h i p s o r D N A mi croarraysare recent
developmentsfor DNA sequencingas result of
a d v a n c em s a d ei n a u to ma ti o and A
n mi ni ari zati on.
l a rg e n u m b e r o f D N A p ro bes,each one w i th
d i ffe re n ts e q u e n c e a, re i m m obi l i zedat defi ned
p o s i ti o n so n th e s o l i d s u rfa ce,made up of ei ther
nylon or glass.The probes can be short DNA
Hybridization
molecules such as cDNAs or synthetic oligo-
nucleotides.
For the preparationof high density arrays,
oligonucleotidesare synthesizedin situ on the
s u rfa c eo f g l a s s o r s i l i c o n . Thi s resul tsi n an
oligonucleotidechip ratherthan a DNA chip.

Technique of DNA sequencing

A D N A c h i p c a rry i n g an array of di fferent
o l i g o n u c l e o ti d ec sa n b e u s e dfor D N A sequenci ng.
For this purpose,a fluorescentlylabeledDNA test
molecule,whose sequenceis to be determined,ls
a p p l i e dto th e c h i p . H y b ri d i zati on occursbetw een
the complementarysequencesof the test DNA
of the chi p. The Hybridizing
m o l e c u l ea n d o l i g o n u c l e o ti des signals
p o s i ti o n so f th e s e h y b ri d izi ngol i gonucl eoti des
can be determined by confocal microscopy.
Ea c h h y b ri d i z i n go l i g o n u cl eoti derepresentsan
B-nucleotidesequencethat is presentin the DNA I
probe.The sequenceof the testDNA moleculecan I
be deduced from the overlaps between the T
s e q u e n c e so f th e h y b ri d i zi ng ol i gonucl eoti des AGTCCCTT-\
(Fig. 7.1e). OTCCCTTO\ Hybridizing
- (8)
fCCCrree\ligonucleotides
Applications of DNA chips CCCTTGGC'
- "' AGTCCCTTGGC - ^- DNAsequence
There have been many successeswith this
relativelynew technologyof DNA chips.Someof Fig. 7.19 : Microarray (or chip) technology in
them are listed. DNA sequencing.
ldentification of genes responsible for the
development of nervous systems.
. Rapid detection of microorganisms for
Detection of genes responsiblefor inflammatory e n v ir o n m e n t a l m o n i t o r in g .
d iseases.
The future of DNA chips
Constructionof microarraysfor every gene in the
genome of E. coli, and almost all the genes of the The maj orl i mi tati onof D N A chi psa t pr esentis
yeast Saccharomyces cerevisi ae. the unavailabilityof completegenomearraysfor
hi ghereukaryotes, i ncl udi nghumans.l t is expect ed
Expressionof several genes in prokaryotes has
thatw i thi nthe nextfew yearssuchD N A chipswill
been ident if ied. t os
Thi sw i l l hel p the bi otechnologist
be avai l abl e.
Det ec t ion and s c r eening o f s i n g l e n u c l e o t i d e capturethe functionalsnapshots of the genomein
poly m or phis m s ( SNPs ) . action for higherorganisms.
 l N C EN ETICE N C E N E E R IN C
Chapt er7 : B A SICT EC H N IQ U ES t09

DMT-O

Advances in the Iaboratorvtechniqueshave

m ade it pos s i b l eto c h e m i c a l l ys y n th e s i zD e NA in
a s hor tper iod T o sf a b o u t100
. h u s ,o l i g o n u c l e o ti d e
bas es c an b e p ro d u c e d i n a b o u t 10 h o urs.
Laboratorysynthesisof DNA (recentlyby use of
DNA synthesizersor gene machines)with specific
s equencof e nu c l e o ti d e sra, p i d l ya n d i n e x p e nsi vel y,
has s ignif ic a n tlcyo n tri b u te d to c l o n i n g .C h e mi cal
synthesis of DNA is basedon the abilityto protect
t he r eac t iv e - 5 ' a n d - 3 ' e n d s b y b l o cki ng Fig. 7.20 : Structure of a phosphoramidite
(protecting) them. (DMT-Dimethoxytrityl; Me-Methyl)

T HE P HO S P H O R AMID IT E M ET H O D
Another nucleotide precursor is now added and
T hephos p h o ra mi d im tee th o di s th e te c h n i queof the reactions described in steps 3 and 4 are
choice,currentlyin use,for the synthesis of DNA reDeated.
The synthesis is carriedout on a solidphasesystem
Coupling, oxidation and deprotection are the
g e g ro w i n gD N A s tra n dto a sol i d
i. e.by at t ac h i n th
three steps in the cvclic reaction for the DNA
s uppor t , in a re a c ti o n v e s s e l .T h e p ro c edure
s y n t h e s i sb y p h o s p h o r a m i d i t e m e t h o d . T h e c y c l e s
involvesthe following stages.
are repeated again and again to synthesise the
.l : desired DNA. At the end, the completed
. Nucleosideattachmentto solid support
The initial nucleoside (base + sugar) is attached at o ligonucleotide is removed from the glasssupport
3' end t o an i n e rt s o l i d s u p p o u
rt, s u a l l yto gl ass and the groups protectingthe basesand phosphates
beadswith uniformpores,referredto as controlled are cleaved
pore glass(CPC)beads.
Applications of synthesized
2. Preparationof phosphoramidites : For each oligonucleotides
one of the four bases (A, C, C and T),
phos phor am i d i tecsa n b e s y n th e s i z e dT. h i s i s C h e m i c a l l y s y n t h e s i z e do l i g o n u c l e o t i d e sh a v e a
(DMT)group number of uses in biotechnology.
achievedby attachingdimethoxytrityl
to the 5'-hydroxyl group of deoxyribose and a 1. The linkers and adaptors are the
g roup
diis opr opy la m i ngero l p to th e 3 ' -p h o s p h i te oligonucleotides commonly employed in the
of the nucleoside. A methylresidueprotectsthe 3'- preparation of recombinant DNA (Chapter 6).
phosphite group. The general structure of
phosphoramidite is depictedin Fig. 7.20. 2. Chemically synthesizedDNAs with known
sequencescan be used for the synthesisof proteins/
3. Coupli n g : A n u c l e o ti d ep re c u rs o r(i.e. a polypeptides.
phos phor ami d i te w)i th i ts 3 ' -p h o s p h o rucso upl es
3 . D N A p r o b e s , p a r t i c u l a r l yt h e s i n g l e - s t r a n d e d
e o u ndto
wit h 5' - hv dr o x vol f th e i n i ti a ln u c l e o s i d b
oligonucleotide probes, can be synthesized in the
a CPC bead.
laboratory.This is based on the codon sequenceof
4 Oxidation and deprotection: The unstable mRNA which in turn is dependent on the amino
trivalentphosphiteis oxidizedto pentavalentstable acid sequence
phosphate. This is followedby the removalof DMT
4 . S i n g l e - s t r a n d e do l i g o n u c l e o t i d e sa r e u s e d a s
that protects(deprotection) 5-hydroxylgroup. For
p r i m e r s i n p o l y m e r a s ec h a i n r e a c t i o n ( P C R ) .
conven ience, oxidation and deprotection are
consideredtogether in Fig. 7.21 also, they are 5. For in vitro mutagenesis, single-stranded
actuallvtwo independentreactions. o l i g o n u c l e o t i d e sa r e e m p l o y e d .
 B IOTE CHNO LO CY
rto

Base1

oH
I
O cPG bead
to a bead
lnitialbaseattached

OH

H\H

HO-QH2 Base2

H
H H/H

N4eo-il-o-cH2
ct;

oH
I
(cPG-Controlled porc gtass;
Fig. 7.21 : Chemicat synthesisof DNA by phosphoramidite method
DMT-DImethoxytrityl; Me-Methyl; N(iP) t-Diisopropylamine)'
 l N C EN ET ICE N C E N E E R IN C
Chapt er7 : B A S ICT E C H N IQU ES ttt

OH OH P 5ott

OH OH OH

Jnnnearino

T4 DNA
ligase

Fig. 7.22 : Chemical synthesis of a gene.

S Y NT HE S I S O F GE N E S A nother w ay of synthesi zi nggenes i s to

produceoverl appi ngol i gonucl eoti des w hi ch on
Cene is a double-stranded DNA; two single-
anneal i ngcontai nl argegaps.Thesegapscan be
strandedcomplementaryoligonucleotides can be
fi l l ed by enzymati csynthesi sof D N A by D N A
synthesized, separately, and on annealingthey form pol ymerase-w hi l e synthesi zi nggenes i n the
double strands.This can be convenientlyachieved laboratory,utmost care should be taken to see
for smaller genes (60-90 bp). However, the that the nucl eoti desare i n the correctsequence
synthesisof longergenes(> 300 bp) is associated (thi s can be checked by D N A sequence
wit h s om epr ac ti c adl i ffi c u l ti e sT.h i s i s m a i n l yd u e techni que).
to the fact that the coupling efficiencyfor the
chemical synthesisof DNA is never 100%. To An lndian born and Americansettledscientist
overcomethis problem,small DNA fragments are Har Cobind Khoranaand his associates were the
synthesizedand assembled (Fig. 7,22). Sealing of first to synthesize an entire tRNA gene for yeast
the nicks is achieved by use of T4 DNA ligase. al anyl IR N A i n 1972.
- l- h" poly m er as e c hain r e a c t i o n ( P C R ) i s a
I laboratory \in vitro) technique for generating
large quantities of a specified DNA. Obviously,
PCR is a cell-free amplification technique for
s y nt hes iz ingm ult iple ident ic a l c o p i e s ( b i l l i o n s ) o f
any DNA of int er es t .Dev elop e d i n 1 9 8 4 b y K a r r y
M ullis ( Nobel laur at e, 199 3 ) , P C R i s n o w
c ons ider ed as a bas ic t ool f o r t h e m o l e c ur a r
biologis t . As is a phot oc opier a b a s i c r e q u i r e m e n t
in an of f ic e, s o is t he PCR m a c h i n e i n a m o l e c u l a r
biology labor at or y !
Pr inc iple of PCR
The double- s t r anded DN A o f i n t e r e s t i s
denat ur ed t o s epar at eint o t w o i n d i v i d u a l s t r a n d s
Eac h s t r and is t hen allowed t o h y b r i d i z e w i t h
a pr im er ( r enat ur at ion) . T h e p r i m e r - t e m p l a t e
duplex is us ed f or DNA s y n t h e s i s ( t h e e n z y m e -
DNA poly m er as e) . Thes e three steps-
denaturation, renaturation and synthesis are
r epeat edagain and again t o ge n e r a t em u l t i p l e f o r m s
of target DNA.
TECHNI Q UE O F PCR
The es s ent ial r equir em ent sf o r P C R a r e l i s t e d
below
.l
A t ar get DNA ( 100 35, 0 0 0 b p i n l e n g t h )
2 Two pr im er s ( s y nt het ic o l i g o n u c l e o t i d e s o f
17- 30 nuc leot ides lengt h) t hat a r e c o m p l e m e n t a r y
t o r egions f lank ing t he t ar get D N A .
112
3 . F o u r c l e o x y r i b o n u c l e o t i d e s ( d ATR d C TR
dCTP, dTTP).
4 . A D N A p o l y m e r a s et h a t c a n w i t hsta n d a t a
t e m p e r a t u r eu p t o 9 5 " C ( i . e , t h e r m o s t a b l e ) .
T h e r e a c t i o n m i x t u r e c o n t a i n s t h e t ar g e t D N A,
t w o p r i m e r s ( i n e x c e s s ) , a t h e r m o s ta b l e D N A
pcrlymerase(isolated from the bacterium Therntus
a q u a t i c u s ( i e . , T a q D N A p o l y m e r a s e) a n d fo u r
d e o x y r i b o n u c l e o t i e sT
. h e a c t u a l t e c h n i q u e o f PC R
i n v o l v e s r e p e a t e dc y c l e s f o r a m p l i f i c a t i on o f ta r g e t
DNA. Each cycle has three stages
.l
Denaturation : On raisingthe temperatureto
a b o u t 9 5 o C f o r a b o u t o n e m i n u t e , t h e D N A g e ts
denatured and the two strands separate.
2. Renaturation or annealing : As the
t e m p e r a t u r e o f t h e m i x u t r e i s s l o w l y co o l e d to
a b o u t 5 5 'C , t h e p r i m e r s b a s e p a i r w i th th e
c o m p l e m e n t a r y r e g i o n s f l a n k i n g t a rg e t D N A
s t r a n d s . T h i s o r o c e s s i s c a l l e d r e n a tu r a ti o n o r
a n n e a l i n g H i g h c o n c e n t r a t i o n o f p r i m e r e n su r e s
a n n e a l i n g b e t w e e n e a c h D N A s t r a n d a n d th e
p r i m e r r a t h e r t h a n t h e t w o s t r a n d so f D NA.
3 . S y n t h e s i s: T h e i n i t i a t i o n o f D N A syn th e sr s
o c c u r s a t 3 '- h y d r o x y l e n d o f e a c h p r i m e r . Th e
p r i m e r s a r e e x t e n d e d b y j o i n i n g th e b a se s
c o m p l e m e n t a r y t o D N A s t r a n d s . T h e syn th e tr c
p r o c e s s i n P C R i s q u i t e c o m p a r a b l e t o th e D N A
r e p l i c a t i o n o f t h e l e a d i n g s t r a n d ( R e f e rCh a p te r 3 ) .
However, the temperaturehas to be kept optimal as
r e q u i r e d b y t h e e n z y m e D N A p o l y m e r a se Fo r Ta q
 C hA P I CT
B : P O L Y M ER AS (D N A A MP LIFIC A TION )
CEH A IN R EA C T ION 113

90
t I Denaturation
J Primerannealing

c
L
g- 80 E
o
1
svntnesis
(g
Jorun
- Originalstrand
ii zn
.(
E -{ ')Long template
,o)
---+...-...F
60 Originalstrand

2345
IJ
t ....-..<--- Longtemplate
Time(minutes)

Fig. 8.1 : The three stages in each cycle of PCR in L

temprates
relation to temperature and time E
).n".
(Each cycle takes approximately 3-5 minutes). 2
L ----+.....-.. Longtemplate

DNA polymerase,the optimum temperatureis

around 75' C (for E. coli DNA polymerase,it is
around 37" C). The reactioncan be stoppedby
raisingthe temperature(to about 95" C).
The 3 stagesof PCRin relationto temperature
and time are depictedin Fig. 8.1. Eachcycle of
P CR t ak es abo u t 3 -5 mi n u te s . In th e n o rm al
practice,the PCR is carriedout in an automated <--
m ac hine. _____________-f

As is evidentfrom the Fig.8.2 (cyclel), the new L

DNA strandjoined to each primer is beyondthe {-

sequencethat is complementaryto the second
--______-|
primer.Thesenew strandsare referredto as long
templatesand they will be used in the second
cy c r e.
For the secondcycle of PCR,the DNA strands
(original+ newly synthesizedlong template)are
denatured,annealedwith primersand subjectedto
DNA synthesis. At the end of secondround, long Fig. 8.2 : The polymerase chain reaction (PCR)
templates,and short templates(DNA strandswith representing the initial three cycles
(- i11flisqtsprimers)
primer sequence at one end, and sequence
complementary to the otherend primer)areformed.
I n t he t hir d c y c l e o f PC R ,th e o ri g i n a l D N A cycle. lt is estimatedthat at the end of 32nd cycle
strandsalongwith long and shorttemplatesare the of PCR, about a million-fold target DNA is,
startingmaterials.The techniqueof denaturation, synthesized(Table 5.1). The short templates
renaturationand synthesisare repeated.This possessing preciselythe target DNA as double-
procedureis repeatedagain and again for each strandedmol ecul esaccumul ate.

Biotechnology[8]
114 B IOTECHNO LO CY

SOURCES OF DNA POLYMERASE

Tlau 8.1 Theorctlcal anpllflcation of
In the ori gi nal techni que of P CR, Klenow
target DI{A by polymerase chain reaction
fragment of E . col i D N A pol ymerase
w as used.This
Cycle number Number of double- enzyme, gets denaturedat higher temperature,
strandedtarget DNA therefore,fresh enzvme had to be added for
.l
eachcycle.A breakthrough occurred(Lawyer 989)
1 0 with the introductionof Iaq DNA polymerase from
2 0 thermophilic bacterium, Thermus aquaticus. Ihe
Taq DNA polymerase is heat resistant, hence it is
z
not necessaryto freshly add this enzyme for each
4 4 cycle of PCR.
( 8 KEY FACTORS FOR OPTIMAL PCR
6 16 P ri mers: P ri merspl ay a si gni ficantr ole in
7 J1
determi ni ngP C R .The pri mers(17-30 nucleot ides)
without secondary structure and without
8 64 compl ementari tyamong themsel ve sar e ideal.
I 128 The complementary primerscan hybridizeto form
primer dimer and get amplified in PCR. This
10 256 preventsthe mul ti pl i cati on
of targetDNA.
1l 512 DNA polymerase: As alreadydescribed,Iaq
12 1024 DNA polymeraseis preferredas it can withstand
high temperature.In the hot-start protocof DNA
13 2048 polymeraseis added after the heat denaturation
14 4096 stepof the first cycle.This avoidsthe extensionof
the mi smatched pri mersthat usual l yo ccur at low
l6 8192
temperatLrre
to 16,384 Iaq polymerase lacksproofreadingexonuclease
17 32,768 (3'-5')activitywhich might contributeto errorsin
the products of PCR. Some other thermostable
18
DNA polymerases with proof-reading
activityhave
19 131,072 been identifiede.9., Ima DNA polymerasefrom
20 242 144 Thermotogamaritama;Pfu DNA polymerasefrom
Pyrococcusfuriosus.
zl 524,288
TargetDNA : In general, the shorterthe sequence
zz 1,048,576 of targetDNA, the betteris the efficiencyof PCR.
2,097j52 However, in recent years, amplification
ZJ
of DNA fragments upto10 kb hasbeenreported. The
24 4,194,304 sequenceof targetDNA is also importantin PCR.
25 8,388,608 Thus,C C -ri chregi onsof D N A strandh inderPCR.
Promotersand inhibitors: Additionof protetns
26 to,t I I,zto
suchas bovi neserumal bumi n(B S Aenhances
) PCR
27 33,544,432 by protectingthe enzymeDNA polymerase. Humic
28 67,108,864 acids,frequentlyfound in archeological samplesof
targetD N A i nhi bi t P C R .
29 134,217,728
30 268,435,456
31 536,870,912
T h e b a s i c t e c h n i q u e o f the PCR has been
1,073,741,824
described. Being a versatile technique,PCR is
 CHAIN REACTION(DNA AMPLIFICATION)
ChapterB : POLYMERASE 1t5

Target
DNA UndesiredDNA
rl
,+
ll
J PCR with
first primers

-t ; +l
PCRwith
secono

I
pnmers

The secondprimers
do not bind to undesired
DNA. hence no PCR occurs

Fig. 8.3 : Nested polymerase chain reaction.

modifiedas perthe specificdemandsof the situation. now cut with another restrictionenzyme. This
Thus,thereare manyvariationsin the originalPCR, cleaves only the known sequence.The target
someof them are discussed, hereunder. DNA so formed contains the known sequence
at both the ends"withtargetDNA at the middle.
NESTED PCR The PCR amplification can now be carried
out. lt may be notedthat the primersare generated
Sequencesimilaritiesbetweenthe target DNA in the oppositedirectionto the normal,sincethe
and relatedDNA are very frequentlyseen.As a ori gi nalsequence i s i nvertedduri ngci rcul ari zati on.
result of this, the primersmay bind to both the
DNAs and thereforeeven the undesiredDNA also ANCHORED PCR
gets amplified in PCR. Use of nested primers
In the anchoredP C R , a,smal l sequenceof
increasesthe specificityof PCR, and selectively
nucleotidescan be attached(tagged)to the target
amplifiestargetDNA. NestedPCRis illustratedin
D N A i .e.,the D N A i s anchored. Thi si s parti cul arl y
Fig. 8.3. In the first cycle of PCR,the productsare
usefulwhen the sequenceSurrounding the target
both from target DNA and undesiredDNA. A
DNA is not known.The anchoris frequentlya poly
secondset of internalprimersis now used.They
C tai l to w hi ch a pol y C pri mer i s used. The
will selectively bind to target DNA and
anchoringcan alsobe done by the useof adaptors.
amplificationproceeds.
As the adaptorspossessa known sequence,the
orimercan be chosen.
INVERSE PCR

ln the inversePCR,amplificationof DNA of the REVERSE TRANSCRIPTION PCR

unknownsequences is carriedout from the known
sequence(Fig, 8.a1. The target DNA is cleaved
with a restriction
endonuclease which doesnot cut
the known sequence but cuts the unknown
sequenceon either side. The DNA fragments
so formed are inverted and get circularized
(DNA ligase is employed as a sealing agent).
The circle containing the known sequencesis
The PCRtechniquecan also be employedfor
the ampl i fi cati onof R N A mol ecul esi n w hi ch
caseit is referredto as reversetranscription- PCR
GT-PCQ. For this purpose, the RNA molecule
(mRNA)must be first convertedto complementary
DNA (cDNA) by the enzyme reversetranscriptase.
The cDNA then servesas the templatefor PCR.
Differentprimerscan be employedfor the synthesis
116 B IOTECHNO LO CY

fl uorescence compoundl i ke ei thi di umbr om ide.The

principleis that the double-stranded DNA molecules
bi nd to ethi di umbromi dew hi ch emi t fluor escence
DNA with knownsequence(in colour) that can be detected,and DNA quantified.

Restriction The synffiesisof genesby PCRand the role of

enoonuclease PCR in site-directed mutagenesisare described
elsewhere(ReferChapter10).

RANDOM AMPLIFIED POLYMORPHIC

I DNA (RAPDI
Normally,the objectiveof PCR is to generate
defined fragmentsof DNA from highly specific
primers.In the caseof RAPD(pronounced as rapid),
@ short oligonucleotide primers are arbitrarily
selected to amplify a set of DNA fragments
throughout
randoml ydi stri buted the ge nom e.This
techni que,randomampl i fi edpol ymorphicDNA is
also known as arbitrarily primed PCR(AP-PCR).
The procedureof RAPD is comparableto the
generaltechni queof P C R .Thi s meth odbasically
i nvol ves the use of a si ngl e pri m er at low
stri ngency.A si ngl eshortol i gonucl eo t ide ( usuallya
9-10 base pri mer) bi nds to many sit es in t he
Primer genomeand the DNA fragments are amplifiedfrom
r----+ them. The stri ngencyof pri mer bi nding can be
increasedaftera few PCRcycles.This allows the
{-r Primer amol i fi cati onof best mi smatches. R APD can be
careful l ydesi gnedso that i t fi nal l yyi e ldsgenom e-
Fig. 8.4 : lnverse PCR. specific band patterns that are useful for
comparati veanal ysi s. Thi s i s possible since
genomicDNA from two differentindividualsoften
of f ir s t s t r and of c DNA. The s e i n c l u d e t h e u s e o f producesdifferent amplified patternsby RAPD.
r andom pr im er s , oligo dT pr i m e r a n d a s e q u e n c e Thus, a particularDNA fragmentmay be generated
specific primer (Fig. 8.5). for one i ndi vi dualand not for the other ,and t his
represents D N A pol ymorphi sm w hi ch can be used
ASYMMETRIC PCF as a geneticmarker.
PCRt ec hnique c an als o be u s e d f o r t h e s y n t h e s i s
of s ingle- s t r anded DNA r n o l e c u l e s , p a r t i c u l a r l y
(A) AAAAA mFINA
us ef ul f or DNA s equenc in g ( C h a p t e r 7 ) . l n t h e
{--<-- <-r{--{--
as y m m et r ic PCR, t wo pr im er s i n a r a t i o o f 1 0 0 : 1 cDNA
are used. After 2O-25 cycles of PCR, one primer is
(B) AAAAA mRNA
exhausted.The result is that in the next 5-1 0 PCR
T T TTT cD N A
cycles, only single-strandedDNAs are generated

REAL.TIME QUANTITATIVE PCR (c) AAAAA mRNA

The quantification of PCR products in different
cycles is not as simple as projected by thereotical
considerations (Table 8.1). In practice, large
v ar iat ionsoc c ur .The m os t c om m o n l y u s e dt e c h n i q u e
f or m eas ur ingt he quant it y of P C R i s b y e m p l o y i n g a
CDNA
Fig, 8.5 : Synthesis of first strand of zDNA in reverse
transcription-PCR with different primers (A) Random
primers (B) oligo dT primer (C) Sequence specific
primer (No,te : The primerp ale shown in colour),
 CHAIN REACTION(DNA AMPLIFICATION)
ChaoterB : POLYMERASE 117

RA P D is wi d e l y u s e d b y p l a n t m o l e c u l ar s / - - - - G A A T T C T T A A- - - - 3/
biologists for the genetic identification of plant 3/----CTTAAG A A T T- _ _ _ 5 /
species (ReferChapter53).Forthispurpose, different GenomicDNA
cornbinations of nucleotides, mostof them random II
oligonucleotide primershavebeendesignedand are I Restrictionendonucleases
. s e a c h ra n d o m p ri mer
com m er c iallyav a i l a b l e A | (EcoRl and Msel)
+
annealsto a differentregionof DNA, manydifferent AATTC T
regionsof loci on the DNA can be identified.RAPD
is thus usefulfor the construction of geneticmaps
and as a m et ho dfo r g e n o mi cfi n g e rp ri n ti n g .

Limitations of RAPD
The main problemof RAPD is associated with
reproducibility. lt is oftendifficultto obtainsimilar
levelsof primerbindingin differentexperiments. lt Additionof first
is thereforedifficultto correlateresultsobtainedby orimerwithone
selectivenucleotide
differentresearchgroupson RAPD. 5'#A
AATTCT CTTA-
A M P LI F I E D F R A G ME N T L E N GT H
POLYMORPHTSM (AFLPI TTTAAGA GAAT
c +5 ,
AFLP is a very sensivemethod for detecting
p oly m or phis min th e g e n o me .l t i s b a s e do n the Additionof second
primerwiththree
p r inc iple of r e s tri c ti o nfra g m e n t l e n g th p o l y- selectivenucleotides
m or phis m( RF L BR e fe rC h a p te r1 4 ) a n d R A PD . 5.-AAC
AFLPmay be appropriately regarded as a diagnostic r AATTCT CTTA-
fingerprintingtechnique that detects genomic TTAAGA GAAT-
restrictionfragments. -
I AAo sN
I n t he A F LP , PC R a mp l i fi c a ti o nra th e r th an
I selective
Southernblotting(mostlyusedin RFLP)is usedfor I amplification
+
the detectionof restrictionfragments.lt may be AACTTA-
notedthatAFLPis employedto detectthe presence -AATTCTTG
or absenceof restrictionfragments,and not the TTGAATE
-TTAAGAAC
lengths of these fragments. This is the major
I
I

differencebetweenAFLPand RFLP.AFLP is very

+
Gel electrophoresis
widely used in plant genetics.lt has not proved
u s ef ulin t he m a p p i n go f a n i ma l g e n o m e ss, i n ce Fig. 8.6 : The technique of amplified fragment
this techniqueis mainly basedon the presenceof length of polymorphism (AFLP).
high ratesof substitutional variationswhich are not
found in anim al sOn . th e o th e rh a n d ,s u b s ti tu ti onal
var iat ionsr es ulti n gi n R F L Psa re m o re c o m m o ni n fragments are ligatedwith EcoRlandMseladaptors.
olant s . These common adaptor sequences (flanking
T he bas ic p ri n c i p l e o f A F L P i n v o l v e s th e genomi csequences) serveas pri merbi ndi ngsi tes
a m plif ic at ionof Su b s e tso f R F L Psu s i n g P CR on the restriction fragments. The DNA fragments
(Fig. 8.6). can be ampl i fi edw i th A FLPpri merseach havi ng
only one selectivenucleotide.ThesePCRproducts
A genomic DNA is isolated and digested
are dilutedand usedas temolatesfor the selective
simultaneouslvwith two different restriction
ampl i fi cati onempl oyi ngtw o new A FLP pri mers
endonucleases - EcoR/ with a 6 base pair
that have 2 or 3 selectivenucleotides.
recognitionsite and Msel with a 4 base pair
recognitionsite.Thesetwo enzymescan cleavethe After the selectiveamplificationby PCR, the
D NA and r es ulti n s ma l lfra g me n ts (< 1 k b ) w h i ch DNA productsare separated on a gel.The resultant
c an be am plif iedb y PC R .F o rth i sp u rp o s e th e D N A DNA fingerprintis identifiedby autoradiography.
118 B IOTECHNO LO CY

AFLPfragmentsrepresentuniquepositionsin the 3 'm R N A

genomes,and hencecan be usedas landmarksto
bridgethe gapsbetweengeneticand physicalmaps
of genomes.In plants,AFLP is usefulto Benerate
high densitymaps,and to detectgenomicclones. Il
I Reverse
RAPID AMPLIFICATION OF cDNA ENDS I transcriptase
(R A C E I +
5/-€'
As a l re a d y d e s c ri b e d( S ee p. 115), reverse 3'fi-r5' ,
transcription, followed by PCR(RT-PCR) resultsin cDNA(
./' I
|
th e a mp l i fi c a ti oonf R N As e q uences i n cD N A form. ) lDenature
But the major limitationof RT-PCRis relatedto E-'-- |
L+
i n c o mp l e te D N A s e q u e n c es in cD N A .Thi sprobl em Qr -
\
"
i s s o l v e db y u s i n gth e te c h n i querapi dampl i fi cati on NNNN
of cDNA ends. RACEis depictedin Fig. 8.7, and I AdddATP
brieflydescribedbelow. | + terminal
transferase
+
ThetargetRNA is convertedinto a partialcDNA 3'AAAAANNNN
b y e x te n s i o no f a D N A p ri mer.Thi s D N A pri mer I Anneal to
I ancnorpnmer
was first annealedat an intervalpositionof RNA, +
not too far from the S'-endof the molecule.Now 5' NNNNNNTTTTT 3'
additiondATP(As) and terminaldeoxynucleotidyl AAAAANNNN
transferase extendsthe 3'-end of the cDNA. This 5' 3',
happensdue to the additionof a seriesof As to the
A
cDNA. TheseAs seriesnow act as the orimer to I_
annealto the anchor primer.A secondstrandof I
I Secondstrand
DNA can be formed by extendingthe anchor I DNAsynthesis
primer.The double-stranded DNA is now readyfor +
amplificationby PCR. -_
The above proceduredescribedis called .f- +
RACE,sinceit is carriedout by amplificationof the PCR
Continue
51endof the startingRNA. Similarprotocolcan be
Fig. 8.7 : Rapid amplification of aDNA
used to carry out 3'-RACEwhen the 3'-end RNA
ends (RACE),
seouenceis desired.

Limitations of RAGE
of PCRare discussed
Other applications
described.
Sincea specificprimeris used,the specificityof at appropriateplaces.
amplificationof RACE may not be very high.
Another disadvantage is that the reverse PCR IN CLINICAL DIAGNOSIS
transcriptase
may not fully reach the S'-endsof
RNA, and this limits the utility of RACE.ln recent The specificityand sensitivityof PCR is highly
years, some modificationshave been done to useful for the diagnosisof various diseasesin
imoroveRACE. humans. These i ncl ude di agnosi so f inher it ed
disorders(geneticdiseases),
viral diseases,bacterial
diseasesetc.

The occurrenceof geneticdiseasesfrequently

identified by restrictionfragment length poly-
The adventof PCRhad, and continuesto have morphism (RFLP)can be employed only when
tremendousimpact on molecular biology. The thereis a mutationresultingin a detectable change
of PCRare too many to be listedhere. in the length of restrictionfragment.Many genetic
applications
Some of them are selectivelyand very briefly diseasesoccur without the involvementof RFLP.
 8 : P O L Y M ER AS
ChA P t CT (D N A A MP LIFIC A TION )
CEH A IN R EA C T ION 119

For all such disorders,PCR technique is a real and ampl i fi ed by P C R . More detai l s on thi s
boon, as it providesdirect informationof DNA. technique,referredto as site-directedmutagenesis,
T his is done b y a mp l i fi c a ti o no f D N A o f the are gi ven el sew here(C hapter10). B y usi ng thi s
relevantregion,followed by the direct analysisof method,coding sequencecan be altered(thereby
PCRproducts. changi ngami no aci ds)to synthesi zeprotei n of
genemani pul ati ons
Prenataldiagnosisof inherited diseases: PCR i nterest. Further, are i mportant
in understanding the effects
of promoters, initiators
is em ploy edi n th e p re n a tadl i a g n o s i so f i n h eri ted
etc., i n geneexpressi on.
dis eas esby u s i n g c h o ri o n i c v i l l u s s a m p lesor
c ells f r om a mn i o c e n te s i sT. h u s , d i s e a s esl i ke PCR is importantin the study of mRNAs,the
s ic k le- c ell a n e m i a , p -th a l a s s e m i a and productsof geneexpression. This is carriedout by
pheny lk et on u ricaa n b e d e te c te db y PC Ri n these reversetranscriotion - PCR.
s am ot es .
Diagnosisof retroviral infections : PCR from PCR IN COMPARATIVE STUDIES OF
to o lfo r d i a g n o s iasn dmo n i tori ng GE N OME S
c DNA is a v al u a b l e
of retroviralinfections,
e.g.,HIV infection. The differences in the genomesof two organisms
Diagnosisof bacterialinfections: PCRis used can be measured by P C Rw i th randompri mers.The
for the detection of bacterial infection e.9., products are separatedby electrophoresisfor
tuberculosisby Mycobacteriumtuberculosis. comparative identification.Two genomes from
closely related organismsare expectedto yield
Diagnosisof cancers: Severalvirally-induced
more si mi l ar bands.For more detai l s,refer the
cancers(e.g., cervical cancer causedby human
techni querandom ampl i fi ed pol ymorphi cD N A
papillomavirus)can be detectedby PCR.Further,
(S eep.116).
s om e c anc er sw h i c h o c c u r d u e to c h ro mosomal
t r ans loc at io(nc h ro m o s o me
1 4 a n d 1 B i n fo l l i cul ar PCRis very importantin the studyevalutionary
lymphoma)involving known genesare identified biology, more specifically referred to as
by PCR. phylogenetics. As a techniquewhich can amplify
even minute quantitiesof DNA from any source
PCR in sex determinationof embryos : Sex of
(hai r,mummi fi edti ssues,bone, or any fossi l i zed
human and live stock embryosfertilizedin vitro,
material),PCR has revolutionizedthe studiesrn
can be determinedby PCR,by using primersand
palaentologyand archaelogy. The movie 'Jurassic
DNA probesspecificfor sexchromosomes. Further,
Park',hascreatedpublicawareness of the potential
this technioueis also usefulto detectsex- linked
appl i cati ons
of P C R !
disordersin fertilizedembryos.

PCR IN DNA SEQUENCING P C R IN FOR E N S IC ME D IC IN E

A s t he P C R te c h n i q u ei s mu c h s i m p l er and A si ngl e mol ecul eof D N A from any source

quic k ert o am p l i fyth e D N A , i t i s c o n v e n i e n tlused
y (bl oodstrai ns,hai r,semenetc.)of an i ndi vi duali s
for sequencing.For this purpose, single-strands of adequate for amplificationby PCR.Thus, PCR is
DNA are required. ln asymmetric PCR (See verf importantfor identificationof criminals
p. 116) ,pr ef e re n ti a lm p l i fi c a ti oonf a s i n g l e - strand The reader may refer DNA finger printing
is carriedout. In anothermethod,strand removal techniquedescribedelsewhere(Chapter14).
can be achievedby digestingone strand(usually
done by exonucleaseby its action on 5'-phos- PCR IN COMPARISON WITH
phorylatedstrand). GE N E C LON IN G

P CR I N G EN E M AN IPU L AT ION A N D PCRhas severaladvantages over the traditional

EXPRESSION STUDIES genecl oni ngtechni ques (C hapter 6). Thesei ncl ude
better efficiency, minute quantities of starting
The advantage with PCRis that the primersneed material (DNA), cost-effectiveness,minimal
not have complementary sequences for the target techni calski l l , ti me factor etc. In due courseof
DNA. Therefore, the sequence of nucleotides in a time, PCRmay takeover mostof the applications of
pieceof the gene(targetDNA) can be manipulated genecl oni ng
Jhe collection of DNA fragments (specifically g e n e l i b r a r i e s )w h i c h e n c o d e f o r p r o te i n s Th e r e i s
I genes ) f r om a par t ic u l a r s p e c i e s r e p r e s e n t s a distinct difference in the genes of prokaryotic and
gene libr ar ies .The c r eat io n o r c o n s t r u c t i o no f g e n e eukaryotic cells. In prokaryotic organisms, the
libraries (broadly genomic libraries) is s t r u c t u r a lg e n e sc o d i n g f o r p r o t e i n sa r e co n ti n u o u s.
ac c om plis hed by is olat in g t h e c o m p l e t e g e n o m e However, in case of eukaryotes,the coding regions
( ent ir e DNA f r om a c e l l ) w h i c h i s c u t i n t o (exons) of structural genes are separated by non-
fragments,and cloned in suitable vectors Then the c o d i n g r e g i o n s ( i n t r o n s ) . F o r t h i s r e a so n / th e
s pec if icc lone c ar r y ing t he d e s i r e d( t a r g e t )D N A c a n constructionof gene librariesfor eukaryotesis more
be identified, isolated and characterized. In this comolicated.
m anner , a libr ar y of genes o r c l o n e s ( a p p r o p r i a t e l y
considered as gene bank) for an the entire genome CREATING A GENE LIBRARY
of a species can be constructed. The sizes of
genomes in different species are variable (Iable The DNA from the source organism is digested
9. 1\ . A c om plet e gene li b r a r y f o r e a c h o r g a n i s m b y r e s t r i c t i o ne n d o n u c l e a s e( e g . , Eco R Il ,to r e su l t
c ont ains all t he genom ic D N A . in fragments.lt is desirable to create conditions so
t h a t p a r t i a l d i g e s t i o n a n d n o t c o m pl e te d i g e sti o n
Biot ec hnologis t sar e p a r t i c u l a r l y i n t e r e s t e d i n
occurs. By this way, all possible DNA fragmentsof
the isolation of genes (and therefore creation of
v a r i a b l e s i z e c a n b e p r o d u c e d .T h e pa r ti a l d i g e sti o n
of a DNA with a restriction endonuclease is
depicted in Fig. 9.1. fhe cleavages occur at
Tlrrr 9.1 Genomesizes of some olganisns different sitesto result in DNA fragmentsof varying
lengths, some of them may be large while others
Organism cenome sizetit tl,b)
are small. ln practice, a combination of restriction
Escherichiacoli 4.0x 103 enzymes are used to digest source DNA to release
Saccharon yces cerevisiae x 104
1.35 a large number of DNA fragments. The desired
fragments can be isolated and cloned.
Tobacco 1.6x106
Wheat 5 9 x 106 Some workers use the term shotgun experiment
DrosophiIa meIanogaster 1.8x 105 (or shotgun approach)for ceation of random clones;
( w i t h o u t n e c e s s a r i l yi d e n t i f y i n g a l l o f th e m ) fr o m a
Mouse 3.3x 106
genomic DNA. In shotgun approach, the DNA is
Human 3.2x 106 subjected to random cleavage by restriction
8 Haptoidwhereappropriate endonucleases.

12l)
 9 : CE N EL IBR AR IES

RE RE RE

SourceDNA

Restrictionendonuclease
(partialdigestion)

2 +3 +4
(a)

1+ 2 3 +4
(b)

1+ 2+ 3 4
(c)

3 +4
(d)

f+z 4
(e)

2+ 3 4
(f)

4
(s)

Fig. 9.1 : Paftial digestion of a DNA by the enzyme restriction endonuclease (RE) at three sites
;
[Note : the coloured fragment represents the desired gene and it is one (shown in f) ;
among several fragments formedl.
I
i
I

Maniatis technique for have 23 differentchromosomes (24 in man),there I

creating gene library
ln the techniquedevelopedby Maniatiset al
*endonucleases
(1978),two restriction are used to
cut the target DNA. Partialdigestionallows the
formationof majority of DNA fragmentswith a
lengthof 10-30 kb. The fragmentsare frequently
overlappingand they can be fractionatedby gel
electrophoresis. The isolated fragments
(approximately20 kb in size) are insertedinto L
phage vector and cloned.
Establishing a gene library for humans
T he hum an c e l l u l a rD N A (th e e n ti reg e n o m e)
may be subjected to digestion by restriction
endonucleases (e.g., EcoRI). The fragmentsformed
on an averageare of about 4 kb size. (i.e.,4000
nitrogenes bases). Each human chromosome,
containingapproximately100,000kb can be cut
into about 25,000 DNA fragments. As the humans
are a total of 575,000 fragmentsof 4 kb length
formed.Among these 575,000 DNA fragmentsis
the D N A or geneof i nterest(sayi nsul i ngene).
Now is the selectionof a vector and cloning
process.E.coli,a harmlessbacteriumto humansrs
most commonly used. fhe plasmids from E. coli
are isolated. They are digested by the same
restrictionenzymeas was usedfor cuttinghuman
genome to form open pl asmi ds.The human
chromosomalDNA fragmentsand open plasmids
are joined to producerecombinedplasmids.These
plasmids contain different DNA fragments of
humans.The recombinedolasmidsare insertedinto
E. coli and the cells multiply(Fig.9.2).The E. coli
cells possessall the human DNA in fragments.lt
must, however be rememberedthat each E. coli
cell contains different DNA fragments. All the
E. coli cells put together collectivelyrepresent
genomic library (containingabout 575,000 DNA
fragments).
 B IOTECHNO LO CY
122

E collcells

+
r-/ "O
.- /)
l-/r-./
a)
l-/
Plasmids

1 rg 76
)c
2w uou )7
DNAfragments

lr.'-\ \
'/-\
tn() A
O,cu,,
'40 /z'l

o,
7-
trJ l)
'O
.o 20
a) (
... (-./a

'( .) ,--{ ,on

^r-\
?l-/
( - \q
\)- t[-/
'.
Genelibrary
 t er g: CE N EL IB R A R IE S

Other vectols for creating Fragment libraries

genomic libraries
Biotechnologistsoften come across minute
I n plac e o f p h a g e s a n d p l a s mi d s , o t her quanti ti esof starti ngmateri al se.g., si ngl e cel l s,
vectorsare in use for constructionof large sized fixed tissues,fossilsetc. lt is quite difficultto apply
DNA libraries.These include cosmids,bacterial tradi ti onaltechni quefor constructi on of genomi c
artificial chromosomes(BACs) and yeast artificial l i brari esfrom such sampl es. P C R i s i deal l ysui ted
chromosomes (YACs). These are considered for i sol ati on and ampl i fi cati onof genes from
as high capacity vectors. Although they are very smal l sampl es,Thus, P C R can be used
ideal for constructionof gene libraries, there for the creation of random genomic fragment
are many practicaldifficultiesassociated with their l i brari es.
us e.
COMPLEMENTARY DNA LIBRARIES
PCR AS AN ALTERNATIVE TO C l oni ng of eukaryoti c genes i s rather
G E NO M I C LI B R AR Y C ON ST R U C T IO N compl i catedand requi resspeci altechni ques. Thi s
i s mai nl ydueto the non-codi ng sequences (i ntrons)
As alreadydescribedin detail (ChapterB), PCR
is a techniquefor amplificationof a specificDNA i n the D N A . In the eukaryoti ccel l as the gene i s
sequence.PCRwith primerscan be usedto isolate transcribed, the RNA undergoes severalchangesin
targetDNA directlyfrom the Senome'Thus, PCR the nucleus(referredto as splicing)to releasea
serves as an alternativeto DNA library (gene matureand functionalmRNAinto the cytoplasm.In
library)constructionby cloning.This is not always this manner,the introns are removed. ln some
possiblesincePCRtechniquecan be employedfor genes,intronsform a major bulk of the gene.For
i nstance,i n the humandystrophi ngene,as much
of s h o rtl e n g thD N A s (u s u a l l y1 -2 kb
am plif ic at ion
wit h a m ax imu m o f 5 k b ). F u rth e r,th e hi gh as 99o/oof the DNA sequenceis composedof
temperature used in PCR, sometimes causes introns.Thereare as many as 79 introns!
damage to bases and generatesnicks in DNA The prokaryotes,particularlythe bacteria,do
strands. not possess the abilityto removethe introns.Hence
the functionalmRNA is not correctlyformed in a
Long PGR prokaryoticcell for an eukaryoticgene. Thus,
W it h s om e m o d i fi c a ti o n si n th e P C R , i t i s cloning of eukaryoticgenes becomesa difficult
now possibleto amplify DNA fragments up to a task.
fengthof 22 kb from the human genomic DNA.
This is achievedby using a combinationof two Synthesis of comPlementary DNA
DNA polymeraseenzymes,besideslowering the C ompl ementaryD N A (cD N A ) i s a doubl e-
reactiontemperature. One of the DNA polymerases
strandedcomplementof an mRNA. cDNA can be
has proof-reading activity to remove the synthesizedfrom mRNA lry reversetranscription.
mismatched bases.Somecommercialcompaniesin An eukaryoticfunctionalmRNA which does not
fact provide enzyme cocktails ideally suited for have intronspossesses a C cap at 5'and a poly (A)
long PCRe.g.,TaqPlusLong PCRsystemmarketed tai l at the 3' end (approxi matel200 y adeni ne
by Stratagene.lt contains laq polymerase and residues).
the thermostable proof-reading enzyme Pfee
polymerase. The requisitemRNA is isolatedand purified
(parti cul arl yfrom cel l s w hi ch are ri ch i n the
Long PCR has been appliedfor the structured speci fi cmR N A e.8., pancreati ccel l s for i nsul i n
analysisof human genesand Senomesof HlV. mRNA).An oligo-dTprimeris addedto bind to the
(by annealing)'
LongPCRis unlikelyto replacethe construction shortsegmentof poly A tail region
primer provides 3'-hydroxyl group for the
of genom iclib ra ri e sT. h i s i s b e c a u s ec re a ti o nof This
DNA libr ar ie si s p e rm a n e n w t h i l e l o n g PC R i s synthesisof a DNA strand. With the additionof the
employed for enzyme reverse transcriptase and four
temporary.Further,PCR can be
DNA fragments (of interest) deoxynucleotides (dATP, dTTP, dGTP and dCTP),
amplifying selected
from genomiclibraries. DNA synthesis proceeds.Forthe basesd G, C and
124 B IOTE CHNO LO G Y

5' G AAAAAA 3'

MR N A
IOligo(dT)primer
I
J
5(l AAAAAA 3'
TTTTTT 5'

AAAAAA A A A A AA

AAAAAA A A A A AA
-._ TTTTTT

CompletecDNA Incomolete
cDNA

Fig. 9.3 : Synthesis of complementary DNA (IDNA).

U in the template (mRNA), Ihe corresponding
complementary bases in DNA respectivelyare
T, C, G and A. The newly synthesizedfirst
DNA strand has a tendencyto fold back on to
itselffor a few nucleotidesto form a hairpin loop
(Fig. e.3).
The loop of the first DNA strandservesas the
templatefor the synthesisof secondDNA strand.
By the additionof E. coli DNA polymerase (Klenow
fragment), the secondDNA strandsynthesis occurs
startingfrom the end of the hairpin loop. On
tre a tm e n tw i th th e e n z v m e R N ase H . mR N A
m o l e c u l e sa re d e g ra d e dT. h e enzymeS l nucl ease
c l e a v e sth e h a i rp i n l o o p s a nd degradessi ngl e-
strandedDNA extensions.The final productsare
c o mp l e m e n ta ry D N A c o p i e s of ori gi nal mR N A ,
some of them are complete while others are
incomplete.
limitation of the technique : The main
disadvantage with the hairpinmethodis the lossof
a s ma l l s e q u e n c ea t th e 5 ' e n d of cD N A due ro
c l e a v a g eb y S l n u c l e a s e .
lrnproved method for cDNA synthesis
To overcomethe limitation describedabove,
some improvementshave been made in cDNA
One such i mprovedtechni queis shown
synthesi s.
in Fig. 9.4.
As the first strandof cDNA is synthesized, it is
tailed with cytidine residueswith the help of the
enzymeterminaltransferase. The mRNA strandis
hydrol ysed w i th al kal i ,and the ful l l eng t hcDNA is
recovered.A syntheticoligo-dC primer is then
anneal edto ol i go-dC .Thi s i n turn en ablest he
synthesisof the secondstrandof cDNA. By this
i mproved techni que, a ful l l ength of cDNA
correspondi ng to mR N A (i n turn the gene) is
obtained. But the efficiency of this method rs
comparativelylower.
Construction of cDNA libraries
The compl ementaryD N A mol ecu lescan be
cl onedi n cl oni ngvector(e.g.,pl asmi d),for cr eat ing
cD N A l i brari es.
ThecD N A i nserti oni nto t he vect or
shouldhavecorrectorientationThis is achievedby
Chapt er9 : CE N EL IBR AR IES 125

AAAA 3' prone, and even a v e r y m i n u t e c o n t a m i n a t i o n

of mRNA (with o t h e r m R N A s ) w i l l g i v e f a l s e
results.

3', T T T T5 '
Once a D N A l i brary or a C D N A l i brary i s
I r"rrin"t transferase created,the clones (i.e., the cell lines) must be
+ocre screenedfor identificationof soecificclones.The
J screeningtechniquesare mostly based on the
AA AA sequenceof the clone or the structure/function of
TTTT its product.

I HydrolysemRNA
S C B E E N IN G B Y D N A H Y B R ID IZA TION
(withalkali)
J The target sequence in a DNA can be
TTTT 5' determinedwith a DNA probe (Fig. 9,5). To start
with, the double-strandedDNA of interest is
converted into single strandsby heat or alkali
(denaturation).
Jo"*' TTTT 5'
The two DNA strandsare keptapart
by bi ndi ngto sol i dmatri xS uchas ni trocel l ul ose
nylon membrane.Now, the singlestrandsof DNA
or

"@ 3'
probe(100-1,000bp) labeledwith radioisotope are
c uuu added. H ybri di zati on(i .e., base pai ri ng)occurs
(/ Double-strandedcDNA betweenthe complementary nucleotidesequences
Primer

Fig. 9.4 : lmproved method for complementary DNA

synthesis.
SourceDNA (double-stranded)

I
the addition of a syntheticlinker to the double- I Denaturation
membranebinding
strandedcDNA.
J
ln a techniquedevelopedby Okayamaand Berg
(1982),the mRNA is first linked to the plasmid
cloningvectorand then cDNA synthesis is carried DNAs
Single-stranded
out.

RT.PCR as an alternative to
cDNA cloning
Reverse transcription followedby PCR(RT-PCR)
can am plif yt he m R N A to g i v e c D N A (F o rd e tai l s
ReferChapterB).
R
RT-PCRis very rapid hence cDNA molecules
can be obtainedin a shortperiod.Further,
eventhe x x )(
long length mRNAscan be convenientlyused in HybridDNAS
RT-PCR.
Fig. 9.5 : Screening by DNA hybridization
also in RT-PCR.
There are some disadvantages ({ indicates radioisotope label in the DNA probe)
The DNA polymeraseused in RT-PCRis error-
 126 B IOTE CHNO LO CY

of the target DNA and the probe. For a stable base 3',
pairing, at least B0% of the basesin the two strands
(target DNA and the probe) should be matching. SourceDNA
The hybridized DNA can be detected by
autoradiography. I uyorioization
primers
Jwith
DNA PROBES
-J
The DNA probes used for screeningpurpose can
be s y nt hes iz edin m any way s

Random primer method

J 5

Radiois ot ope labeled DNA p r i m e r s c a n b e

produced by this technique (Fig. 9.6). The double-
s t r anded DNA c ont aining t he s e q u e n c e n e e d e d t o
serve as a probe is denatured . A mixture of
synthetic oligonucleotides, with all possible
combinations of hases (A, C, C and 71, with a
length of 6 nucleotides each serve as primers. +_r.V_ Primer
Some of these primers with complementary * extension
s equenc eswill hy br idiz e wit h t h e t e m p l a t e D N A .
This oc c ur r enc e is ent ir elv b v c h a n c e a n d t h e _-{A,t+ _IL+ lX_+
probability is reasonablygood.

By the addition of four deoxyribonucleotides

(one of them is radiolabeled)and in the presenceof
the enzyme DNA polymerase of E. coli (Klenow
fragment),the primers are extended on the template
DNA. Sinc e a r adioac t iv e labe l i s u s e d , t h e n e w l y
synthesized DNA fragments are labeled at
appropriate places, and these are the DNA probes.
A number of labeled DNA probes can be produced
from an unlabeled template DNA.

Non-isotopic DNA probes

For the production of non-isotopic DNA probes,

one of the four deoxynucleotides (used for primer
extension described above) is tagged with a label
(e.9., biotin). The label of the DNA probes can be
detected by use of chemical and enzymatic
reactrons.
SCREENI NG BY CO LO NY
HYBBI DI ZATI O N
The DNA s equenc e in t he t r a n s f o r m e dc o l o n r e s
can be detected by hydridization with radioactive
DNA probes (some times labeled RNA probes can
als o be us ed) . Colony hy br idi z a t i o n t e c h n i q u e r s
also referred to as replica plating by some authors.
The technique depicted in Fig. 9.7 is briefly
described.
Fig. 9.6 ; Synthesis of radioisotope labeled DNA
probes (Note : Non-isotopic DNA probes can be
prepared by tagging with chemical labels e.g. biotin)
The transformedcells are grown as colonies on
a master plate. Samples of each colony are
transferredto a solid matrix such as nitrocellulose
or nylon membrane.The transferis carefully carried
out to retain the oattern of the colonies on the
m a s t e r p l a t e . T h u s , t h e n i t r o c e l l u l o se p a p e r
contains a photocopy pattern of the master plate
c o l o n i e s . T h e c o l o n y c e l l s a r e l yse d a n d
deoroteinized. The DNA is denatured and
i r r e v e r s i b l yb o u n d t o m a t r i x . N o w a r a d i o l a b e l e d
D N A p r o b e i s a d d e d w h i c h h y b r i d i z e s w i th th e
Ch a pt er9 : CE NEL IB R A R IE S r27

hasthe completesequenceof the targetgene,data

observedfrom the restriction
endonuclease
analysis
w i l l be hel pful .

Modifications of colony hybridization

technique
Several improvements in the colony
hybridizationtechnique,describedabove, have
been made in recent years. In the plaque Iift
technique,nitrocellulose paper is directly applied
on the uppersurfaceof masteragarplatemakinga
direct contact.By this way, plaquescan be lifted
and severalidenticalDNA printscan be madefrom
Solid matrix Subculture a singleplate.This techniqueincreasesreliability.
frommasterolate
More recently,screening of DNA librariesis carried
out by automatedtechniques.

Fig. 9.7 : Screening by colony hybridization

(Note : Step 4 is carried out lor the colony
identified in step 3).

complementarytarget DNA. The non-hybridized

probe moleculesare washed away. The colony
with hybridized probe can be identified on
The cells of this colony (from the
autoradiograph.
masterplate)can be isolatedand cultured.
Many a timesmultiplecoloniesare detectedon
Fig. 9.8 : lmmunological assay lor screening
hybridizationby a DNA probe. This is due to a gene library.
To identifywhich colony
overlappingsequences.
724 BIOTECHNOLOCY

SCREENING BY PCR anti bodyw hi ch speci fi cal l ybi nds to t he pr ot ein

(actsas an antigen),encodedby the targetDNA.
Polymerase chain reaction(PCR)is as good as
Afterremovingthe unboundantibodyby washings,
h y b ri d i z a ti o n te c h n i q u e for screeni ng D N A
a second antibody is added which specifically
libraries.But adequateinformation(on the franking
binds to the first antibody.Again the unbound
sequencesof target DNA) must be availableto
antibodiesare removedby washings.The second
prepareprimersfor this method.The coloniesare
antibody carries an enzyme label (e.9., horse
m a i n ta i n e di n m u l ti w e l l pl ates, each w el l i s
raddishperoxidase or alkalinephosphatase) bound
screened by PCR and the positive wells are
to it. The detectionprocessis so devisedthat as a
i d e n ti fi e d .
colourless substrateit is actedupon by thisenzyme,
a coloured product is formed. fhe colonieswhich
SCREENIl{G BY IMMUNOLOGICAL
give positive result (i.e., coloured spots) are
ASSAY
identified. The cells of a specific colony can be
l mmu n o l o g i c atel c h n i q uescan be usedfor the subculturedfrom the masterplate.
detectionof a proteinor a polypeptide, synthesized
by a gene (through transcriptionfollowed by S C R E E N IN G B Y P Ii OTE IN FU NCTI O N
translation). The procedureadoptedfor immuno-
lf the'targetDNA of the gene libraryis capable
logical assay and hybridization technique
of synthesizing a protein(particularlyan enzyme)
(described already)arequitecomparable. Screening
that is not normally producedby the host cell,
procedureby immunologicalassayis depictedin
the protein activity can be used for screening.
Fig. 9.8, and brieflydescribedhereunder.
A specific suhstrateis used, and ifs utilization by
The cellsare grownas colonieson masterplates a colony of cells indicates the presence of
which are transferredto a solid matrix (i.e., an enzyme that ,acts on the substrate. For
nitrocellulose). The coloniesare then subjectedto instance, the genescodingfor enzymesct-amylase,
lysisand the released proteinsboundto the matrix. and B-glucosidasecan be identified by this
These proteinsare then treated with a primary techni oue.
 ith the adv anc es in genet ic engineer i n g ,
genes can be isolated (from any organism)
an d used for th e s y nt hes isof nat ur ally oc c ur r i n g
proteins. Some of these proteins serve as enzymes
(i.e., biocatalysts),and a selectedfew of them have
industrial applications. Thus, of the 2000 natural
enzymes, around 20 are used in food, and
pharmaceutical industries e.g., cr-amylase,
cellu lase, p ap ain The indus t r ial enz y m es enjoy a
special status in biotechnology.
The n atu ral e nz y m es ar e not well s uit ed fo r
ind ustrial a pp lic at ions due t o unnat ur al an d .
un ph ysiolo gical env ir onm ent s s uc h as hig h
temperature,unsuitable pH, the presenceof certain
chemicals (organic solvents)etc. Many attemptsare
made to improve the efficiency of natural enzymes
fo r in du stria l u s e e. 9. , im m obiliz at ion ( Re f e r more theoreticalthan practical, some achievements
Cha pte r 21 ), ch em ic al m odif ic at ions . But t he s e have been made for more efficient use of industrial
have very limited impact
STRATEGIES TO IMPROVE V'TRO
ACTIVITIES OF ENZYMES 'IV
Se ve ral p oss ibilit ies c an be t hought of t o
improve the in vlfro activities of the enzymes.
Some of them are listed below.
. lncreasing substrateaffinity to enzyme.
. Making the enzyme thermal tolerant (active at
high temperature)and/or pH stable.
. En ha ncingthe iubs t r at e s pec if ic it y by m odif y in g T h e n e t r e s u l t i n s i t e - d i r e c t e d m u t a g e n e s i s i s
the sub stra tebinding s it e of t he enz y m e.
Biotechnology [9]
. D e s i g n i n g t h e e n z y m e t o m a k e i t r e s i s t a n tt o
proteolytic degradation.
. S y n t h e s i z i n ge n z y m e t h a t i s s t a b l e a n d a c t i v e i n
n o n - a q u e o u ss o l v e n t s
. Changing the enzyme in order to make it
independent of cofactor(s)for its function.
o E l i m i n a t i n ga l l o s t e r i c s i t e s i n t h e e n z y m e s o t h a t
it is not controlled by feedback inhibition.
. lmproving the stability of the enzyme to heavy
metals.
Fusing the enzymes (making a multienzyme
complex) needed in the reactionsto give a final
oroduct.
Although the above possibilities appear to be
enzymes. By subjecting the proteins to chemical
modifications, suitable changes can be made in
enzyme activity. Chemical manipulation of
enzymes are non-specific, harsh and should be
done repeatedly,hence not preferred.
Modifications in the DNA sequenceof a gene
are ideal to create a protein with desired properties.
Site-directed mutagenesis is the technique for
generating amino acid coding changes in the DNA
(gene). By this approach specific (site-directed)
change (mutagenesis) can be made in the base (or
bases) of the gene to produce a desired enzyme.
incorporation of a desired amino acid (of one's
129
130 B IOTECHNO LO CY

choice) in place of a specific amino acid in a M13 DNA

/---."rr-
protein or a polypeptide. By employing this /\
technique,enzymesthat are more efficient and ll
rarsetDNA
m o re s u i ta b l e th a n th e natural l y occurri ng \-Z-
counteroarts can be created for industrial DNA
Single-stranded
applications.But it must be remembered that site- l/ - -
directedmutagenesis is a trial and error method ['/ Oligonucleotide
I Pnmer
that may or may not result in a better protern.
Further,the detailedinformationon the structure
and functions of a protein are desirable to
undertakesite-directed mutagenesis. lt is customary
to give emphasisfor the creation of enzymes
by this technique becauseof their commercial
v a l u e th ro u g h i n d u s tri a lu se. The fact i s that
a n y n a tu ra l l yo c c u rri n g protei n w i th i mproved
functions can be synthesizedby site-directed
mutagenesis.
Thereare manymethodsemployingsite-directed
A selectedfew of them are briefly
mutagenesis.
described.
DNAstrand
I
E coli
lTransform
+

In th e te c h n i q u eo f ol i gonucl eoti de-di rected

mutagenesis, the primer is a chemically
synthesized oligonucleotide (7-20 nucleotides
long).lt is complementary to a positionof a gene
around the site to be mutated.But it contarns
mismatchfor the baseto be mutated. Wild type DNA MutanttYPeDNA
The basic techniqueis depicted in Fig. 10.1.
The startingmaterialis a single-stranded DNA (to Fig. 10.1 : Oligonucleotide-directed mutagenesis
be mutated)carried in an M1, phagevector.On
m i x i n gth i s D N A w i th p ri m er,the ol i gonucl eoti de
hybridizeswith the complementarysequences,
introduced into E. coli bv transformation.The
except at the point of mismatchednucleotide.
infectedE. coli cells produceM.', virus particles
H y b ri d i z a ti o n(d e s p i tea s ingl ebasemi smatch)i s
possibleby mixing at low temperature containing eitherthe originalwild type sequence
with excess
or the mutant sequence. The virusparticleslysethe
o f p ri me r, a n d i n th e presenceof hi gh sal t
cel l sand form pl aques.
concentration.
Theoretically,it is expectedthat half of the
By the addition of 4-deoxyribonucleoside
phage Mr s particles should carry wild t!,pe
tri p h o s p h a te sa n d D N A pol ymerase (usual l y
sequencewhile the other half mutant sequence
Klenow fragment of E.coli DNA polymerase)
(sincethe DNA replicatessemiconservatively). But
n c c u rsT
re p l i c a ti o o pri mer
. h u sth e ol i gonucl eoti de
in actual practice,due to technicalreasons,only
is extendedto form a complementarystrandof
1-5% of the viruses with mutated sequencesare
the DNA. The ends of the newly synthesized
recovered.
DNA are sealedby the enzyme DNA ligase.The
d o u b l e -s tra n d e dD N A (i .e .,M,, phagemol ecul e) The double-stranded DNAs of M', are isolated.
containing the mismatched nucleotides is The mutatedgenesarecut with restriction enzymes,
C hapt er10 : s I T E-D | R E C T E
MUD T AC EN ESAN
T S D P R OTE TN
E N C TN E E R TN C 131

small position of target DNA to be deleted

(A)
(Fig. 10.2Q.

(B)
(c)
Fig. 10.2 : Variations in oligonucleotide-directed
mutagenesis (A) Multiple point mutagenesis
(B) lnsertion mutagenesis (C) Deletion mutagenesis.
CASETTEE MUTAGENESIS
In casetteemutagenesis, a syntheticdouble-
strandedoligonucleotide(a small DNA fragment
i.e., casettee)containing the requisite/desired
mutant sequence is used. lt replaces the
correspondingsequencein the wild type DNA.
Casetteemutagenesisis possibleif the fragment of
the gene to be mutated lies between two
restriction enzyme cleavagesifes. This intervening
sequencecan be cut and replacedby the synthetic
(w i th mutati on).
ol i gonucl eoti de
The outline of the casettee mutagenesis
techniqueis depicted in Fig. 10.3. The plasmid
DNA is cut with restriction enzymes(suchas EcoRl
and H i nd111l . The syntheti c ol i gonucl eoti de
containingmutant sequenceis also cleavedand
l i gatedto pl asmi dD N A . The recombi nant
pl asmi ds
that multiplv in E. coli are all mutants.

ligatedto a plasmidvectorof E. coli. The altered

protein is producedin the E. coli which can be
is olat edand pur i fi e d .
Oligonucleotide-directed mutagenesisby using
PlasmidDNA
plasmidDNA (insteadof M13) is also in use..This
technique reduces the number of steps 6ince,
mutagenesisand cloningthe targetDNA are in the
same vector.

Variations in oligonucleotide-directed
mutagenesis
There are some variations in use in the
oligonucleotide-d
irected mutagenesis, as the
situationdemands.
1. Multiple point mutagenesis : Oligo-
nucleotide-directed
mutagenesis can be used to
createDNAswith multiplepoint mutationswith the
requisitenumberof basemismatches (Fig. 10.2A).
2. I ns er t ionm u ta g e n e s i s : In th i s c a s e , th e
mutant oligonucleotidecarriesa sequenceto be
inserted (sandwichedbetween two sites with
complementary sequences). This can bind with the
target DNA on either side (Fig. 10.28).
3. Deletion mutagenesis : The mutant
oligonucleotidebinds to two separatesites on Fig. 10.3 : Casettee mutagenesis.
either side of the target DNA. This enables a
 B IOTECHNO LO CY
132

Tube 1

Normalprimers

Primerswith a

l:)
TargetDNA nucleotidemismatch
Originalnucleotide

Strand C

StrandD

LinearDNA

Changed
nucleotide

StrandB
Strand D

(---+-Normat primers; -")-Primers with a

Fig. 10.4: PCR-amptified oligonucteotide-directedmutagenesis
singlenucteotidemismatch;o.originatnucteotides;-}:--changednucleotides)'

a plasmid
Casettee mutagenesis is a simpletechniquewith the targetDNA (gene)is cloned on to
The vector and distributed into two reaction tubes'To
almost 1007oefficiencyto get mutant genes'
two primers (oligonucleotides
drawback is the requirementof restrictionsites eachtube are added
target fragment of DNA svnthesized by using PCR).One primer (A in tube
specificallyflanking the
is complementary to a regionin
a n d th e l i m i ta ti o nto g e t desi redol i gonucl eoti des'1 and C in tube 2)
one strand of the cloned gene except for one
nucleotide mismatch (i.e., the one targetedfor a
PC R .A M PL IF IE D OLIGON U GLE OTID E .
DIRECTED MUTAGENESIS change).The other primer (B in tube 1 and D in
tube 2) is fully complementary to a sequencein the
The polymerasechain reaction(PCR)and its otherstrand,within-oradjacentto the clonedgene'
importancehavebeendescribed(ReferChapterB)' The placementof primersfor hybridization(with
T h e P C Rte c h n i q u ec a n be usedi n ol i gonucl eoti de-the DNA strands)in eachtube is done in opposite
directed mutagenesis.This avoids the use of a direction.Now the PCRtechniqueis carriedout
bacteriophage (M13) system, besides enrichment for amplificationof the DNA molecules'These
of mutated gene. DNAs are actually linear. In Fig. 10.4' they are
for understanding
The PCR-based mutagenesis technique shown as discontinuouscircles
commonlyemployedis depictedin Fig. 10.4' First subsequent steps.
 ISN D P R OTE INE N C IN E E R IN C
ED T AC EN ESA
Chapt er10 : S IT E -D IR EC T MU 133

The productsof PCR in the two reactiontubes cysteineresiduesto respectively

form one, two or
ar e m ix ed. Th e D N A m o l e c u l e s u n d e rgo threedi sul fi debonds.
denaturationand renaturation. A strandfrom one In the wild type (native)T4 lysozyme,thereare
reaction tube (strand A) hybridizes with its two cysteineresiduesat positions54 and 97 which
complementarystrand from other reaction tube howeverare not held togetherby a disulfidebond.
(strandC). They form circles with nicks (Baps).
By oligonucleotide-directed mutagenesis, cysteine
T he c ir c ular pl a s m i d s(c o n ta i n i n gm u ta n t D NA )
residuescreateddisulfidebondsbetweenpositions
with nicks are introduced into E. coli by (numbered form N -termi nalend) 3 and 97, 9 and
transformation. The nicksare sealedin vivo by host 164, and 21 and 142 of the enzyme.Introduction
cetll enzymesand the plasmidsare propagated of disulfidebonds increasesthe folded structure
indef init ely . and thermostabilityof the enzyme. Thus, T4
Iysozyme with three disulfide bonds is very stable
with good biological activity. (Note : T4 lysozyme
has no i ndustri alor therapeuti appl
c l t rs
i cati ons.
di scussedhere due to the successachi evedi n
One of the most exciting aspectsof genetic protei nengi neeri ng).
engineeringis to design, develop and produce
proteins with improved operating characteristics Xylanase
(increased stability,improvedkineticsetc.),besides
sometimes creating even novel proteins. The This is an enzyme used in the industryfor
techniquessuch as site-directed mutagenesis and manufactureof paper from wood pulp. Xylanase
genec loningar e u ti l i z e dfo r th i s p u p o s e . has to be catalyticallyactiveat high temperature.
Introductionof disulfide bonds (one, two or three)
I NCB E A S I NG T H E ST A BIL IT Y AN D makes it thermostable,and substanti ally improves
BIOLOGICAL ACTIVITY OF PROTEINS its functional efficiency.

By increasing the half-livesor thermostability of CHANG'NG ASPABAG'NE TO OTHER

enzymes/proteins, their industrialapplicationsor AM'NO AC'DS
therapeuticusescan be more appropriatelymet.
Some of the approachesfor producing proteins At high temperature,the amino acidsasparagine
with enhancedstabilityare describedbelow. and also glutamine are likely to undergo
deamidation(releasingammonia)to respectively
ADDITTON OF D'SULF'DE BONDS form asparti c aci d and gl utami c aci d. These
alterations are oftenassociated with changesin the
Significant increase in the thermostability protein folding and loss of biological activity.
of enzymes is obse.rved by adding disulfide
bonds. However, the additional disulfide bonds Triosephosphate isomerase
should not interfere with the normal enzyme
Thi si s a di meri cenzymew i th i denti calsubuni ts,
f unc t ion. which are
eachone havingtwo asparagine residues
In genera[, the new protein with added thermosensi ti ve (undergo deami dati on).Ol i go-
dis ulf idebond s d o e s n o t re a d i l y u n fo l d a t hi gh nucleotide-directedmutagenesiswas used to
temperatures,and further it is resistant to introduce threonine or isoleucine in place of
at n o n -p h y s i o l o g i ccaol n d i ti o n s(h i gh asparagineat positions 14 and 78. The new
denat ur at ion
pH, presence of organic solvents). These enzyme was found to be thermostable.On the
characteristicsare particularly important for other hand, when the asparagineresidueswere
industrialapplicationsof certainenzymes. replacedby asparticacid,the enzymewas unstable
even at low temperature, with reducedactivity.
T4 Lysozyme
BEDUC'NG THE FNEE
This an enzyme of bacteriophageT4. Cood
SULFHYDRYL GROUPS
successhasbeen achievedin introducingdisulfide
bondsin T 4 ly s o z y m eT. h i sw a s d o n e b y c h a n gi ng Sometimes,the presenceof free sulfhydryl
two, four or six aminoacids(in closeproximity)to groups(contributedby cysteineresidues)in more
134
(A)
(B) Valine
(358)
No cleavaoe
Elastase
Fig. 10.5 : (A) The cleavage of a,-antitrypsin by binding to elastase
(B) No cleavage occurs when methionine (358) is replaced by valine.
num ber s m ay be r es pons ib l ef o r t h e l o w a c t i v i t y o f
the protein. In such a case, the protein or enzyme
stability and its activity can be increased by
reducing the number of sulfhydryl groups
l{urrran ij-inter-feron
The ant iv ir al ac t iv it v o f h u m a n B - i n t e r f e r o n
( lFN B) pr oduc ed in E. c oli b y g e n e t i c e n g i n e e r i n g
was f ound t o be only 1 0 % o f t h e o r i g i n a l
glycosylatedform. Further,IFN p was found to exist
as dim er s and oligom er swh i c h a r e a l m o s t i n a c t i v e .
In fact, the cysteine residues were involved rn
int er m olec ular dis ulf ide bo n d i n g r e s u l t i n g i n t h e
' f or m at ion
of dim er s and o l i g o m e r s . T h i s h a p p e n s
in E. c oli c ells and not i n h u m a n c e l l s . T h i s
pr oblem was s uc c es s f ullyo v e r c o m e b y r e p l a c i n g
cysteine residues by serine lt may be noted that
the structures of these two amino acids are
s im ilar ex c ept t hat s er ine h a s o x y g e n i n p l a c e o f
sulfur in cysteine. Consequently, introduction of
serine in place of cysteine reduces free sulfhydryl
Sroups.
By the above process, IFN B with increased
s t abilit y and good biolo g i c a l a c t i v i t y c a n b e
produced. Increasedstability is in fact required for
storage and therapeutic use of proteins such as
i nterfero n s.
B IOTECHNO LO CY
_,.- Serine(359)
394
.-----_-... -
^,--Methionine
(358)
S'AIGT.E AMINO ACID CHANGES
Some of the recombinantproteins can be
improvedin their stabilityand biologyactivityby a
second generation variants. These have been
frequentlachi
y evedby a si ngl eami noacidchange.
crr-Antitrypsin
inhibitsthe action of neutrophil
a.,-Antitrypsin
elastase(elastase is an enzymethat damagesthe
l ung ti ssues,often resul ti ngi n emphysem ai. e.
abnormaldi stensi on of l ungsby ai r).ct l- Ant it r ypsin
binds to elastaseand preventsits action. In this
process,a,-antitrypsingetscleavedbetweenserine
(359 resi due)and methi oni ne(358 r esidue)The .
freemethionineis oxidizedto methioninesulfoxide,
maki ngo,,-anti trypsianpoor i nhi bi to rof elast ase.
By replacing methionine at 358 position
by valine, an oxidative'resistant variant of
ar-antitrypsin has been created (Fig. lO.5). This
new enzymei s parti cul arl yi mportantin t r eat ing
patientswith geneticdeficiencyof ct,-antitrypsin.
l nsul i n
l n the neutralsol uti on,therapeut icinsulin is
presentmostly as zinc-containing hexamer.By
i ntroduci ng insulins
si ngl eami noaci dsubsti t ut ions,
Chaot er10 : SIT E -D IR EC T M
ED A N D P R OTE INE N C IN E E R IN C
U T A C E N E SIS 135

were found to be in monomeric state with good (K m, speci fi ci ty etc.) through ol i gonucl eoti de-
stabilityand biologicalactivity. directedmutagenesis. This is particularlyrequired
for enzymes w i th i ndustri al and therapeuti c
Tissue plasminogen activator (tPAf appl i cati ons.
Tissueplasminogenactivatoris therapeutically
usedto lysethe blood clotsthat causemyocardial Subtilisin
infarction.Due to its shorter half-life (around 5 Subtilisinis a major industrialenzymesecreted
minutes),tPAhasto be repeatedly administered. By by Cram-positive bacteria(Bacillusspecies).
lt is a
replacing asparagine residue(at position 120) with serine proteasevery widely used as an enzyme
glutamine, the half-life of tPA can be substantially detergent(cleaningagent in laundries).However,
increased. This is due to the fact that glutamineis the l argescal ei ndustri aluseof nati vesubti l i si ni s
lessglycosylated than asparagine and this makesa restricteddue to the oxidativeinactivationof this
differencein the half-lifeof tPA. enzyme.Thi soccursi n a manneranal ogous to that
already discussedfor cx,-antitrypsin. The reason
Hir udin beingthat methionine(at position222) lying close
Hirudin is a proteinsecretedby leech salivary to the active site gefs oxidized, making the enzyme
(i
gland,and is a s tro n gth ro m b i ni n h i b i to r .e .,
acts inactive. Logically,replacementof methionine (at
as an anticoagulant).By replacing asparagine(at position 222) by other amino acids will solve the
47 position) with lysine, the potency of hirudin can problem.This has been done, and in fact, all the
be increasedseveral-fold. 19 otherami noaci dshavebeentri edas substi tutes
for methi oni ne, w i th varyi ngsuccess.
Dihydrof olate reductase i s an enzymethathasbeenextensi vel y
S ubti l i si n
The enzyme dihydrofolatereductase(DHFR) exploitedfor geneticmanipulationsover the past
catalysesthe conversionof 7, B-dihydrofolate to 5, 15 years.The resultis that abouf50% of the native
6, 7, 8-tetrahydrofolate.The lattef coenzyme is amino acidsof this enzyme have been changedby
closelyinvolvedin one carbonmetabolism, which in vitro mutagenesis. And almostevery propertyof
ultimatelyresultsin the synthesisof nucleic acids subti l i si n has been al tered. These i ncl ude i ts
and am ino a c i d s . T h e i n h i b i ti o n o f DH FR stability,substratespecificity,thermaland alkaline
(conventionallyby folate analogues such as i nacti vati on.
methotrexate) will restrictthe growthof tumorcells.
Modifying metal requirementof subtilisin : The
Thereforethe enzymeDHFRhas sometherapeutic enzyme subti l i si n bi nds to cal ci um and gets
applic at ion s . B ut i n many i ndustri es w heresubti l i si n
stabi l i zed.
By employing site-directed mutagenesis, is used,metal-chelatinB agentsare also employed.
replacementof glycine (at position 95) by alanine They bind to calcium and the enzyme becomes
was found to produce DHFR which is completely inactive.This problem can be solved by oligo-
inactive. nucleotide-directed mutagenesis. The nucleotide
sequencein the DNA encoding for the amino
T4 lysozyme aci ds (resi dues 75 to 83) that bi nd to cal ci um i s
Replacement of glycine by any other amino removed. Then, several other amino acids are
acid in the protein structure, in general, decreases modi fi ed i n subti l i si nby tri al and errormethod.In
the stability.On the other hand, proline residues facl, success has been achieved in creating
increase protein stability. In f4 lysozyme, cal ci um-freesubti l i si nw i th good stabi l i ty and
substitutionof glycine(at position77) by alanine, activity.
and alanine(at position82) by prolineare found to
increasethe enzymestability. Asparaginase
This is an enzyme used in the control of
I M P RO V I N G K IN E T IC P R OP ER T IES Ieukemia (uncontrolledgrowth of white blood
OF ENZYMES cells). Intravenousadministrationof asparaginase
It is possibleto improvethe functionalactivities cleaves asparagineto aspartate (the reduced
of enzymes,by improvingtheir kinetic properties avai l abi l i ty
of asparagi ne cel l prol i fi rati on).
restri cts
136 B IOTECHNO LO CY

Surprisingly,the asparaginasesfrom different By using protein engineering technique, the

sourcesexhibiteddifferences in their effectiveness existing restriction endonucleaseshave been
to c o n tro l e u k e mi aT. h i s is due to the vari ati onsi n suitably modified to produce rare cutters.
the kineticproperties. Thus,the asparaginase with
a low K- value (i.e., with high affinity for P R OTE IN E N GIN E E R IN G B Y USE O F
asparaginehence more breakdown)has to be GE N E FA MILIE S
selected for therapeutic use in the control of
leukemia. The recentdevelopmentin proteinengineering
is DNA shuffling, also known as molecular
Tyrosyl t-RNA synthetase breeding (developedby Nesse in 2000). This
methodcan be appliedto a proteinif it belongsto
The enzyme tyrosyl tRNA synthetasefrom a know n protei nfami l y.The techniquepr im ar ily
Bacillusstearothermophilus hasbeen modifiedwith involvesisolationof genesfrom each species,and
regard to substratebinding i.e., K^ value. This then creationof hybridsin differentcombinations.
enzymecatalysesthe two reactionsgiven below to While applyingthis approachto subtilisin,genes
fi n a l l yg i v e ty ro s i n et-R N A . {rom 26 specieswere mixed to createa library'
Tyrosine+ ATP--+ Tyrosyladenylate + PPi Among these, 4 enzymeswere found to have
improvedproperties.
Tyrosyladenylate + t-RNA -----+
TyrosineGRNA+ AMP
P R OTE IN E N GIN E E B IN G TH RO UG H
Replacement of threonine (at position 51), by C H E MIC A L MOD IFIC A TION S
eitheralanine or proline, variantsof tyrosyl I-RNA
synthetasehave been produced.Alanine variant A l though w i th l i mi ted succ ess, chem ical
has a two fold binding affinity (low K,n)for ATP. modifications of proteinshave been attemptedto
.l increase the stabilityof proteinsto hightemperature
And for proline variantATP binds about O0-fold
more tightly (very low K.). (Note : The proline in and organic solvents. The amino acid lysine
generaldistortsthe helical structureand reduces residuescan be cross-linkedby use of chemical
the enzyme affinity to the substrate.The one Iinkers.
mentionedhere is surprisingly, an exception). The most extensivelyused protein cross linker
is glutaraldehyde. lt stabilizes the proteins in
Restriction endonucleases solutions.By usingglutaraldehyde, certainproteins
The p ro te i n e n g i n eeri ng studi es can be (hemoglobin, insulin,phosphofructokinase,lactate
successfullyemployed for modifying enzyme dehydrogenase) have been stabilized.
specificities.This is what has been achieved in
oligonucleotide-directed mutagenesis with respect P R OTE IN E N GIN E E R IN G_A N EVER
to restrictionendonucleases. E X P A N D IN G FIE LD
So far,about2500 restrictionendonucleases are
Selectedexamplesof protein engineeringare
known. However,since most of these enzymes
describedabove only to highlight the scope of
recognizethe samesequenceon the DNA for their protein engineering.The techniquesof enzyme
action, there are only about 200 different
engineering are rapidly expanding and several
recognitionsites.Thus there is an overlap in the
newer developmentsare in the offing to develop
recognitionsitesof severalrestrictionenzymes.As proteins
/ enzymesfor industrialand therapeutic
such, there are two types of restriction purposes.
endonucleases-the frequent cutters which
recognizea sequenceof 4-6 bp and rare cutters The question before biotechnologists
recognizinga sequenceabove B bp. The rare specialized in protein engineering is not /what
cuttersare more usefulfor producinglarge DNA modifications are possible', but'how do I achieve
frasments. the modifications I wish to make'.
Jhe
p rep ara t ion of r ec om binant DNAs , t h e r r
I in se rtion in t o v ec t or s ,and t he c loning m et h o d s
have been described (Chapter6). The expressionof
clon ed g en es in t he hos t or ganis m sis dealt wit h r n
this chapter.
SEL ECTION O F HO ST CELLS FO R
GENE EXPRESSI O N
The n atu re o f a hos t c ell or an or ganis m is a s
important as the nature of a vector. The most
important requirementsof a good host include its
suita ble cu ltiv at ion in t he labor at or y , bes i d e s
incorporating the vector's genetic material. Several
prokaryotesand eukaryotesare employed as hosts
to expressforeign genes (Refer Chapter 6).
Prokaryotic hosts
The bacterium Escherichia coli was the frrsr
o rga nism to be us ed in r ec om binant D N A
technology experiments,and continues to be a fiosf
of choice for commercial production of proteins.
The extensiveuse of E. coli is mainly due to its high
ra te of rep rod uc t ion ( t he c ells double in num b e r ,
every twenty minutes),besidesgood knowledge on
its biochemistry,physiology and molecular biology.
There are a few disadvantagesalso in using F. coll
as a host. These include a relatively poor export
system for proteins and the production of
endotoxins which are often difficult to remove
(from other useful products)
The other host bacterium in use is Bacillus
subtilis. This organism is widely employed for
commercial production of antibiotics, industrial
e n z y m e s , i n s e c t i c i d e se t c
Eukaryotic hosts
I t i s o f t e n d e s i r a b l et o u s e e u k a r y o t i c o r g a n i s m s ,
w i t h a w e l l d e f i n e d n u c l e u sa n d c e l l u a r o r g a n e l l e s ,
as hosts. The main advantage with eukaryotes rs
that they bring about several post-translational
modifications to make viable and functional
proteins Further, use of eukaryotic hosts is not
associatedwith the generation and interferenceof
toxins which is the case with some prokaryotes.
Thus, eukaryotic gene expression systems are
preferred for the production of proteins and
therapeutic agents that are useful for humans and
a ni m a l s .
The yeast Saccharomyces cerevisiae is widely
used as a host for the exoression of cloned
eukaryotic genes.The other yeastsin use include
Kluveromyces lactis, Schizosaccharomyces pombe
and Picha pastoris.
The insect cells which are infected bv
baculovirus are in use as hosts in recent years. The
b a c u l o v i r u ss y s t e mc a n c a r r y a n d e x p r e s sh u n d r e d s
o f g e n e s i n i n s e c t c e l l s . A n o t h e r a d v a n t a g ei s t h a t
the safety factor since baculovirusesdo not infect
h u m a n s , o t h e r v e r t e b r a t e so r p l a n t s .
Mammalian cells such as mouse cel/s can be
used as hosts to produce complex proteins with
optimal biological functions. But the limitations
137
138 B IOTE C HNO LO CY

w i th m a m m a l i a nc e l l sa re th a t t he techni ques are foreign proteins(i.e., fusion proteins).The fusion

tedious,often difficult,and also expensive. proteinsin fact protectthe proteolyticdegradation
T h e ma n i p u l a ti o n o f g e n e e xpressi on,
as i t i s of cloned gene product.The synthesisof fusion
carriedout, in prokaryoticand eukaryoticcells is protei ns i s achi eved by l i gati ng the coding
b ri e fl yd i s c u s s e idn th e fo l l o w i ngpages. sequencesof two genes (cloned gene and host
gene).However,it is absolutelyessential to ensure
that clonedgenecontainsthe correctsequencefor
the synthesisof the targetprotein.
Cleavage of fusion proteins : The fusion
proteins, as such, interferewith the biological
The prime objectiveof gene cloning is to finally activity of the target protein. Therefore,these
result in the large scaleproduction of proteins tor proteinsshould be cleavedto releasethe specific
a v a ri e ty o f p u rp o s e s(i n d u s tri al ,commerci al , desi redfuncti onalprotei ns.
h u m a nh e a l tha n d w e l fa re ). T h i si s achi evedby the Uses of fusion proteins.: The purificationof
m a x i m a l e x p re s s i o no f c l o n e d genes through recombinantproteinsis much easierin the form of
m a n i p u l a ti o n sT. h e fo l l o w i n g are the i mportant fusion proteins.Fusionproteinsare also usefulfor
featuresof geneexpression that can be considered generatingantibodiesagainsttargetproteins.
fo r m a n i o u l a ti o n .
. The presenceof regulatablepromoters. Tandem gene arrays
. The numberof copiesof cloned genes. In general i,ncreasei n the numberof plasm ids
r The location of the cloned genes whether (containing clonedgene)proportionately increases
insertedinto a plasmidor integratedinto host the productionof recombinantprotein.This has a
genome. drawback.As the plasmid number increases, the
also increase.
genestoding for antibioticresistance
. The translationefficiencyof the host.
The overall effect is that the regularrnetabolic
. The cellular locationof the foreignproteinand activitesof the host cell are disturbedfor the
i ts s ta b i l i tyi n th e h o s tc e l l . synthesisof plasmid proteins.Consequently, the
Someof the strategies that are employedfor the yield of cloned gene productis not optimum.
manipulationof gene expressionin E. coli are An alternativeapproach is to clone multiple
discussedhereunder. copies of the target gene on a single plasmid
(i nsteadof a si ngl egene on a pl asmi d ) .I n t his
Regulatable pvomoters manner/tandemarraysof a Benecan be created.
The presenceof a strongregulatablepromoter However,eachsequenceof the genesshouldbe in
sequenceis essential for an effectiveexpression of correctorientationfor transcription and translation.
a cloned g e n e .
T h i s i s a c h i e v edby empl oyi ngthe
promoters of E. coli /ac (lactose)operon or trp Efficiency of translation
(tryptophan) operon.Thesepromotershave strong The quantity of the cloned gene product
affinityfor RNA polymerase, and consequently the produceddependson the efficiencyof translation.
dounstreamregion(of cloned gene)is transcribed. In general the , bi ndi ngabi l i tyof mR N A wit h t he
The promotersthus providea switchfor turningon ri bosomalR N A , at transl ati onal i ni ti ati o nsignal
or turningoff the transcriptionof a.cloned gene. called ribosome binding site determines
translation. Thus, the efficiency of translation is
Fusion proteins betterif the bindingof mRNAto rRNA is stronger.
fhe combination of a foreign protein (encoded The actual binding between mRNA and rRNA
by a cloned gene)with the host protein is referred occurs by complementarybase pairing of a
to as a fusionprotein. sequenceof 6-8 nucleotides.
In general,the foreignproteinssynthesized are To achieve maximum translation,the E. coli
rapidly degraded. This can be reduced by expression vectorsare designedto possess
a strong
covalently linking a stable host protein to the ri bsomebi ndi ngsi te.
Chapt er11 : M AN IPU L AT ION
OF C EN EE XP R E S S ION
l N H OS TC E LLS 139

Stability of proteins
T he half - liv eosf re c o m b i n a nptro te i n sa reh i g hl y '.{- Plasmid
v ar iable, r ang i n g fro m mi n u te s to h o u rs . The
stabilityof proteinscan be increasedby adding
amino acidsat the N-terminalend of the proteins.
T hus ,by at t ac h i n gme th i o n i n es, e ri n ea n d a l a n i ne Chromosomal
DNA
to the N-terminal end, the half-life of
B-galactosidase can be increased from 2 minutesto
20 hours!Frequently, a singleamino acid addition
at N-terminalend stabilizesthe protein The yield
of recombinantDNA proteinscan be enhancedby
inc r eas inghalf -li v e s .

Secretion of proteins
The stabilityof a protein and its secretionare Fig. 11,1 : lntegration of a cloned DNA into
interrelated.An amino acid sequence (signal chromosomalDNA.
peptide)may be attachedto a protein to facilitate
its secretionthroughcell membrane.Recombinant
proteinssecretedinto the growth medium can be metabolicload.Theseincludeincreasedutilization
eas ilypur if ied . of energy for replication and maintenanceof
plasmids,overproductionof proteins(also drains
Integration of cloned DNA into the amino acids, tRNAs),and interference of foreign
host chromosome proteinson the host cell function.
The use of plasmids for transcriptionand
translationof cloned DNA imposesa metabolic
load (discussed later)on the host.In addition,there
is of t en a c han c eo f l o s i n gp l a s mi d sd u ri n g cel l
multiplication.Theseproblemscan be overcome
by integratingthe cloned DNA directly into host
chromosomal DNA. Once the cloned DNA Expression of cloned genesin eukaryoteshas
becomesa part of genome, it can be maintained certain advantages.The most important being
for severalgenerations. the ability of eukaryotic organisms to bring about
post-translational modifications--glycosylation,
Cloned DNA integrationinto the host DNA is
phosphorylation, correctdisulfidebond formation,
possible only when .there is a complementary
proteolytic cleavage etc. Eukaryoticexpression
sequenceof about 50 nucleotidesbetweenthem.
systemsproduce stable and biologically active
The exchangeof DNAs occursby a recombination
proteins.This is in contrastto the prokaryotic
process(Fig. 11.1\.The cloned DNA lies in the
expression of cloned genes.
middle of plasmidDNA. On physicalcontactwith
chromosomalDNA, base pairing occurs between In general,the eukaryoticexpression of cloned
plasmid DNA (x and y) and chromosomalDNA genes is quite comparableto that occurs in the
(x' and y'). And the cloned DNA is transferred to prokaryotes. However, from the technical
host chromosomalDNA by a physicalexchange perspective,it is more difficult to conduct
i.e., recombination. experimentswith eukaryoticcells. Many a times,
vectorswith two distinctoriginsof replicationare
Metabolic load used.They serveas shuttlevectorsand functionin
prokaryoticas well as eukaryotichosts.
The presence of cloned DNA alters the
metabolism and cellular functions of the host The inserlionof a foreign DNA into bacterial
organism.Suchmetabolicchangesare collectively and yeast cells is referred to as transformation
referredto as metabolic load, metabolic drain or (ReferChapter6). The term transfecfionis usedfor
metabolic burden. Thereare severalcausesfor the the introduction of a foreignDNA into animalcells.
 14|J BIOTECHNOLOCY

The insert DNA in the eukaryotic cells may be IO

associatedwith vector or integrated into the host LTC
chromosomal DNA.

SACCH ABOMYC ES C EB EV'S' AE_

ForeignDNA
THE YEAST IN EXPRESSING
C L ON ED GE N E S

The common yeastSaccharomyces cerevisiaeis

widely usedas a hostfor the expression
of cloned LTC RT
genes.There are many justifiablereasonsfor its
Yeastartificialchromosome
extensiveuse.

. S. cervisiaeis single-celledthat can be easily Fig, 11.2 : Construction of yeast artificial chromosome
geneticsand physiology (LT-Left teIomere; C-Centromere; RT-Rig ht teIomere).
grown. lts biochemistry,
are quite known.
r lt has a naturallyoccurringplasmidand strong tandemgenearrayshasnot met with success,
since
promotersfor efficient expression. they are unstable.
r 5. cerevisiae can bring about many post- 2. Integratingvectors : They are basicallythe
changesin proteins.
translational integrationof cloned genes with chromosomal
o The secretedrecombinantproteinscan be easily DNA. These are not frequentlyused, since the
proteinproductionis low.
isolated, since very few host proteins are
secreted. 3. Yeast artificial chromosome (YAC) :
. The U.S. Food and Drug Administrationhas lntroduced in 1987, YAC is a fragmentof yeast
certified S. cerevisiaeas a generally recognized DNA that will accept a foreign DNA of about
as a safe (GRA9 organism. 250-500 kb in length.In fact, the yeastDNA is
only about 1% of the total DNA which however,is
As such,S. cerevisiaehasbeen in usefor several very important,since it containsthree essential
decades in baking and brewing industries. genesrequiredfor replication.Theseare the genes
Biotechnologists work quite comfortablywith this for telomere (that protects DNA from nuclease
yeast to produce a large numher of recombinant degradati on and thus mai ntai ns st abilit y) ,
proteins. These include insulin, q-antitrypsin, centromere(forms spindlesduring cell division)
hepatitis B virus surface antigen, platelet and the origin of replication (where DNA
derived growth factor, fibroblast growth factor polymeraseinitiatesreplication). YAC behavesjust
and HIV-l antigens.These products are in use like a chromosomeand replicates.
as diagnosticagents,vaccines,and therapeutic
agents. The construction of the veast artificial
chromosomeis depictedin Fig. 11.2.Two opposite
ends of a yeast chromosomenamely the left
Vectors for S. cerevisiae
telomereand right telomereare chosen.The left
Thereare three typesof vectorsfor S. cerevisiae: telomereis then attachedto a centromere. A large
segmentof the foreign DNA is added and all the
1. Episomalor plasmidvectors. three are ligated.Unlike the plasmidvectors,the
stabilityof YAC increases as the sizeof insertDNA
2. Integrating vectors.
IncreaSes.
3. Yeastartificialchromosomes (YACs).
YACs have not been used for commercial
1. Plasmid vectors : Among the vectors, productionof recombinant proteins.However,they
plasmidswith singleclonedgenesare widely used. have been employed successfullyfor physical
Manipulationwith growth conditionsincreasethe mappi ngof genomi cD N A s,parti cul arly in hum an
vector stabilityand expressionefficiency.Use of genomeproject,(Fordetails,ReferChapter12).
ChA O t CT OF C E N EEX PhE S S ION
11 : M A N IP U L A T IO N IN H OS TC E LLS 141

Post.translational modifications by
S, cerevisiae Table 11.1 Selectedexanples of reconbinant
proteins producedby baculovlrusexpyesslon
The heterologous proteins synthesized by vectoY systen
S. cerevisiaeundergo post-translational changes
while they are beingexportedinto the extracellular Adenosine deaminase
environment.To facilitate protein secretion, a phosphatase
Alkaline
single (leader) peptide is attached to the Amyloidprecursor protein
protein. This peptide is removed by the yeast Anthraxantigen
endoprotease. DNApolymerase a
Erythropoietin
OTHER YEAST EXPRESSION SYSTEMS HIV-lenvelope protein
(a, B)
Interferons
Despitethe very successfuluse of 5. cerevisiae lnterleukin-2
for generatingrecombinantproteins, there are Malariaproteins
certainlimitations.Theseincludea very low or a
Pancreaticlipase
of someproteins
limitedyield,difficultyin secretion
Poliovirusproteins
and hyperglycosylation. Attemptsare being made
Rabiesvirusproteins
to explore the utility of other yeasts for the
production of hepatitis B virus surface antigen Rhodopsin
(HBsAg) and bovine lysozyme. The yeast, Simianrotaviruscapsidanligen
Hansenula polymorpha, is employed for the Tissueplasminogen activator
synthesis of a- and p-globin chains of human
hemoglobin.
cloned gene expresses,and large quantitiesof
recombinantproteinsare produced.Becauseof a
INSECT CELL EXPBESSION SYSTEMS close similarity in the post-translational
Culturedinsectcells are in use for expressing modifications between insects and mammals,
cloned DNAs. Baculovirusesexclusively infect biologicallyactive proteins can be producedby
insect cells. The DNA of these virusesencode this approach.
And in fact, by usingbaculovirusas
for several products and their productivity in an expressionvector system, a good number of
cellsis very high to the extentof morethan 10,000 mammalian and viral oroteins have been
(Table11.1).
t im es c om oare d to ma mma l i a n c e l l s . Be si oes synthesized
carrying a large number of foreign genes, the
baculoviruses can effectivelyexpressand process Baculovirus expression vector system
the productsformed.Anotheradvantage with these The most commonly used baculovirus is
viruses is that they cannot infect humans, other Autographa californica multiple nuclear
vertebratesor plants.Thus, baculovirusesare safe polyhedrosis virus (AcMNPV). lt can grow on the
vectors. insectcell lines(e.g.,derivedfrom fall armyworm)
and produce high levels of polyhedrin or a
Polyhedrin gene of baculovirus recombinantorotein.
The organizationof a baculovirus(AcMNPV)
The polyhedrin gene is responsiblefor the
transfervectoris shown in Fig. 11.3A.lt consistssf
synthesisof a matrix protein-polyhedrin.This
protein is synthesizedin large quantities by an E. coli-basedplasmid vector along with the
D N A of bacul ovi rus.
Thi s i n turn has A cMN P V
baculovirusduring the infectioncycle. Polyhedrin
protects the virus from being inactivated by DNA, a polyhedrinpromoterregion,cloning site
for insertDNA and polyhedrinterminationregion.
environmental agents.The promoterfor polyhedrin
gene is very strong. However,the life-cycleof When the insectculturecells,transfectedwith
baculovirusdoes not depend on the presenceof AcMNPVare mixedwith transfervectorcarryinga
this gene. Polyhedringene can be replacedby a clonedgene,a doublecrossover occurs.The result
cloned gene, and the genetically engineered is that the cloned gene with polyhedrinpromoter
baculovirus can infectthe culturedinsectcells.The and termination sequencesgets integratedinto
142 B IOTE CHNO LO CY

VectorDNA S'-ACMNPV Pp Cs Pt 3'-AcMNPV VectorDNA

, DNA DNA

( B)
Transfer
vector

5' Pp Clonedgene pt 3'

AcMNPV

Recombinant
AcMNPV

Fig. ll,3 : Baculovirus expression vector system (A) Organization of baculovirus transfer vector (B) Replacement
of the polyhedrin gene of baculovirus with a cloned gene from a transfer vector (AcMNPll-Autographa californica
multiple nuclear polyhedrosis virus; Pp-Polyhydrin gene promoter; Cs-Cloning site; Pt-Polyhedrin gene
termination; Note : The coding region of polyhedrin gene not shown in A).

AcMNPV DNA (Frg. 11.38). In this process/ Modifications in the production of

poly hedr in Bene is los t . T h e r e c o m b i n a n t recorr?binant baculovirus
bac ulov ir us c ont aining c loned g e n e i s i s o l a t e d ,
The ori gi nal method of creati ngre com binant
The hos t ins ec t c ult ur e c e l l s , o n i n f e c t i o n w i t h bacul ovi rus has undergone several changes.
recombinant baculovirus, produce heterologous Incorporati onof a uni que B su 361 r qst r ict ion
proteins. A large number and a wide variety of endonucl ease si teon the pol yhedri ngen eincr eases
r ec om binant pr ot eins ( ar ou n d 5 0 0 ) h a v e b e e n the yi el dof recombi nant product iont o
bacul ovi i us
synthesized in the laboratory. A majority of them about 30% from the normal l 7o.
(>95%) have the requisite post-translational Bacmid : This is shuttlevector for E. coli ano
modifications. A selected list of recombinanr i nsect cel l bacul ovi rus. C onstruct ion of a
proteins is given in Table 11.1. recombinantbacmid is a novel approachto carry
 l N H OS TC E LLS
O F C EN EE XP RE S S ION
- - : c t er 11 : M A N IP U L A T IO N 143

termi natethe transcri pti on.A mpi ci l l i n resi stant

Ampt
OriE
p pa
mcs
Fig. 11,4 : A diagrammatic representation of
nammalian exprcssion vector (p-Promoter sequence;
Pa-polyadenyl ation seq uence; mcs-M ultiple cloni ng
site; sm-Selectablemarker gene; OrPuk4rigin of
eukaryotic replication; OrF-Origin ol E. coli replication;
Ampt-Ampicilin rcsistant marker gene).
marker gene can be used for selecting the
transformedE. coli cells.
Markers for mammalian expression
vectors
Thereareseveralmarkersin usefor the selection
of transformedmammaliancells.The bacterialgene
(Neor)that encodesfor neomycin phosphotrans-
feraseis frequentlyused.The other markersare the
genesthat encode for the enzyme dihydrofolate
reductase(DHFR),and glutaminesynthetase (CS).

o ut all t he gen e ti cma n i p u l a ti o n si n c l u d i n gt he

expression of baculovirusvector in E. coli.
Useof yeastcells: The geneticmanipulations of
A c M NP Vgenom ec a n b e d o n e i n y e a s tc e l l sw ith
yeast-insect shuttlevector.Then the recombinant
is in tro d u c e di n to i n s e c tc e l l s .
bac ulov ir us
Several proteins have been produced by
mammalianexpression vectors.Theyield of protein
synthesisin some cases,is increasedby inserting
an intron betweenthe oromoterand the cloneo
gene. However,the reasonfor this is not clear.

M A M M A LI A N C E L L EX PR ES SION Goordinated expression

VECTORS By coordinating the expression of a clonedgene
Mammalianexoressionvectors are useful for and a selected marker. the production of
the production of specific and authentic recombinant protein can be increased.
recombinant proteins (Ior use as therapeutic The coordinatedexpressionof DHFR (marker)
agent s ) .ln add i ti o n , th e y a re a l s o h e l p fu l for and a cloned gene is illustratedin Fig. 11.5.The
n f ma mma l i an DHFRgene is insertedcloseto a clonedgeneand
s t udy ingt he f unc ti o na n d re g u l a ti o o
g enes . I n gene ra l , th e ma mma l i a n e x p re s si on both of them are under the control of a single
vectorsare quite comparableto other eukaryotic promoter and termination (polyadenylation)
expression vectors. However, large-scale sequences. The DHFR gene is flanked by intron-
production of recombinant proteins with removingsequence.As the genesare expressed,
engineer ed m am ma l i a nc e l l s i s c o s tl y . D H FR i s synthesi zedby pri marytranscri ptw hi l e
A diagrammaticrepresentation of mammalian the recombinant protein is formed from spliced
vectoris shownin Fig. 11.4.lt containsa eukaryotic mR N A .
or iginof r eplica ti o n fro m a n a n i ma lv i ru ss u c h as
S im ianv ir us40 (SV 4 0 a ) n d a p ro k a ry o tioc ri g i nof Expression of two cloned genes
replication(from E. colrJ.The mammalianvector Some proteinsare composedof two or more
hasa m ult iplec l o n i n gs i tea n d a s e l e c ta b lma e rker subuni ts.For exampl e,hemogl obi ni s a tetramer
gene. Both of them are under the control of w i th tw o copi esof each subuni ti .e., a2B r. E ach
eukaryotic promoter and polyadenylation subunit can be separatelysynthesizedby the
sequences.These sequencesare obtained from correspondingcloned gene, and they are then
eit heranim alv iru s e s(S V4 0 ,h e rp e ss i m p l e xv i rus) mixedto form the multimericproteinin vitro.This
or m am m ali a n g e n e s (g ro w th h o rmone, method is not very satisfactory since the in vitro
metallothionein). The promotersequences facilitate assemblyof multimeric proteinsis not efficient.
the transcriptionof cloned genes(at the multiple Techniques havebeendevelopedin recentyearsto
cloning site)and the selectable marker genes. On produce two differentproteins(i.e.,subunitsof a
the other hand, the polyadenylationsequences mul ti meri cprotei n)i n the samecel l si mul taneousl y.
144 B IOTECHNO LO G Y

genescan be placedunderthe independentcontrol

of promoters and polyadenylationsequences
(Fig, 11.V to producethe assembledproteinwith
two subunits.This method, however,does not
ensure production of equal quantities of two
subun its.

Dicistronic expression vector

A dicistronicvectorcan be constructed with two

cloned genesjoined togetherto a small sequence
of DNA that containsan internal ribosomal entry
sife (lRE9. lRESs,found in mammalianviruses,
al l ow a si mul taneous
transcri pti on
and t r anslat ion
to producethe assembledprotein(Fig. l1.B). The
transcription of gene-IRES-gene is underthe control
of a si nge promoterand termi nati onsignals.By
usingdicistronicexpression vector,fhe synthesisof
equal amounts of the protein subunits can be
achieved.

n
tl oI mRNA
spticed
Dihydrofolate
reductase
ITranslation
I
J
As the recombinantproteinsare producedby
the clonedgenes,they startaccumulating. The next
task is to collect and . purify the specific gene
Recombinantprotein product i.e., the requisiteprotein.This is not an
easy job since many a times the recombinant
Fig. 11.5: Coordinatedexpressionof DHFRand a proteinis foreignto the host cell which possesses
cloned gene (DHFB-Dihydrofolatereductase; an enzyme machinery to degrade the outside
p-Pronoter Sequenee:pa-Polyadenylation6equence). protei ns.Thus, human i nsul i n producedin t he
bacterial host cells can be degraded by the
proteases. This problem can be solved by using
Two.vector expression system bacterialstrains(e.9.,E. coli) deficientin proteases.
Two expression vectors,each carryinga cloned But there is a disadvantage sincethe proteases are
genefor each subunit,are co-transfected into host defensiveenzymes and hence the new strains
cells (Fig. 71.6). These genes encode for the lacking these enzymesare susceptiblefor easy
c o rre s p o n d i npgro te i ns u b u n i tsTheythenassembl e destruction.
to form a functional protein. The two-vector
ln an alternative approach, Ihe recombinant
expression systemhas certainlimitations - lossof
proteins are fused with the native host proteins
one vector/overexpression of clonedgenein one of
(Referfusion proteinsp. 138).The fusion proteins
th e v e c to rs .T h i s c a u s e sa n i mbal ancei n tne
are resistant to protease activity. Some times,
fo rm a ti o na n d a s s e m b l o y f s ubuni ts.
foreign proteinsaccumulateas aggregates in the
Two-gene expression vector host organi sm, mi ni mi zi ng the pr ot ease
degradation. The problemwith proteinaggregates
A single vector carryingtwo cloned genes is is that the biologicalactivity may be lost while
used to overcomethe above problem. The twcr extractingthe proteinfrom the aggregates.
 IN H OS TC E LLS
OF C E N EEX PR E S S ION
11 : M A N IPU L AT ION
ChA P t CT 145

Clonedgenea

r t
jpa
Jpa RNA

I
+ J
E
Protein
,
subunit
oProtein
subunitp

EO
Assembledorotein
with2 subunits

Fig. 11.6: Two-vector expression system for producing a dimeric protein (p-Promoter sequence;
pa-P olyadenylation sequence)

Cloned Cloned
gene cl geneB
I
+
I
+

rJpa rJpa p IRES pa

I
+
RNA-

IJ - RNA

TO
Protein I I Protein Protein I I Protein
subunit subunit
B subunislt ------T---- l subunipt
----l--- +
flo
EN Assembledprotein
with two subunits
Assembledprotein
with two subunits
Fig. 11.8 : Dicistronic expression vector for producing
Fig, 11.7 : Two3ene expression vector for producing a dimeric protein (p-Promoter sequence;
a dimeric protein (p-Promoter sequence; pa-Polyadenylation sequence; IRES-lnternal
pa-Polyad enylation sequence). ribosomal entrv site).

Biotechnology [10]
146 B IOTECHNO LO CY

Export and secretion of reqombinant

proteins

The yield of productionof recombinantproteins

is efficientif they arequicklyexportedand secreted
i n to th e e n v i ro n me n t (s urroundi ngmedi um).
Further,the recoveryand purificationof foreign
proteins is easier from the exported proteins. Signal
Seriouseffortshavebeenmadeto developmethods sequence
for increasing the exportof recombinantproteins.
Someof the speciesof the bacterium,Bacillus
subtilis normally secrete large quantities of
/-----\__.
extracellularproteins. A short DNA sequence, | +.-t
called signal sequence from such species is | | Signal
peptide
introducedinto other B. subtilis.These bacteria -
produce recombinantDNA tagged with signal Recombinant
proteintagged
peptide, which promotes export and secretion
(Fig.11.9).The signalpeptidecan be removedafter
purificationof foreignprotein.
t_
I Ennanceo
expon
andsecretion
I
S i g n a ls e q u e n c e a n d s i g nalpepti dehave been, J
in fact, effectivelyused for the production of f------]--'
re c o m b i n a ni tn s u l i n .
I t--
P U R IF IC A T IO N OF R E COMB IN A N T
PROTEINS I Purification
and tag removal
I
There are severaltechniquesin use for the
purificationof recombinantproteinsfrom a mixture
+
of secretedproteins. I I r-------r
I I sis."l
peptide
Fusion proteins and purification Recombinant
protein
The production of fusion protein has been
d e s c ri b e d(Se e p . 1 3 8 ). B esi desreduci ng the
Fig. 11.9 : Role of signal peptide in the collection
degradation, fusion proteins simplify the
of recombinant Drotein.
purificationof recombinantproteins.A couple of
p u ri fi c a ti o n te c h n i q u e s a re bri efl y di scussed
hereunder.
The DNA sequence thatencodesfor six histidine
resi dues(H i su)i s l i gatedto a cl oned gene. The
Affinity tagging fusion protein (i.e., recombinantprotein tagged
with Hisu)can be isolatedby passingthe mixtureof
In th i s te c h n i q u e ,a s mal l D N A sequence
cell extract througha column packedwith nickel-
encoding for a peptide (a short amino acid
triacetic acid agarose beads. The desired
sequence)is ligated to the cloned gene. This
protei n through i ts he xahist idine
peptide, in turn, has an affinity to bind to a recombi nant
resi duesbi ndsto the ni ckeli ons(N i 2+)The
. r estof
compound or a macromoleculeor even an
the proteinscan be easilyelutedfrom the columrt.
element.
The bound fusion proteinscan be then eluted by
The taggedaminoacid sequence, which formsa loweringthe pH of the buffersolution.Alternately,
part of the recombinantprotein acts as an affinity the fusion proteinscan be selectivelyremovedby
tag or identification tag. The use of hexahistidine bi ndi ng of heaxahi sti di ne tag to a com pet it or
tag is brieflydescribedin the next column. comooundsuch as imidazole.
 IN H OS TC E LLS
OF C EN EEX PR ES S ION
11 : M A NI PU L AT ION

e- The next step is to femove the hexahistidinetag.

I l__l T h i s c a n b e d o n e b y d i g e s t i o nw i t h p r o t e a s e sw h i c h
t\
Marker I specifically act at the site of the tag There is a
peptide Interleukin-2 need fo remove hexahistidine residues only if
Other proteins the recombinant protein is being used as a
Fusionprotein
therapeutic agent. For all other purposes/
hexahistidine tag is acceptable, since it does not
(- PolYProPYlene interferein the normai structureand function of the
suppod Drotetns.
- Anti-marker
PePtide lmmunoaffinity purif ication
antibodY
column
lmmunoaffinitY T h e i m m u n o a f f i n i t yc h r o m a t o g r a p h i cp u r i f i c a t i o n
I
I Proteinmixture technique of fusion proteins with special reference
I loaded t o h u m a n i n t e r l e u k i n - 2i s d e s c r i b e d .
Y

Interleukin-2 gene joined to a small DNA

s e q u e n c e e n c o di n g a m a r k e r p e p t i d e ( t h a t
- MarkerPeptide
of fusionProtein synthesizes B amino acids, Asp-Tyr-Lys-Asp-
bindsto antibody Asp-Asp-Asp-Lys) produces a fusion protein tn
yeast (5. cerevisiae). The marker peptide has a
1
> ?A
dual function-reduces the degradation of
/2* 6<- Other Proteins
- \= O pass througn interleukin-2, besides helping in its purification
I column (Note : The eight-amino acid marker peptide is
I
+ commercially available under the brand name
n
> lll FusionProteins FIag peptide).

eluted The fusion protein, interleukin-2 joined to a

\ ||
marker peptide can be purified by immunoaffinity
'-l) chromatography (Fig. 11.10). The specific

t ?_-. II
monoclonal antibodies (MAb) against the marker
peptide, are immobilized on a polypropylene

t o<---------------
I-

L_\
lnterleukin-2
MarkerPePtide
support. These MAb serve as the ligand (an[-
marker peptide antibodies) and selecfively bind to
fusion proteins tagged with marker peptide'
However, the remaining proteins pass through the
Fig. 11.iO : tmmunoaffinity chromatographic immunoaffinity column. The immunopurified
purification of a'fusion Protein fusion proteins can be eluted later from the
column.

<-
 Humqn Proiecl
Oenome
he m os t im por t ant f e a t u r e s o f a D N A
m olec ule ar e t he nuc le o t t c l es e q u e n c e s a
, nd the
ident if ic at ion of genes an d t h e i r a c t i r r i t i e s .S i n c e
1920, s c ient is t shav e bee n n , o r k i n g t o d e t e r r r r i n e
t l. r es equenc esof piec es o f D N A . T h i s w a s f u r t h e r
ex t ended ior t he c onr plete s e q u e r - r cdee t e r m i n a t i o n
of genonr e of c er t ; r in lor v e r o r g a n i s r r se . g p l a s m i d
pBR 322 in 1979 The r r i t o c h o n d r i a l g e 'n o m er 'v a s
s equenc ec lin 1981
The hum an genom e p r o j e c t ( H C P I w a s
c onc eiv ed in 1984, and o f f i c i a l l y b e g u n i n e a r n e s t
in O c t ober 1990. The pr i n r a r y o b j e c t i v e o f H C P
was t o det er m ine t he nu c l e o t i d e s e q u e n c e o f t h e
ent ir e hum an nuc lear ge n o m e . I n a d d i t i o n , H C P
was als o ent r us t ed t o el u c i d a t e t h e g e n o m e s o f
severalother model organismse g, Escherichiacoli,
Saccharontyces cerevisiae (ycast), Caenorhabditis
ele5lans (roundu,ornr), Mus musculus (rirouse)
J anr esW at s on ( who eluc .i d a t e dD N A s t r u c t u r e )w a s
t he f ir s t Dir ec t or of HCP
ln 1997, Unit ed St at e se s t a b l i s h e dt h e N a t i o n a l
Human Genome Research Institute (NHGRI). The
HCP was an int er nati o n a l v e n t u r e i n v o l v i n g
r es ear c h gr oups f r om s i x c o u n t r i e s U S A , U K ,
Fr anc e, Cer m any , J apan a n d C h i n a , a n d s e v e r a l
indiv idual labor at or ies a n d a l a r r g c n u m b e r o f
s c ient is t sand t ec hnic iansf r o m v a r i o u s d i s c i p l i n e s .
This c ollabor at iv e v en t u r e \ v a s n a m e d a s
International Human Genome Sequencing
144
Consortium (IHGSC) and was headed by Francts
Collins. A krtal expenditure of $3 billion, and a
t i m e p e r i o d o { 1 0 1 5 y e a r s i o r t h e co m p l e ti o n o f
H C P w a s e x p e c t e d . A s e c o n d h um a n g e n o m e
project was set up by a privaie company - Celera
G e n o m i c s , o f M a r y l a n d U S A i n 1 9 9 8 . Th i s te a n 'r
was led by Craig Venter.Very rapid and urrexpected
fjrogressoccurred in HCP r,r,ithgood cooperation
between the two teams of workers and improved
r n e t h o c l si n s e q u e n c i n g .
The date 26th June 2000 will be rememberedas
one of the most important dates in the history of
s c i e n c e o r e v e n m a n k i n d . l t w a s o n th i s d a y,
F r a n c i sC o l l i n s a n d C r a i g V e n t e r ,t h e l e a d e r so f th e
t w o h u m a n g e n o m e p r o j e c t s ,i n t h e p r e se n ceo f th e
P r e s i d e n to f U S . , j o i n t l y a n n o u n c e d th e w o r ki n g
d r a f t s o f h u m a n g e n o m e s e q u e n ce . Th e d e ta i l e d
r e s u l t s o f t h e t e a m s w e r e I a t e r p u b l i sh e d r n
F e b r u a r y2 0 0 1 i n s c i e n t i f i cj o u r n a l s Na tu r e ( IH C SC )
a n d S c i e n c e ( C e l e r aC e n o m i c s )
The human genome project results attracted
w o r l d w r d e a t t e n t i o n .T h i s a c h i e v e me n tw a s h a i l e d
w i t h m a n y d e s c r i p t i o n si n t h e m e d i a
. The mystery of life unravelled.
. The library of life.
. The periodic table of life.
. T h e H o l y g r a i l o f h u m a n g e n e t i cs.
 C hapt er12 : HUM A N GE N OMEP R OJ EC T
Wcytogeneticmap
_k' "l_ ^
H Ge n e lin l( a g e m a p
Gene I Ge n e
I techniques are also useful t,or the detection of
v
I n o r m a l a n d d i s e a s eg e n e s i n h u m a n s .
- - - - Restriction
fragment
n""trl"tionlr[n,"r,t"-
Physicalmap
Basesequence
Fig. 12.1 : Different types of genome maps.
It mav however. be noted that the draft human
genome sequences were not complete, and may
rep resen ta rou nd 9 0% . The r em aining 10% is m ad e
up of sequence where few genes are located.
MAPPING OF THE HUM AN G ENO M E
The most important objective of human genome
project was to construct a series of maps for each
chro mosome . ln Fig. 12. 1, an out line of t he
different types of maps is given
1. Cytogenetic map : This is a map of the
chromosorne in which the active genes respond to
a chemical dye and display themselvesas bands on
the chromosome.
2. Gene linkage map : A chromosome map In
which the active genes are identified by locating
closely associated marker genes. The most
commonly used DNA markers are restriction
fragment length polymorphism (RFLP), variable
number tandems repeats (VNTRs) and short
tandem repeats (STRs). VNTRs are also called as
minisatellites while STRs are microsatellites.
3. Restriction fragment map : This consists of
the random DNA fraqments that have been
sequ en ce o.
4 . Ph ysica l ma p : This is t he ult im at e m ap of
t he chro mosome wit h highes t r es olut ion bas e
sequ en ce . The met hods f or DNA s equenc ing ar e
give n in Ch ap ter 7 Phy s ic al m ap depic t s t he
location of the active genes and the number of
bases between the active genes.
149
APPROACHES FOR GENOME
SEQUENCING
A list.of different methods used for mapping of
human genomes is given in Tablc 12.1. fhese
For elucidating human genome, different
approacheswere used by the two HCP groups.
IHGSC predominantly employed map first and
sequence later approach. The principal method
was heirarchical shotgun sequencing. Th is
technique involves fragmentationof the genome
i n t o s m a l l f r a g m e r r t s( 1 0 0 - 2 0 0 k b ) , i n s e r t i n gt h e m
into vectors (mostly bacterial artificial
chromosomes, BACs) and cloning. The cloned
fragmentscould be sequenced.
Celera Genomics used whole genome shotgun
approach. This bypasses the mapping step and
savestime Further,Celera group was lucky to have
high-throughput sequenators and powerful
computer programmes that helped for the early
completion of human genome sequence.
Whose genome was sequenced?
One of the intriguing questions of human
genome project is whose genome is being
s e o u e n c e da n d h o w w i l l i t r e l a t et o t h e 6 b i l l i o n o r
s o p o p u l a t i o n w i t h v a r i a t i o n si n w o r l d ? T h e r e i s n o
simple answer to this question. However, looking
from the positive side. it does not matter whose
genome is sequenced, since the phenotypic
differences between individuals are due to
variations in just 0.1% of the total genome
s e q u e n c e sT. h e r e f o r em a n y i n d i v i d u a l g e n o m e sc a n
be used as source material for sequencing.
Much of the human genome work was
performed on the material supplied by the Centre
for Hunran Polymorphism in Paris, France This
institute had collected cell lines from sixty different
French families, each spanning three generations.
The material supplied from Paris was used for
human Senome sequencing
HUMAN GENOME SEOUENGE.RESULTS
SUMMABISED
The information on the human genome prolects
i s t o o v a s t , a n d o n l y s o m e h i g h l i g h t sc a n b e g i v e n
(Table 12.2). Some of them are briefly described.
 15|, BIOTECHNOLOCY

Tmu 54.2 Major htghllghtsof humangenonre

mapping of genomes(and also normal and
diseasegenes in hunans) The draltrepresents about90%of the entirehuman
genome. parts
thatmostof the important
lt is believed
Method Comments
havebeenidentified.
DNAsequencing Physicalmapof DNAcanbe
a Theremaining10%ofthegenome areatthe
sequences
identified
withhighest
(i.e.telomeres)
veryendsof chromosomes andaround
resolution.
thecentromeres.
I lco nf nrnhoe
vu v v,
P,vvvs
To identify
RFLPS,
STSand
SNPs. a Human genome iscomposedof3200Mb(or3.2Gb)i.e.
Radiation
hybrid genome basepairs(3,200,000,000).
3.2billion
mappingFragment intolarge
piecesandlocatemarkersand 1.1to 1.5%of the genome
Approximately codesfor
genes.Requires
somatic ce 0roterns.
hybrids.
24%of thetotalgenome
Approximately is composed ol
Fluorescence
rn sltu a geneon
Tolocalize
introns (exons),
thatsplitthecodingregions andappear
(FISH)
hybridization cnr0m0s0me.
as repeating
sequences withno specific
functions.
Sequence
taggedsite to anypartof DNA
Applicable
(STS)mapping sequenceif somesequence Thenumberof protein genesis in therangeof
coding
information
is available 30,000-40,000.
Expressed
sequence A variant
of STSmapping; An averagegeneconsistsof 3000bases,the sizes
tag(EST)mapping expressedgenesareactually gene is the larget
howevervary greatly.Dystrophin
mapped andlocated. knownhuman genewith2.4millionbases.
gel
Pulsed-field Fortheseparationand
Chromosome 1 (thelargethuman contains
chromosome)
(PFGE) isolation
electrophoresis of largeDNA
the highestnumber0f genes(2968),whilethe Y
fragments.
chromosomehasthelowest. Chromosomesalsodifferin
in vectors
Cloning To isolateDNAfragments
of theirGCcontent andnumber of transoosable
elements
(plasmids,
phages, variable
lengths.
cosmids,
YACs,BACs) Genesand DNA sequences associatedwith many
diseasessuch as breastcancer,musclediseases,
Polymerase
chain To amplify
genefragments
andblindness
deafness havebeenidentified,
(PCR)
reaction
Chromosome
walking Usefulforcloning
of appearto havebeencopied
About100codingregions
overlappingDNAfragments and moved by RNA-basedtransposition (retro-
(restricted
to about200kb). transoosons).
jumping
Chromosome DNAcanbe cutintolarge i. Repeated about50%ofthehuman
constitute
sequences
fragments for
andcjrcularized gen0me.
usein chromosome
walking.
of the genome(- 97%)hasno known
A vastmajority
Detection
of cytogenetic Certain
genetic diseases can
functions.
abnormalities be identified
by cloning
the
affectedgenese.g.Duchenne Belweenthehumans,
theDNAdiffers
onlyby 0.2%or
muscular dystrophy. onein 500bases.
Databases Existing facilitate
databases polymorphisms
Morethan3 millionsinglenucleotide
geneidentification
by (SNPs)havebeenidentified.
comparisonof DNAand
proleinsequences :a HumanDNA is about 98% identicalto that of
(RFLP-Restriction fragmentlengthpolymorphism cnrmpanzees.
; STS-sequence
tagged site; SNP-Singlenucleotidepolynorphisn; YAC-Yeast
About200genesarecloseto thatfoundin bacteria.
artificial chromosome; BAc-Bacteri
al artificial chronosome)
C hapt er12 : HU MA N C E N OMEPR O J EC T 151

Genesand related
genesequences
12 00M b

Fig. 12.2 : An overview of the organization of human genome (LlNEs-Long interspersed nuclear elements;
S/NEs-Shorf interspersed nuclear elements; LTR-long terminal repeats).

rt,r,rtt,l Most of the genoflne sequer"e*e frs

Tmrr 12.3 Some interesting analogs/sidellghts identified
about hunan genome
About 90% of the human genome has been
in human
Thebasesequence genome wouldfillabout s e q u e n c e d .l t i s c o m p o s e d o f 3 . 2 b i l l i o n b a s e p a i r s
booksof 1000pageseach.
200telephone (3200 Mb or 3.2 Cb). lf written in the format of a
telephone book, the base sequence of hunran
lf the genomeis recitedat the rateof one baseper g e n o m e w o u l d
fill about 200 telephone books of
secondfor 24 hoursa day,it wouldtakea century to
1 0 0 0 p a g e s e a c h . S o m e o t h e r i n t e r e s t i n ga n a l o g s /
recitethebookof life. genome
sidelights of g,iven in Table 12.3.
are
ll a typisttypesat therateof 60 wordsperminute(i.e.
Individual differenc€s in genomes : lt has to be
360letters) for8 hoursa day,he/she
wouidtakearound
remembered that every individual, except identical
50 yearsto typehumangenome. twins, have their own versions of genome
lf theDNAsequence is typedin lines10cmconlainingsequences.The differencesbetween individuals are
basesand printed
60 nucleotide the humangenome largely due ro single nucleotide polymorphisms
sequence(lroma singlecell)wouldstretch of (SNPs). SNPs represent positions in the genome
a distance
5000km. where some individuals have one nucleottoe
{i . e . a n A ) , a n d o t h e r s h a v e a d i f f e r e n t n u c l e o t i d e
lf theDNAin theentirehuman bodyis putendto end, (i.e. a C). The frequency of occurrence of
it wouldreachto the sun and backover600 times SNPs is estimated to be one per l oOO base
(Note: Thehuman bodycontains cells;the
100trillion
pairs. About 3 million SNPs are believed to
lengthof DNAin a cellis 6 feet;thedistancebetween
be present and at least half of them have been
thesunandearthis 93 million miles).
identified.
Thetotalexpenditure{orhumangenome project
was$3
billionThemagnitude of thishugeamount hasto be Organization of human gellouffie
lf onestartscounting
appreciated. rateof
at a non-stop
An outline of the organization of the human
a dollarper second,it wouldtakeabout90 yearsto genome is given in Fig. 12.2. Of the 3200 Mb, only
comolete.
a small fraction (4S Mb) represents the actual
152 B IOTECHNO LO CY

Up"tr""t t

tt
Start of End of biological
biologicalinformation information

Fig. 12.3 : A diagrammatic representation of a typical structure of an average human gene

genes , while t he r es t is d u e t o g e r l e - r e l a t e d As alreaddy described, a huge portion of the

s equenc es ( int r ons , ps eud o g e n e s )a n d i n t e r g e n i c genome is composed of introns, and intergenic
DNA ( long int er s per s edn u c l e a r e l e m e n t s , s h o r t s e q u e n c e s( j u n k D N A ) .
int er s pr eadnuc lear elem en t s ,n r i c r o s a t e l l i t e sD
, NA
The major categoriesof the proteins encoded by
transposons etc.). Intergenic DNA represents the
human genes are listed in Table 12.4. The function
pa(s of the genome that lie between the genes
of at least 4O"h of these proteins are not known.
whic h hav e no k now n f u n c t i o n . T h i s r s
appropriately regarded as junk DNA.
Marked differences in individual
chromosomes
Genes present in human genome
T h e l a n d s c a p e o f h u m a n c h r o m o so m e s va r i e s
The two genome projects differ in their estimates
w i d e l y . T h i s i n c l u d e s m a n y f e a t u r e ssu ch a s g e n e
of t he t ot al num ber of g e n e s i n h u m a n s . T h e i r
number per megabase,CC content, density of SNPs
figures are in the range of 30,000-40,000 genes.
and number of transposableelements.For instance,
The main reasonfor this variation is that it is ratner
chromosome 19 has the richest gene content (23
dif f ic ult t o s pec if ic ally r e c o g n i z e t h e D N A
g e n e s p e r m e g a b a s e )w h i l e c h r o m o so m e 1 3 a n d Y
s equenc eswhic h ar e gene s a n d w h i c h a r e n o t .
chromosome have the least gene content (5 genes
Before the resultsof the HCP were announced, per meSaDasej.
the best guess of human genes was in the range of
80,000-1 00,000. This estimate was based on the Other interesting / important features of
fact that the number of proteins in human cells are human genome
80,000-1 00.000, and thus so many genes
For more interestingfeaturesof human genome,
expected. The fact that the number of genes is
refer Table 12.3.
much lower than the proteins suggeststhat the RNA
editing (RNA processing)is widespread, so that a
s ingle m RNA m ay c ode f or m o r e t h a n o n e p r o t e i n .

A diagrammatic representation of a typical

structure of an average human gene is given in
Fig. 12.3. lt has exons and introns.

A broad categorization of human gene catalog

in the tbrm of a pie chart is depicted in Fig. 12.4.
About 1 7.5"h of the genes participate in the general h
o-
\>
biochemical functions of the cells, 23"/" in the
m aint enanc e of genom e , 2 1 % in signal
23.2% s,
s"{
t r ans duc t ionwhile t he r em a i n i n g 3 \ o / o a r e i n v o l v e d
in the production of structural proteins, transport
pr ot eins , im m unoglobins e t c . .$'
Human genes encoding proteins
Fig. 12.4 : A pie chart showing a broad categorization
I t is now c lear t hat only 1 . 1- 1 . 5 '/ " o f t h e h u m a n
of the human gene catalog (About 13000 genes
genom e c odes f or pr ot e i n s . T h u s , t h i s f i g u r e
whose functions arc not known are not included).
1 .1-'l.5"k representsexons of genome.
Chapt er12 : H U M AN C E N OMEP R OJ EC T 153

model closest to human has been sequenced. A

Tlru 12.4 Dlfferent categoyiesof proteins selected list of genomes that have been sequenced
encoded by hunan genes lbased on the Human is given in Table 12,5.
Genome ProJect report, 20O71
BENEFITS/APFLICATIONS QF [*UIVIAN
Categ,oryof Percentag,e Actual number
G E N O M E S E Q I 'E N C I N G
proteins af genes
It is expected that the sequencing of human
Unknownfunctions 41.jfo 12,809 g e n o m e , a n d t h e g e n o m e s o f o t h e r o r g a n i s m sw i l l
Nucleic
acidenzymes 75% 2,308 dramatically change our understanding and
p e r c e p t i o n so f b i o l o g y a n d m e d i c i n e . S o m e o f t h e
Transcription
factors 6.0% 1,850
benefits of human Benome project are given.
Receptors 5.0% 1,543
ldentification of human qenes and
Hydrolases 4.0o/o
their functions
proteins
Regulatory 3.2Yo 988
(G-proteins,
cellcycle Analysis of genomes has helped to identify the
regulators genes, and furrctions of some of the genes. The
etc.)
functions of other genes and the interaction
Protooncogenes 2.9o/o 902 between the gene products needs to be further
proteins
Structural of 2.9fo 6/O elucidated.
cytoskeleton
Kinases 2.8% 868
Tlalr 12.5 A selected llst of genomes that
(Note: Thistableis basedon the toughdraftof humangenone
have been sequenced
repoftedby CeleraGenomics.Thdpercentages are derivedfron a
totalof 26,383genes)
fVarirr' r,lf t/;::
species (Mb/kb) (yEar)
o lt is surprisingto note that the numberof genes Bacteriophage QXl74 5.38kb Firstgenome
found in humansis only twice that presentin the sequenced (1977).
r oundwor m( 1 9 ,0 9 9 )a n d th ri c eth a t o f fru i t fl y Plasmid
pBR322 4.3kb Firstplasmid
( 13, 001) . sequenced (1979).
. Around 200 genesappearto have been derived Yeaslchromosome lll 315kb Firstchromosome
from bacteria by lateral transfer.Surprisingly, sequenced (1992).
noneof thesegenesare presentin non-vertebrate Haemophilus influenzae 1.8Mb genome
First of
eukaryotes. cellular
organism
o The proteinsencodedby humangenesare more to besequenced
comolexthan that of invertebrates. (19e5)
aronyces cerevisiae 12MB Firsteukaryotic
. The flood of the dataof humangenomeprojects Sacch
organism to be
will be high l y u s e fu l fo r b i o i n fo rm a ti c sand (1996).
sequenced
biotechnology.
Arabidopsis
thaliana 125MB Firstplant
genome to be
G E NO M E S O F S O ME O T H ER sequenced (2000).
O RG A NI S M S SE O U EN C E D
(human) 3200MB
Honosapiens Firstmammalian
Sequencingof genomes is not confined to genome to be
hum ans .F or o b v i o u s re a s o n sa n d s i g n i fi c ance, sequenced (2001).
hum an genom es e q u e n c i n ga ttra c te dw o rl d wi de )ryza sativa(rice) 430MB Firstcropplant
attention. genome to be
sequenced (2002).
In fact, the first genome sequenceof the
(mouse)
musculis 3300lvlB Animal model
e X 1 7 4 w a s d e te rm i n e di n 1 977. Mus
bac t er iophagQ
closestlo human
Yeast was the first eukaryoticorganismto be
(2003).
sequenced(1986).Recently, the mouse,an animal
154 B IOTECHNO LO CY

Understanding of polygenic disorders Knowledge on mutations

The bioc hem is t r y and gen e t i c s o f m a n y s i n g l e -
gene disordershave been elucidated e g. sickle-cell
anemia, cystic fibrosis,retinoblastoma.A majority of
the common diseases in humans, however, are
poly genic in nat ur e e. g. c a n c e r , h y p e r t e n s i o n ,
diabetes At present,w'e have very little knowledge
about the causesof these diseases.The informatron
on t he genom e s equenc e w i l l c e r t a i n l y h e l p
t o unr av el t he m y s t er ies s u r r o u n d i n g p o l y g e n i c
d iseases.
lmprovements in gene therapy
At present,human gene therapy is in its infancy
{ or v ar ious r eas ons .Cenom e s e q u e n c e k n o w l e d g e
will certainly help for more effective treatment of
Many events leading to the mutations can be
uncovered with the knowledge of genome.
of developmental
Better understanding
biology
By determining the biology of human genome
and its regulatory control, it will be possible to
understand how humans develop from a fertilized
eggs to adults.
Comparative genomics
C e n o m e s f r o m m a n y o r g a n i s m s h a ve b e e n
s e o u e n c e d . a n d t h e n u m b e r w i l l i n c r e a se i n th e
coming years.The information on the genomes of
d i f f e r e n t s p e c i e s w i l l t h r o w l i g h t o n th e m a j o r
s t a g e si n e v o l u t i o n .

Benetic diseasesby gene therapy. Development of biotechnologY

T h e d a , t ao n t h e h u m a n g e n o m e s eq u e n ce w i l l
lmproved diagnosis of diseases
spur the developrnent of biotechnology in various
In the near future, probes for many genetic spneres.
dis eas eswill be av ailable f or s p e c i f i c i d e n t i f i c a t i o n
and appropriate treatment. ETHICS AND HUMAN GENOME
The researchon human genomeswill make very
sensitive data available that will affect the personal
Development of pharmacogenomics
a n d p r i v a t e l i v e s o f i n d i v i d u a l s . F o r i n sta n ce ,o n ce
The dr ugs m ay be t ailor ed t o t r e a t t h e i n d i v i d u a l it is known that a person carries genes for an
pat ient s .This will bec om e po s s i b l ec o n s i d e r i n gt h e incurable disease,what would be the strategyof an
v ar iat ions in enz y m es and o t h e r p r o t e i n s i n v o l v e c l i n s u r a n c e c o m p a n y ? H o w w i l l t h e s o ci e ty tr e a t
in dr ug ac t ion, and t he m e t a b o l i s m o f t h e him/her? There is a possibility that individuals with
ind iv iduals . substandard genome sequences may be
discriminated. Human genome results may also
Genetic basis of psychiatric disorders p r o m o t e r a c i a l d i s c r i m i n a t i o n c a t e go r i zi n g th e
people with good and bad genome sequences.
By s t udy ing t he genes in v o l v e d i n b e h a v i o u r a l
Consideringthe gravity of ethics relatedto a human
patterns,the causation of psychiatric diseasescan
genome, about 3% of the HCP budget was
be under s t ood. This will h e l p f o r t h e b e t t e r
earmarked for ethical research.
treatment of these disoroers.
In the 1990s, there was a move by some
scientiststo patent the genes they discovered.This
Understanding of complex social trait
c r e a t e d a n u p r o a r i n t h e p u b l i c a nd sci e n ti fi c
W it h t he genom e s eque n c e n o w i n h a n d , t h e community. Fortunately,the idea of patentinggenes
comolex social traits can be better understood. For ( o f h u m a n g e n o m e s e q u e n c i n g )w a s d ro p p e d . Th e
ins t anc e, r ec ent ly genes c on t r o l l i n g s p e e c h h a v e fear still existsthat genetic information will be used
been ident if ied. for commercial purposes.
 13 C ene Therapy 157
14 D N A i n D i seaseD i agnosi sand
Medi cal Forensi cs 173
15 P harmaceuti calP roductsof D N A
Technol ogy 189
16 R ecombi nantV acci nes 199
17 Monocl onal A nti bodi es
(H ybri domaTechnol ogy) 213
18 A ssi stedR eproducti ve
Technology 227

MEDICAL/
PHARMAGEUTICAL
BIOTECHNOLOGY
IBToTEGHNOLOGY
rN HEALTHCABEI

e (e, ?"{
o.,- o
;oo
) G)

155
 d va nces i n bioc hem is t r y and nr olec u l a r
I
/ \ bio log y ha v e helped t o under s t andt he gen e t r c
ba sis of in he rit ed dis eas es .lt was a dr eanr of t h e
researchersto replace the defective genes with
good ones, and cure the genetic disorders.
Gene therapy is the process of inserting genes
into cells to treat diseases.The newly introduced
ge ne s will en c ode pr ot eins and c or r ec t t h e
d eficien cie s tha t oc c ur in genet ic dis eas es .Th u s ,
gene therapy primarily involves genetic
manipulations in animals or humans to correct a
d ise ase,an d keep t he or ganis m in good healt h.T h e
initia l expe riment son gene t her apv ar e c ar r ied o u t
in a nima ls, a nd t hen in hum ans O bv ious ly , t h e
g oa l of the re s ear c her sis t o benef it t he m ank i n d
a nd imo rove their healt n.
An overview of gene therapy strategies rs
depicted in Fig. I 3.1 . ln gene augmentation
therapy, a DNA is inserted into the genome to
replace the missing gene product In case of gene
inhibition therapy, the antisensegene inhibits the
e xp ressio nof th e dom inant gene.
APPROACHES FO R G ENE THERAPY
There are two approaches to achieve gene
therapy.
.l
. Somatic cell gene therapy : The non-
reproductive (non-sex) cells of an organism are
referred tcl as somatic cells. These are the cells of
an org an ism o ther t han s per m or eggs c ells , e . 9 . ,
b o n e m a r r o w c e l l s , b l o o d c e l l s ,s k i n c e l l s , i n t e s t i n a l
c e l l s A t p r e s e n t ,a l l t h e r e s e a r c ho n g e n e t h e r a p y i s
directed to correct the senetic.defects in somatic
c e l l s . I n e s s e n c e ,s o m a t i c c e l l g , e n et h e r a p y i n v o l v e s
the insertion of a fully functional and expressible
gene into a target somatic cell to correct a genetic
d i s e a s ep e r m a n e n t l y .
2. Germ cefl gene therapy ; fhe reproductive
(sex) cells of an organism constitutegerm cell line
Cene therapy involving the introduction of DNA
into germ cells is passed on to the successrve
generations. For safety, ethical and technical
reasons, germ cell gene therapy is not being
attempted at present
T h e g e n e t i c a l t e r a t i o n si n s o m a t i c c e l l s a r e n o t
carried to the next generations.Therefore, somatic
cell gene therapy is preferred and extensively
studied with an ultimate objective of correcting
human diseases.
Development of gene therapy in humans for any
specific disease involves the following steps. In
fact, this is a general format for introducing any
therapeuticagent for human use.
1 . ln vitro experiments and research on
l a b o r a t o r y a n i m a l s ( p r e - c l i n i c a lt r i a l s )
2 . P h a s eI t r i a l s w i t h a s m a l l n u n r b e r ( 5 - 10 ) o f
human subjects to test safety of the product.
3 . P h a s e l l t r i a l s w i t h n r o r e h u m a n s u b j e c t st o
assesswhether the product is helpful.
157
 158 B IOTECHNO LO CY

(A)

(B)

4.
Dominant
functionalgene Inhibitoryaction

Fig. 13.1 : Overview of two major gene therapy strategies

(A) Gene augmentation therapy (B) Gene inhibition therapy.

4. Phas e lll t r ials in lar ge h u m a n s a m p l e sf o r a

f inal and c om pr ehens iv eana l y s i so f t h e s a f e t y a n d
efficacy of the product.
The ex vivo gene therapy can be applied to only
As s uc h, gene t her apy in v o l v e s a g r e a t r i s k . s e l e c t e d t i s s u e s ( e . g . , b o n e m a r r o w ) wh o se ce l l s
There are several regulatory agencies whose can be cultured in the laboratory
per m is s ionm us t be s ought be f o r e u n d e r t a k i n ga n y
work related to gene therapy. Recombinant The technique of ex vivo gene therapy involves
DNA Advisory Committee (RAC) is the supervisory the following sleps (Fig. 13.2)
body of t he Nat ional In s t i t u t e o f H e a l t h , 1 . l s o l a t e c e l l s w i t h s e n e t i c d e f ect fr o m a
U. S. A. , t hat c lear s pr opos a l s o n e x p e r i m e n t s o a t i e n t .
inv olv ing gene t her apy . A l a r g e n u m b e r o f
genet ic dis or der s and ot her d i s e a s e sa r e c u r r e n t l y 2 Crow the cells in culture.
at various stages of gene therapy trials. A 3. lntroduce the therapeutic gene to correct
selected list of some important ones is given in gene defect.
Table 13.1.
4. Select the genetically corrected cells (stable
There are two types of gene therapies. transformants)and grov,.

l. Ex vivo gene therapy : This involves the 5 . T r a n s p l a n tt h e m o d i f i e d c e l l s t o th e p a ti e n t.

transfer of genes in cultured cells (e g., bone
m ar r ow c ells ) whic h ar e t hen r e i n t r o d u c e di n t o t h e
Dattent.
lI. In vivo gene therapy :fhe direct delivery of
genes into the cells of a particuiar tissue is referred
to as in vivo gene therapy.
The procedure basically involves Ihe use of the
patient's own cells for culture and genetic
correction, and then their return back to the
patient. This technique is therefore, not associated
w i t h a d v e r s e i m m u n o l o g i c a l r e s p o n se s a fte r
transplanting the cells. Ex vivo gene therapy is
Chaot er13 : G E N ET H ER AP Y 159

Tlrrr 13.1 Humangenetherapytrials

Disease Gene therapy

Severecombined (SCID)
immunodeficiency Adenosine deaminase(ADA).
Cystic
fibrosis Cysticfibrosis
transmembrane (CFTR).
regulator
Familial
hypercholesterolemia Lowdensitylipoprotein
(LDL)receptor.
Emphysema cr,-Antitrypsin
Hemophilia
B FactorlX
Thalassemia a- or B-Globin
Sickle-cell
anemia p-Globin
Lesch-Nyhansyndiome phosphoribosyltransferase
Hypoxanthine-guanine (HGPRT).
Gaucher's
disease Glucocerebrosidase
Peripheral
arterydisease Vascular growthfactor(VEGF)
endothelial
Fanconi
anemia Fanconianemia C
Melanoma Tumornecrosisfactor(TNF)
Melanoma,renalcancer (lL-2)
Interleukin-2
(brain
Glioblastoma AIDS,ovarian
tumor), cancer Thymidine
kinase
(herpes virus)
simplex
Headandneckcancer n53
Y

Breastcancer Multidrug
resistance
I
A I DS rev andenv
Colorectal melanoma,
cancer, renalcancer Histocompatability (HLA-87)
locusantigen-B,
Duchennemusculardystrophy Dystrophin
Shortstature* Growthhormone
Diabetes* Glucose (GLUT-2),
transporter-2, giucokinase
uria*
Phenylketon Phenylalanine
hydroxylase
Citrullinemia* Arginosuccinate
synthetase
* Mostlyconfined
to animalexperiments

efficient only, if the therapeutic gene (remedial
ge ne ) is sta bly inc or por at ed and c ont inuou s l y
expressed.This can be achieved by use of vectors.
VECTORS IN GENE THERAPY
rhe carrier particles or molecules used to deliver
genes to somatic cells are referred to as vectors. The
important vectorsemployed in ex vivo gene therapy
are listed below and briefly described next.
o Viruses
o Hu man artificial c hr om os om e
r Bo ne marro w c ells .
VIBUSES
The vectors frequently used in gene therapy are
viruses,particularlyretroviruses.RNA is the genetic
material in retroviruses.As the retrovirusenters the
host cell, it synthesizesDNA from RNA (by reverse
transcription) The so formed viral DNA (referredto
as provirus) gets incorporated into the DNA of tne
host cell. The proviruses are normally harmless.
However, there is a tremendous risk, since some of
the retroviruses can convert normal cells inrr-r
cancerous ones. Therefore, it is absolutely essential
to ensure that such a thing does not happen.
Making retroviruses harmless
Researchers employ certain biochemical
methods to convert harmful retrovirusesto harmless
ones, before using them as vectors. For instance,by
artificially removing a gene that encodes for the
viral envelope, the retrovirus can be crippled and
made harmless. This is because, without tne
160
ooo
\:/\J
Geneticallvtransformed
-selected
cells
Fig. 13.2 : The procedure for ex vivo gene therapy.
envelooe, retroviruscannot enter the host cell. The
pr oduc t ion of a lar ge num b e r ( b i l l i o n s ) o f v i r a l
particles can be achieved, starting from a single
envelope defective retrovirus (Fig. 13.3). This ls
m ade pos s ible by us ing h e l p e r v i r u s e s w h i c h
contain normal gene for envelope formation. Along
with the helper virus, the vector (with defectrve
envelope gene) can enter the host cell and both of
t hem m ult iply . By r epeat ed m u l t i p l i c a t i o n i n h o s t
c ells , billions of v ec t or an d h e l p e r v i r u s e s a r e
produced. The vector virusescan be separatedfrom
t he helper v ir us es and pur if i e d . l s o l a t i o n o f v e c t o r
v ir us es , t ot ally f r ee f r om h e l p e r v i r u s e s , i s
abs olut ely es s ent ial. Cont a m i n a t i o n o f h e l p e r
viruses is a big threat to the health of the patients
undergoing gene therapy.
Retroviruses in gene therapy
The genetic map of a typical retrovirus is
depicted in Fig. 13.4A. In general, the retrovirus
particle has RNA as a genome organized into six
regions. lt has a 5'-long terminal repeat (5'-LTR),a
non- c oding s equenc e r equir e d f o r p a c k a g i n g R N A
designated as psi (Y), a gene gag coding for
B IOTECHNO LO CY
&8%
ooo_
in vitro cullure
o 6)
\_-/-
o"
structural protein, a gene pol that codes for reverse
transcriptase, a gene env coding for envelope
protein and a 3-LTR sequence.
For use of a retrovirusas a vector, the structural
genesgag and pol are deleted (besides env gene, as
describedabove).Thesegenes are actually adjacent
to Y region. In addition, a promoter gene is also
included (Fig. 13.a8). This vector design allows the
synthesis of cloned genes. A retroviral vector can
carry a therapeutic DNA of maximum size of
B kb.
A retroviral vector DNA can be used to
transform the cells. However, the efficiency of
delivery and integration of therapeutic DNA are
very low. In recent years, techniques have been
developed to deliver the vector RNA to host cells at
a high frequency. For this purposes, packageo
r e t r o v i r a l R N A p a r t i c l e s a r e u s e d . T h is te ch n i q u e
allows a high efficiency of integration of
pharmaceutical DNA into host genome
Several modified viral vectors have been
developed in recent years for gene therapy. These
include oncoretrovirus. adenovirus, adeno-
 Hostcell

$
Vectorvirus
(withdefective
envelopegene)
@(r@
;9
@@r
@ Veciorviruses

Host cell

@@@ Helperviruses

Fig. 13.3 : Large scale production of vector viruses by using helper viruses.

associated virus, herpes virus and a number of A I D S v i r u s 'i n gene therapy?

hybrid vectors combining the good characters of
It is suggestedthat the human immunodeficiency
the oarental vectors.
virus (HlV) can bb used as a vector in gene
transfer. But this is bound to create public uproar.
Murine leukemia viruses in gene
Some workers have been successful in creating
therapy
a harmless HIV (crippled HIV) by removing
This is a retrovirusthat causesa type of leukemia all the genes related to reproduction. At the
in mice. lt ca n r eac t wit h hum an c ells as well a s same time, the essential genes required tbr
th e mo use cell s , due t o a s im ilar it y in t he s ur f a c e gene transfer are retained. There is a distinct
re ce pto r pro tei n. M ur ine leuk em ia v ir us ( M LV ) r s advantage with H lV when compared with
frequently used in gene transfer. MLV. MLV is capable of bringing out gene

(A) 51LTR Y gag pol env 3,-LTR

(B) 5'-LTR Y p 3'-LTR

Fig. 13.4 : A retrovitus used in gene therapy. (A) General map of a typical retrcvirus (B) Gene map of a
modified retrovirus for use in gene therapy (LTR-Long terminal tepeat; Y-Packaging signal sequence; gag-
Coding sequence for structural protein; pol-Coding sequence for reverse transcriptase; env-Envelope protein
coding sequence; y-Therapeutic gene; p-Pramoter gene).

Biotechnology [11]
162 B IOTECHNO LO CY

t r ans f er only in div iding c e l l s . H I V c a n i n f e c t

ev en non- div iding c ells ( e. g . , b r a i n c e l l s ) a n d d o Taslr 13.2 Selected list of genetic discases that
the job of gene transfer effectively. However, are likely to be cured by using bone marrow
it is doubtful whether HIV can ever be used as a cells (potential candidatesfor gene therapy)
veclor. Severecombined (SCID)
immunodeficiency
anemia
Sickle-ceii
HUMAN ART'FTCIAL CHBOMOSOME
Fanconianemia
The det ails of hum an a r t i f i c i a l c h r o m o s o m e Thalassemia
(HAC) are described elsewhere (Chapter6). HAC is
disease
Gaucher's
a synthetic chromosome that can replicate with
Hunter
disease
ot her c hr om os om es , bes ide s e n c o d i n g a h u m a n
pr ot ein. As alr eady dis c u s s e d a b o v e , u s e o f Hurler
syndrome
retrovirusesas vectors in gene therapy is associated Chronicgranulomatous
disease
with a heavy risk. This problem can be overcome Infantile
agranulocytosis
if HAC is us ed. Som e s uc c es sh a s b e e n a c h i e v e d r n Osteoporosis
t his dir ec t ion.
X-linked
agammaglobulinemia
BONE MARROW CELLS

Bone marrow contains totipotent embryonic Severe combined

stem (ES) cells. These cells are capahle of dividing
and differentiating into various cell types (e.g., red
blood cells, platelets, macrophages, osteoclasts,
B- and T-lymphocytes). For this reason, bone
marrow transplantation is the most widely used
technique for severalgenetic diseases.And there rs
every reason to believe that the genetic disorders
that respond to bone marrow transplantation are
likely to respond ro ex vivo gene therapy also
(Table 13.2). For instance, if there is a gene
mutation that interferes with the function of
er y t hr oc y t es( e. g. ,s ic k le- c ella n e m i a ) ,b o n e m a r r o w
t r ans olant at ionis done. Bon e m a r r o w c e l l s a r e t n e
potential candidatesfor gene therapy of sickle-cell
anem ia. Howev er , t his is n o t a s s i m p l e a s
theoretically stated
immunodeficiency (SClDl
T h i s i s r a r e i n h e r i t e d i m m u ne d i so r d e r
associated with T-lymphocytes, and (to a lesser
extent) B-lymphocytes dysfunction. About 50%
of SCID patients have a defect in the gene
(located on chromosome 20, and has 32,000 base
o a i r s a n d 1 2 e x o n s ) t h a t e n c o d e s f or a d e n o sr n e
deaminase. In the deficiency of AD A,
deoxyadenosine and its metabolites (primarily
d e o x y a d e n o s i n e 5 '- t r i p h o s p h a t e ) a ccu m u l a te
and destroy T-lymphocytes. T-Lymphocytes are
e s s e n t i a lf o r b o d y 's i m m u n i t y B e s i d e spa r ti ci p a ti n g
directly in body's defense, they promote
the function of B-lymphocytes to produce
patients
antibodies. Thus, the of SCID
(lacking ADA) suffer from infectious diseases
and die at an young age. Previously,the children
suffering from SCID were treated with conjugated
b o v i n e A D A , o r b y b o n e m a r r o w t r a n sp l a n ta ti o n .

Technique of therapy for

ADA deficiency
THERAPY FOR ADENOSINE
The general scheme of gene therapy adopted for
D E AMIN AS E D EF IC IEN C Y
introducing a defective gene in the patient has
T h e fi rs t a n d th e m o s t p u bl i ci sedhuman gene
been depicted in Fig 13.2. fhe same procedure
therapywas carriedout to correctthe deficiencyof
w i t h s u i t a b l e m o d i f i c a t i o n sc a n a l s o b e a p p l i e d fo r
th e e n z y mea d e n o s i n de e a m i nase (A D A ).Thi sw as
other gene therapies.
done on September 14, 1990 by a teamof workers
led by Blaeseand Andersonat the NationalInstitute A plasmid vector bearing a proviral DNA is
o f H e a l th ,U SA(T h eg i rl ' sn amei s A shanti4, years selected.A part of the proviral DNA is replaced by
o l d th e n ). t h e A D A g e n e a n d a g e n e ( C 4 1 8 ) co d i n g fo r
C h a pt er13 : CE NET H ER AP Y r6 3

VectorDNA

Human
IADA+gene
Retrovirus
containing
ADA'
gene

Transfection

/'tr"-\
-
t-:'/
LCt)

@@
Lymphocytes
with
viralDNAandADA+gene

Childwith SCID
(ADA- gene)

J
Synthesisof
ADA
II
+ expressioninto Cell culturesto
Correction
of patient verifyexpression
SCID of ADA+transgene

Fig. 13.5: Treatment of adenosine deaminase (ADA) deficient patient by somatic ex vivo gene therapy
(SCID-Severe combined immunodeficiency disease).

antibiotic resistance,and then cloned. The Consequently, the ability of the patientto produce
antibioticresistancegene will help to selectthe antibodies is increased.However, there is a
desiredcloneswith ADA gene. limitation.The lymphocyteshave a short life span
(iustlive for a few months),hencethe transfusions
A diagrammaticrepresentation of the treatment
haveto be carriedout freouentlv.
of ADP deficientpatientis depictedin Fig. 13.5.

Circulatinglymphocytesare removed from a Transfer of ADA gene into stem cells

patientsufferingfrom ADA deficiency.Thesecells
are transfectedwith ADA gene by exposingto
b i l l i onsof r et r ov ir u s ecsa rry i n gth e s a i dg e n e .T h e
genetically-modified lymphocytesare grown in
culturesto confirm the expressionof ADA gene
and returnedto the patient.These lymphocytes
persist in the circulation and synthesizeADA.
ln 1995, ADA gene was transfered into
the stem cells, obtainedfrom the umbilical cord
blood,at the time of baby'sdelivery.Fourdaysafter
birth, the infant receivedthe modifiedcells back.
By this way, a permanentpopulationof ADA gene
produci ngcel l sw as establ i shed.
164
THERAPY FOR FAMILIAL
HYPERCI{OLESTEROLEMIA
The pat ient s of f am ilial h y p e r c h o l e s t e r o l e m i a
Iack the low density lipoprotein (LDL) receptors
on t heir liv er c ells . As a r es u l t , L D L c h o l e s t e r o l i s
not m et abolis ed in liv er . T h e a c c u m u l a t e d L D L -
c holes t er olbuilds up in t he c i r c u l a t i o n , l e a d i n g t o
arterial blockage and heart diseases.Attempts are
being m ade by gene t her ap i s t st o h e l p t h e v i c t i m s
of f am ilial hy per c holes t er o l e m i a I. n f a c t , t h e r e i s
s om e s uc c es sals o. I n a wo m a n , 1 5 % o f t h e l i v e r
was removed. The hepatocytes were transduced
with retrovirusescarrying genes for LDL receptors.
These genetically modified hepatocytes were
infused into the patient's liver. The hepatocytes
es t ablis hed t hem s elv es i n the liver and
pr oduc ed f unc t ional LDL- r e c e p t o r s .A s i g n i f i c a n t
im pr ov em ent in t he pat ient 'sc o n d i t i o n , a s a s s e s s e d
by es t im at ing t he lipid par a m e t e r si n b l o o d , w a s
observed Further, there were no antibodres
pr oduc ed agains t t he LD L - r e c e p t o r m o l e c u l e s ,
c lear ly s howing t hat t he gen e t i c a l l y m o d i f i e d l i v e r
cells were acceoted.
THERAPY FOR LESCH.NYHAN
SYNDROME
Les c h- Ny han s y ndr om e i s a n i n b o r n e r r o r I n
purine metabolism due to a defect in a gene that
encodes for the enzyme hypoxanthine-g,uanine
phosphoribosyl transferase (HGPRT). In the
absenceof HCPRT, purine metabolism is disturbeo
and ur ic ac id lev el builds u p , r e s u l t i n g i n s e v e r e
gout and k idney dam age T h e v i c t i m s o f L e s c h -
Ny han s y ndr om e ex hibit s y m p t o m s o f m e n t a l
retardation,besidesan urge to bite lips and fingers,
c aus ing s elf - m ut lat
i ion.
By using retroviral vector system, HCPRT
producing genes were successfully inserted into
c ult ur ed hum an bone m ar r o w c e l l s . T h e m a i o r
pr oblem in hum ans is t he i n v o l v e m e n t o f b r a i n .
Ex per im ent sc onduc t ed in an i m a i s a r e e n c o u r a g i n g .
However, it is doubtful whether good successcan
be achieved by gene therapy for Lesch-Nyhan
s v ndr om e in hum ans , in t he n e a r f u t u r e .
THERAPY FOR HEMOPHILIA
Hemophilia is a genetic disease due lack of a
gene that encodes for clotting factor IX. lt is
c har ac t er iz ed by ex c es s iv e b l e e d i n g . B y u s i n g a
retroviral vector system, genes for the synthesisof
B IOTECHNO LO CY
factor lX were inserted into the liver cells of dogs.
These dogs no longer displayed the symptoms of
hemophilia.
EX V'VO GENE THERAPY WITH
NON.AUTOLOGOUS CELLS
The ex vivo gene therapiesdescribed above are
basedon the iransplantationof geneticallymodified
cells for the production of desired proteins.
However, there are several limitations in using the
patient's own cells (autologous cells) for gene
t h e r a p y . T h e s e i n c l u d e l a c k o f e n o u gh ce l l s fr o m
target tissues,defective uptake of genes and therr
inadequate expression. To overcome these
problems, attempts are on to develop methods to
use non-autologous cells (i.e., cells from other
i n d i v i d u a l s o r a n i m a l s ) . T h e o u t li n e o f th e
procedure is briefly described below.
T i s s u e - s p e c i f i cc e l l s c a p a b l e o f g r o w i n g i n
culture are selected.These include fibroblastsfrom
skin, hepatocytesfrom liver, myoblastsfrom muscle
and astrocytesfrom brain. These cells are cultured
a n d g e n e t i c a l l ym o d i f i e d w i t h t h e t h e r ap e u ti cg e n e .
They are then encapsulatedin artificial membrane
composed of a synthetic polymer (e.g., polyether
s u l f o n e , a l g i n a s e - p o l y L - l y s i n e - a lg i n a te ) . Th e
p o l y m e r i c m e m b r a n e s a r e n o n - i mm u n o g e n i c,
therefore the patient can accept non-autologous
e n c a p s u l a t e dc e l l s . F u r t h e r , b e i n g s e m i p e r m e a b l e
in nature, these membranes allow the nutrients to
enter in, and the encoded protein (by the
therapeutic gene) to pass out.
Experimentsconducted in animals have shown
some encouraging resultsfor using non-autologous
c e l l s i n g e n e t h e r a p y . T h e e n c a p s u l a te dce l l s w e r e
found to proliferate and produce the required
protein. However, the successhas been very limited
in human trials.
fhe direct delivery of the therapeutic gene
(DNA) info the target cells of a particulartissue
of a patient constitutesin vivo gene' therapy
(Fig. 13.6). Many tissues are the potential
for thi s approach.Thesei ncludeliver ,
candi dates
muscl e,ski n,spl een,l ung, brai n and blood cells.
Cene deliverycan be carriedout by viral or non-
 :apt er 1l : G E NET H ER AP Y 165

Replication defective virus particles can De

produced from the plasmovirus.

As such, for the delivery of genes by retroviral

vectors,the target cells must be in a dividing stage.
But majority of the body cells are quiescent. In
Tarqet
tiss'ue recent years, viral vectors have been engineeredto
t--=-
.. I P infect non-dividing cells. Further,attemptsare on to
-.... include a DNA in the retroviral vectors (by
I _-___---+
engineeringenv gene)that encodesfor cell receptor
protein. lf this is successfully achieved, the
retroviral vector will specifically infect the target
Therapeutic
gene t i s s ue s .

Adenoviral vector system

Adenoviruses (with a DNA genome) are
considered to be good vectors for gene delivery
because they can infect most of the non-dividing
Fig. 13.6 : Diagrammatic representation of in vivo human cells. A common cold adenovirus is a
gene therapy. (p-Promoter gene specific frequently used vector. As the target cells are
infected with a recombinant adenovirus, the
therapeutic gene (DNA) enters the nucleus and
expresses itself. However, this DNA does nor
viral vector systems. The success of in vivo gene integrate into the host genome. Consequently,
therapy mostly depends on the following adenoviral based gene therapy required periodic
Darameters a d m i n i s t r a t i o no f r e c o m b i n a n t v i r u s e s .
. The efficiency of the uptake of the remedial The efficiency of gene delivery by adenoviruses
(therapeutic)gene by the target cells. can be enhanced by developing a virus that can
specifically infect target cells. This is possible by
. I ntra ce llula r d eg radat ion of t he gene and it s
incorporating a DNA encoding a cell receptor
uptake by nucleus.
protein.
. The expressioncapability of the gene.
Adeno-associated virus vector system
In vivo gene therapy with special reference to
Adeno-associated virus is a human virus
gene de live ry syste m s( v ir al, non- v ir al)wit h s uit able
.l9.
examp les is d escrib ed. that can integrate into chromosome lt is a
single-stranded, non-pathogenic small DNA
GE NE DEL IVERY BY VI RUSES virus (4.7 kb). As the adeno-associated virus
enters the host cell, the DNA becomes do;ubte-
Many viral vector systemshave been developed stranded, gets integrated into chromosome and
for gene delivery. These include retroviruses, exoresses.
adenoviruses,adeno-associatedviruses and herpes
simplex viru s. Adeno-associated viruses can serve as
good vectors for the delivery of therapeutic
genes. Recombinant viruses are created by using
Retrovirus vectoi system
two plasmids and an adenovirus (i e., helper
Replication defective retrovirus vectors that are virus) by a special technique. Some attempts were
harmlessare being used (Seep. 159). A plasmid in made to use therapeutic genes for the treatment of
association with a retrovirus, a therapeutic gene t h e h u m a n d i s e a s e s - h e m o p h i l i a( f o r p r o d u c t i o n o f
and a promoter is referred to as plasmovirus. The blood clotting factor lX) and cystic fibrosis (for
plasmovirus is capable of carrying a DNA synthesisof cystic fibrosis transmembraneregulator
therapeuhc gene) of size less than 3.4 kb. p rotein) by empl oyi ng adeno-associated viruses.
166 BIOTECHNOLOCY

Therapy for cystic fihrosis h e r p e s s i m p l e x v i r u s ( H S V ) t y p e l , w hi ch i n fe cts

a n d p e r s i s t si n n o n - d i v i d i n g n e r v e c e l ls. H SV i s a
Cystic fibrosis (CF) is one of the most common
human pathogen that causes (though rarely) cold
(frequence 1 : 2,500) and fatal genetic diseases.lt
sores and encephalitis.
is characterized by the accumulation of sticky,
dehydratedmucus in the respiratorytract and lungs. These are a large number of diseases(metabolic,
Patients of CF are highly susceptible to bacterial neurodegenerative, immunological, tu m o r s)
inf ec t ions in t heir lungs an d m o s t o f t h e m d i e associated with nervous system. HSV is considered
before reaching the age of thirty. as an ideal vector for in vivo gene therapy of many
nervous disorders.
Cystic fibrosiscan be traced in Europeanfolklore,
the follorving statementused to be said "Woe to that The HSV has a double-stranded DNA of about
c hild whic h when k is s ed o n t h e f o r e h e a d t a s t e s 152 kb length as its genome. About 30 kb of HSV
salty. He is be witched and soon must die". genome can be replaced by a cloned DNA without
loss of its basic characteristics (replication,
Biochemical basis : In the normal oersons the
infection, packaging etc.). But there are some
c hlor ide ions of t he c ells ar e p u s h e do u t t h r o u g h t h e
t e c h n i c a l d i f f i c u l t i e s i n d e a l i n g w i t h l a r g e - si ze d
participation of a protein called cysfic fibrosis
DNAs in genetic engineering experinrents. Some
transmembrane regulator (CFTR). In the patients of
modified HSV vectors with reduced genomic sizes
cystic fibrosis,the CFTRprotein is not produced due
have been developed.
t o a gene def ec t . Cons eque n t l y ,t h e c h l o r i d e i o n s
c onc ent r at ewit hin t he c ells w h i c h d r a w w a t e r f r o m Most of the rruork on the gene therapy, related
the surroundings.As a result, the respiratorytract to the use of HSV as a vector, is being conducted
and t he lungs bec om e dehy d r a t e dw i t h s i c k y I n u c u s , i n e x p e r i m e n t a l a n i m a l s . A n d t h e r e su l ts a r e
an ideal er r v ir or r nr ent
f or bac t e r i a l i n f e c t i o n s . q u i t e e n c o u r a g i n g . H S V v e c t o r s c ou l d d e l i ve r
therapeutic genes to the brain and other parts
Cene therapy : As the defective gene for cystic
of nervous system These genes are well
f ibr os is was ident if ied in 1 9 8 9 , r e s e a r c h e r s
expressedand maintained for long periods. More
im m ediat ely s t ar t ed wor k ing o n g e n e t h e r a p y f o r
research, however, is needed before going for
this disease.Adenoviral vector systemshave been
human trials.
us ed, alt hough t he s uc c es sh a s b e e n l i m i t e d . T h e
major drawback is that the benefits are short-lived, lf successful, HSV may help to treat
since the adenovirusesdo not integratethemselves many neurodegenerative syndromes such as
int o hos t c ells . M ult iple a d m i n i s t r a t i o n o f Parkinson's disease and Alzheimer's disease by
r ec om binant adenov ir us ca u s e d i m m u n o l o g i c a l gene therapy.
responsesthat destroyed the cells.
GENE DELIVERY BY
By using,adeno-associatedvirus vector system, NON.VIRAL SYSTEMS
some encouraging results were reported in the
There are certain limitationsin using viral vectors
gene therapy of CF. ln the phase I clinical trials
in gene therapy.In addition to the prohibitive cost of
with CF patients,the vector persistedfor about 70
maintaining the viruses, the viral proteins often
days and some improvement was observed in the
patrents. induce inflammatory responses in the host.
Therefore,there is a continuoussearchby researchers
Some researchersare trying to insert CF gene to find alternativesto viral vector svstems.
int o t he dev eloping f et al c e l l s ( i n e x p e r i m e n t a l
anim als s uc h as m ic e) t o p r o d u c e C F T R p r o t e r n . Pure DNA constructs
Bur a major breakthrough is yet to come.
The direct introduction of pure DNA constructs
into the target tissueis quite simple. However,the
Herpes simplex virus vector system
efficiencyof DNA uptake by the cells and its
The retroviruses and adenoviruses (discussed expressionare rather low. Consequently,large
above) employed in in vivo gene therapy are quantitiesof DNA haveto be injectedperiodically.
engineered to infect specific target cells. There are The therapeuticgenesproducethe proteinsin the
s om e v ir us es whic h hav e a n a t u r a l t e n d e n c y t o targetcells which enter the circulationand often
inf ec t a par t ic ulart y pe of c el l s T h e b e s t e x a m p l e i s get degraded.
Chapt er13 : C E N ET H E R A PY 167

Lipoplexes
-Therapeutic
fhe Iipid-DNA complexesare referred to as
or morecommonlyliposomes.
lipoplexes Theyhave Poly L-
t Cell
gene

a DNA construct surrounded by artificiallipid layers. lysine receptoi

A large numberof lipoplexeshave been prepared
and used.Theyarenon-toxicand non-immunogenic.
The majorlimitationwith the useof lipoplexes is that
as the DNA is takenup by the cells,mostof it gets
degradedby the lysosomes. Thus,the efficiencyof
genedeliveryby lipoplexis very low.
S om ec linic a ltri a l su s i n gl i p o s o m e -C F TgRene
complexshowedthat the geneexpression was very
short-lived.

DNA.molecular coniugates
conJugate
The useof DNA-molecular conjugatesavoidsthe
lysosomalbreakdownof DNA. Anotheradvantage
of using conjugatesis that large-sizedtherapeutic
DNAs (> 10 kb) can be deliveredto the target
tissues. The most commonly used synthetic
conjugateis poly-L-lysine,boundto a specifictarget
The therapeutic
cell receptor. DNA is then madeto
combinewith the conjugateto form a complex(Fig.
13.4. fhis DNA molecular conjugatebinds to
specific cell receptoron the target cells. lt is
engulfedby the cell membraneto form an endosome
which protectsthe DNA from beingdegraded. The
DNA released fromthe endosome entersthe nucleus
wherethe therapeutic gene is expressed.

Human artificial chromosome

Humanartificialchromosome(HAC)which can
cafiy a largeDNA (oneor more therapeuticgenes
with regulatory elements) is a good and ideal
.veclor. Studiesconductedin cell culturesusing
HAC are encouraging.But the major problem is
the delivery of the large-sizedchromosomeinfo
the target cells. Researchersare working to
-cells
produce containinggeneticallyengineered
Fig. 13.7 : DNA-molecular conjugate in the delivery
HAC. There exists a possibilityof encapsulating of therapeutic gene.
and im plant in gth e s ec e l l s i n th e ta rg e tti s s u e B
. ut
a long way to go!
2. The expressionof the therapeutic gene is for
Efficiency of gene delivery by a very short period, consequently there is no
non-viral vectors effective treatment of the disease.
Althoughthe effortsari: continuouslyon to find
suitable non-viral vectors for gene delivery,the GENE THERAPY STRATEGIES
s uc c eshas . h i si s ma i n l yd ueto
s bee nv e ryl i mi te d T FOR CANCER
the followingtwo reasons. Cancer is the leading cause of death throughout
1. The efficiencyof transfectionis very low. the world, despite the intensivetreatment strategies
168 B IOTECHNO LO CY

------* DNA svnthesis

Nucleotide

/^=.--1 \ l'\ lnhibitsDNA DNA synthesis

\\/--==-J / V ' polymerase blocked

Falsenucleotide II
J
Cancer
cells die

Fig. 13.8 : The action of ganciclovir mediated by thymidlne kinase to inhibit the growth of cancer cells.

(surgery, chemotherapy, radiation therapy). Cene
therapy is the latestand a new approach for cancer
treatment. Some of the developments are briefly
described hereunder.
Tumor necrosis factor gene therapy
Tumor necrosis factor (TNF) is a protern
produced by human macrophages. TNF provides
defense againstcancer cells. This is brought out by
enhancing the cancer-fighting ability oI tumor-
infiltrating lymphocytes (TILs), a special type of
im m une c ells .
The tumor-infiltrating lymphocytes were
transformed with a TNF gene (along with a
neomycin resistantgene) and used for the treatment
of malignant melanoma (a cancer of melanin
pr oduc ing c ells , us ually oc c u r s i n s k i n ) . T N F a s
s uc h is highly t ox ic , and f o r t u n a t e l y n o t o x i c s i d e
effects were detected in the melanoma patients
injec t ed wit h genet ic ally a l t e r e d T l L s w i t h T N F
gene. Some improvement in the cancer patients
was observed.
Suicide gene therapy
The gene encoding the enzyme thymidine
kinase is often referred to as suicide gene, and is
used for the treatment of certain cancers.
Thym id ine kinase (f K) phosphorylates Several new strategies are being developed to
nuc leos idest o f or m nuc leot i d e sw h i c h a r e u s e d f o r
t he s y nt hes isof DNA dur ing c e l l d i v i s i o n . T h e d r u g t h r o u e h o u t a t u m o r .
ganciclovir (GCV) bears a close structural
r e s e m b l a n c et o c e r t a i n n u c l e o s i d e s( t hym i d i n e ) .By
mistake, TK phosphorylates ganciclovir to form
triphosphate-CCV a false and unsuitablenucleotide
{or DNA synthesis. Triphosphate-GCV inhibits
DNA polymerase (Fig. 13.0. fhe result is that the
e l o n g a t i o n o f t h e D N A m o l e c u l e a b r up tl y sto p s a t
a p o i n t c o n t a i n i n g t h e f a l s e n ucl e o ti d e ( o f
ganciclovir). Further,the triphospate-CCVcan enter
a n d k i l l t h e n e i g h b o u r i n g c a n ce r ce i l s, a
phenomenon referred to as bystander effect. fhe
u l t i m a t e r e s u l t i s t h a t t h e c a n c e r ce l l s ca n n o r
m u l t i p l y , a n d t h e r e f o r e d i e . T h u s, th e d r u g
g a n c i c l o v i r c a n b e u s e d t o k i l l t h e c a n ce r ce l l s.
Canciclovir is frequently referredro as a prodrug
and this type of approach is called prodrug
activation gene therapy. Ganciclovir has been used
for treatment of brain tumors (e.9., glioblastoma, a
c a n c e r o f g l i a l c e l l s i n b r a i n ) , a l t h o u g h w i th a
limited success.
ln the suicide gene therapy, the vector used is
herpes simplex virus (HSV) with a gene for
t h y m i d i n e k i n a s e ( T K ) i n s e r t e d i n i ts g e n o m e .
N o r m a l b r a i n c e l l s d o n o t d i v i d e w h i l e th e b r a r n
t u m o r c e l l s g o o n d i v i d i n g u n c h e c k e d . Th u s, th e r e
i s a c o n t i n u o u s D N A r e p l i c a t i o n i n t u m o r ce l l s. By
using CCV-HSVTK suicide gene therapy, some
reduction in proliferatinBtumor cells was reported.
i n c r e a s et h e d e l i v e r y o f H S V T K g e n e t o a l l th e ce l l s
Chapt er13 : C E N ET H ER AP Y 169

Two-gene cancer therapy Researchershave used HIV lacking rev and env
genesfor therapeuticDUrposes. T-Lymphocytesfrom
For treatment of certain cancers/ two Sene
HIV-infected patients are removed, and mutant
systemsare put together and used. For instance,IK
viruses are inserted into them. The modified
suicide gene (i.e., CCV-HSVTK) is clubed with
T-lymphocytesare cultivated and injected into the
interleukin-2 gene (i.e. a gene promoting
patients.Due to lack of essentialgenes,the viruses
immu no the rap y ) .I nt er leuk in- 2pr oduc ed m obili z e s
(HlV) cannot multiply, but they can stimulatethe
immu ne re sp ons e. lt is believ ed t hat c er t a i n
production of CDu (cluster determinant antigen B)
proteins are releasedfrom the tumor cells on their
c e l l s o f T - l y m p h o c y t e s .C D u c e l l s a r e t h e k i l l e r
death. These proteins, in associationwith immune
lymphocytes. It is proved in the laboratory studies
ce lls, re ach the t um or and init iat e im m unolog i c a l
that these lymphocytes destroy the HIV-infected
reactions directed against the cancer cells.
c e lIs .
Two-gene therapies have been carried out in
expe rimen tal a nim als wit h c olon c anc er and li v e r Genes of HIV proteins
ca ncer, a nd th e r es ult sar e enc our aging.
Some genes synthesizing HIV proteins are
attached to DNA of mouse viruses. These
Gene replacement therapy
g e n e t i c a l l y - m o d i f i e dv i r u s e s a r e i n j e c t e d t o A I D S
A gene named t'3 codes for a protein with a p a t i e n t sw i t h c l i n i c a l m a n i f e s t a t i o n so f t h e d i s e a s e .
molecular weight of 53 kilodaltons (hence ps3).ps3 I t i s b e l i e v e d t h a t t h e H I V g e n e s s t i m u l a t e n o r m a l
is considered to be a tumor-suppressor gene, since b o d y c e l l s t o p r o d u c e H I V p r o t e i n s . T h e l a t t e r i n
the pro tein it en c odes binds wit h DNA and inhi b i t s t u r n s t i m u l a t et h e o r o d u c t i o n o f a n t i - H l V a n t i b o d i e s
re plicatio n. Th e t um or c ells of s ev er al t is s u e s w h i c h p r e v e n tt h e H I V r e p l i c a t i o n i n A I D S p a t i e n t s .
(breast, brain, lung, skin, bladder, colon, bone)
were found to have altered genes of p53 (mutated Gene to inactivate gpl 2O
psr, synthesizing different proteins from the
o rigin al. Th ese alt er ed pr ot einsc annot inhibit D N A gpl 20 is a glycoprotein (molecular weight 120
replication. lt is believed that the damaged ps3 kilodaltons) present in the envelope of HlV. lt is
gene may be a causative factor in tumor absolutely essentialfor binding of virus to the host
develooment. c e l l a n d t o b r i n g r e p l i c a t i o n . R e s e a r c h e r sh a v e
synthesized a gene (called F105) to produce an
Some workers have tried to replacethe damaged antibody that can inactivate gp12o. In the anti-
ps3 gene by a normal gene by employing adenovirus AIDS therapy, HlV-infected cells are engineered to
vector systems (See p. 165). There are some produce anti-lllV antibodies when injected into
encouragingresultsin the patientswith liver cancer. t h e o r g a n i s m .

The antisense therapy for cancer is discussed Studies conducted in experimental animals
e lse whe re (See p. 170) . showed a drastic reduction in the synthesis of
gp120 due to anti-A|DS therapy. The production of
GENE THERAPY FOR AIDS
HIV particles was also very reduced. There are
AIDS is a global dis eas e wit h an alar m i n g some attemptsto prevent AIDS by antisensetherapy
increasein the incidence every year. lt is invariably (Seep. 171).
fatal, since there is no cure. Attempts are
being made to relieve the effects of AIDS by gene
therapy. Some of the approaches are discussed
hereunder.

rev and env genes

A mutant strainof human immunodeficiencyvirus
(HlV), lacking rev and env genes has been
developed.The regulatoryand envelope proteins of
HIV are respectivelyproduced by revand envgenes.
Due to lack of thesegenes,the virus cannot replicate.
In general, Bene therapy is carried out by
introducing a therapeutic gene to produce the
defective or the lacking protein. But there are
certain disorders (cancer, viral and parasitic
infections, inflammatory diseases)which resuit in
an overproduction of certain normal proteins. lt is
17(J BIOTECHNOLOGY

possible to treat these diseases by blocking (A) Fs.olrA} ]-=---l+1--=,-l

--orun
transcription using a single-strandednucleotide '--T-- |
sequence(antigene oligonucleotide) that hybridizes
with the specific gene, and this is called
I lTranscription
IJ
antigene therapy. Antisensetherapy refers to the
inhibition of translationby using a single-stranded
nucleotide(antisense oligonucleotide). Further,it is
als o o o s s i b l eto i n h i b i t b o th transcri oti onand
translationby blocking(with oligonucleotides) the
transcription factorresponsbile for the specificgene
expressron.
Nucleic acid therapy refersto the use of DNA
or RNA molecules for therapeutic purposes, as
statedabove.The naturallyoccurringsequences of
DNA and RNA (with suitablemodifications) or the
syntheticones can be employed in nucleic acid
therapy.Theoretically,there is a vast potential for
useof nucleicacidsas therapeutic agents.But most
of the w.orkthat is beingcarriedout relatesto the
useof RNA in antisense therapy.Someo{ theseare
described below (Note : Some authors use
antisense therapy in a broad sense to reflect (B)
antigene therapy as well as antisensetherapy, -
AntisenseRNA
discussedin the previousparagraph).

ANTISENSE THERAPY FOR CANCEB

Oncogenesare the genes responsiblefor the
causation of cancer. The dominantly acting
oncogenescan be targetedin antisensetechnology
by usingantisensetransgenes or oligonucleotides.
Antisense oligonucleotidesare used for the
treatmentof myeloidleukemiain as earlyas 1991.
AntisenseRNA moleculesare more frequently
usOdin cancer therapy.This approach is effective
only if the antisenseoligonucleotide(antisense
mRNA) specificallybinds to the target mRNA
(translation).
and blocks protein biosynthesis This
can be achieved in two ways, as illustratedin
Fig. 13.9.
The antisensecDNA can be cloned and
transfected into cells. Antisense mRNA is
synthesizedby transcription.
This can readilybind
with the specific mRNA and block translation
(Fig. 13.94).The mRNA is actuallyformed by a
gene containing exons and introns through
followedby processing.
transcription,
The otherway to block translationis to directly
intro d u c e a n ti s e n s eR N A i n to the cel l s. Thi s
hybridizeswith targetmRNAand blockstranslation
(Fig. 13.98).
mRNA-antisense
RNA comolex
12
I
+
No translation
Fig. 13.9 : lnhibition of translation by antisense RNA
(A) The cloned AS cDNA introduced into cells to
produce antisense RNA (B) Antisense HNA direetly
introduced into cell$. (AS aDNA -- Antisense
c0mplemadary DNA; Ep Er.fixons in a ge:ne; I-lntron)
The antisense mRNA therapy was tried for the
treatment of a brain tumor namely malignant
glioma and the cancer of prostate glarrd. In case
m a l i g n a n t g l i o m a , t h e p r o t e i n i n s u l i n - l i k e g ro w th
(lCF-l) prostate
factor | is overproduced, while in
c a n c e r , i n s u l i n - l i k eg r o w t h f a c t o r l r e c e p t o r ( l CF- l R )
protein is more synthesized.For both these cancers,
the. respectivi: antisense cDNAs can be used to
s y n t h e s i z e ,a n t i s e n s e m R N A m o l e c u l e s . T h ese i n
--r aot er13 : CE N ET H E R A PY 171

-:n. are used to block translation,as briefly

-rescribedabove and illustratedin Fig. 13.9.

ANT I S E NS E T H ER AP Y F O R AID S
{ttempts are also being made to preventHIV
I Targetcell
rrection through antisensetherapy. The basrc ,geneticallyengineered)
crincipleis brieflydescribed(Fig. 13.10)., |
\r_
The targetcellsfor HIV infectionare genetically
engineeredto contain a gene that can expressa
Jlnfection
complementarycopy of HIV genome.This gene -=--=--------Vi ral RNA
producesantisense RNA.Whenthe cellscontaining
antisenseRNA are infectedby HIV it bindsto viral
RNA forminga double-stranded RNA-RNAhybrid Double-stranded
m olec ulesT. his d o u b l e -s tra n d emo
d l e c u l ec a n n ot RNA-RNAhybrid
be used by the enzyme reversetranscriptase.
AntisenseRNA
Consequently, a DNA copy of the HIV genome
cannotbe madeand incorporated into the genome. Geneticallymodified
targetcell infectedwith HIV
A NT I S E NS E OL IGON U C L EOT ID F S A S
THERAPEUTIC AGENTS
Fig. 13.10 : Antisense therapy to prevent AIDS
It is found that oligodeoxynucleotides (with (HI V-Human immunodeficiency virus)
a b out 15- 20 uni ts ) c a n h y b ri d i z ew i th s p e c i f i c
mRNAand blocktranslation with an ultimateeffect
of reducingthe specificprotein.Oligonucleotides Possible inhibition of smooth muscle cell
can hybridize with different types of RNA proliferation: Proliferation of smoothmusclecells
transcripts-mRNAs, intron-exons,double-stranded (along with secretion of extracellular matrix)
RNAs. The major limitation of using naturally has been implicated in hypertension,failure of
occurringoligonucleotides is thatthey aredegraded coronary bypass grafts, hypertension and
b y int r ac ellular
nu c l e a s e s . atherosclerosis.There exists a possibility of
Certainmodificationsin the nucleotides(bases controlling smooth muscle cell proliferationby
or sugars, phosphate)without affecting their usingantisense therapy.
hybridizationabilityhavebeen prepared. However,
these modified oligonucleotidesare resistantto C H IME R IC OLIGON U C LE OTID E S IN
degradationby nucleases.The most r,r,idelyused GE N E C OR R E C TION
antisenseoligonucleotidehas a sulfur group In Many geneticdiseasesare due to single base
place of the free oxygen in phosphodiester bond. pair mutations.A strategyhas been devisedto
The modified bond is referred to as correct such defects by using a chimeric
phosphorothioate linkage. These phosphorothioate oligonucleotides composed of RNA-DNA
antisense oligonucleotides are resistant to ol i gonucl eoti dew i th 6B uni ts. Thi s chi meri c
degradationby nucleases,besidesbeing water ol i gonucl eoti de has hai rpi ncaps and methyl ated
so luble. S om e o th e r mo d i fi c a ti o n so f o l i g o- ribose sugars(at 2nd carbon).The hairpin caps
nucleotidesare with phosphoramidite Iinkageand protect the molecule from the degradationby
2-O-methylribose(Fig. 13.11). The modified exonucleaseswhile methylated ribose prevents
antisenseoligonucleotidesare in fact used as digestionby RNase.
therapeuticagents in the clinical trials for the The correctionof a singlebasepair mutationby
treatmentof certaincancers,bowel disease,AIDS, usi ng a chi meri c ol i gonucl eoti de i s depi ctedi n
m alar iaand v ir al i n fe c ti o n s . Fi g. 13.12.The R N A -D N A mol ecul es(chi meri c
Thefreeoligonucleotides do not readilyenterthe oligonucleotides)
bind more readily w'ith duplex
cells.Therefore, they are encapsulated in liposomes DNA and bringaboutthe correctionin the mutated
for efficientdeliveryto inhibitthe mRNAtranslation. basepair.
172 B IOTECHNO LO CY

Base Base T
I I A
Sugar Sugar
I I TargetDNA with single
o o base pair mutation
I I .....",.TT
O:P-S TT_,_._.._.._..-G-
I I TT T-f
o o
I I Chimericolioonucleotide
Sugar' Sugar
i I Fig. 13.12: Chimericoligonucleotide in gene
Base Base
correction(The dotted lines representribonueleotides
Phosphodiester Phosphorothioate
linkage linkage while the continuouslines correspondto
deaxyribanucJeotidea; the calouredbase:pair is the
Base conecf one tb r€plaaptli?, nutated Wset pair) '
I
Sugar
I THE FUTURE O F G E N E T H E R A PY
N
I
O:P-O Theoretically, gene therapy is the permanent
solution for genetic diseases.But it is not as simple
o a s i t a p p e a r ss i n c e g e n e t h e r a p y h a s s e ve r a l i n b u i l t
I c o m p l e x i c i t i e s . C e n e t h e r a p y b r o a d l y i n vo l ve s
Sugar
I i s o l a t i o n o f a s p e c i f i c g e n e , m a k i n g i ts co p i e s,
Base inserting them into target tissue cells to make ihe
Phosphoramidite desired protein. The story does not end here. lt is
linkage
absolutely essential to ensure that the gene is
h a r m l e s s t c t h e p a t i e n t a n d i t i s a p p r o p r i a te l y
Fig. 13,11 : Certain modifications in antisense expressed(too much or too little will be no good).
oligonucleotides. Another concern in gene therapy is the body's
immune systemwhich reactsto the foreign proteins
produced by the new genes.
APTAMERS AS THERAPEUTIC AGENTS
A special type of oligonucleotides that can The public, in general, have an exaggerated
specifically bind to target proteins and not to expectations on gene therapy. The researchers, at
nucleic acids are referredto as aptamers.Aptamers least for the present, are unahle to satisfy them. As
are useful as therapeutic agents. For instance, an per the records, by 1999 about 10C0 Americans
apt am er t hat c an bind t o t hr o m b i n i n h i b i t s b l o o o h a d u n d e r g o n ec l i n i c a l t r a i l s i n v o l v i n g va r i o u s g e n e
c lot t ing. Suc h oligonuc leot i d e s c a n b e u s e d i n therapies. Unfortunately, the gene therapists are
s ur gic al pr oc edur es . u n a b l e t o c a t e g o r i c a l l yc l a i m t h a t g e n e th e r a p y h a s
permanently cured any one of these patients!Some
RIBOZYMES AS THERAPEUTIC AGENTS p e o p l e i n t h e m e d i a ( l e a d i n g n e w s pa p e r s a n d
magazines) have openly questioned whether it rs
RNA molecules that can serve as enzymes worth to continue researchon gene therapy!!
(biocatalysts)are referred to as ribozymes. The
nat ur allyoc c ur r ing r iboz y m e sa r e t o b e m o d i f i e d s o It may be true that as of now, gene therapy due
t hat t hey c an s pec if ic ally h y b r i d i z e w i t h m R N A to several limitations, has not progressedthe way it
s equenc es and bloc k protein biosynthesis should, despite intensive research. But a hreak-
( t r ans lat ion) . Ther e is a lim i t a t i o n h o w e v e r , i n through may come anytime, and of course, this rs
dir ec t ly ins er t ing r iboz y m es i n t o t a r g e t c e l l s , a s o n l y p o s s i b l e w i t h p e r s i s t e n tr e s e a r c h .An d a d a y
the,riare easily degraded This can be overcome by may come (it might take some years) when almost
binding r iboz y m e wit h an o l i g o d e o x y n u c l e o t i d e . every disease will have a gene therapy, as one of
Ther e is a pos s ibilit yof t r eat in gc e r t a i n c a n c e r sa n d the treatment modalities. And gene therapy will
v ir al dis eas esby genet ic allye n g i n e e r e dr i b o z y m e s . r e v o l u t i o n i z et h e p r a c t i c e o f m e d i c i n e !
 ia gn osis of dis eas es due t o pat ho g e n s
(ba cte ria, v ir us es , f ungi et c . ) or due t o
inherent genetic defects is necessaryfor appropriate
treatment Traditional diagnostic methods for
o ara site inf ec t ions inc lude m ic r osc o p r c
examination, in vitro culture, and detection of
a ntib od ies in s er um . And f or t he genet ic dis e a s e s ,
the orocedures such as estimation of metabolites
(b loo d/u rine ) and enz y m e as s ay s ar e em plo y e d
The se lab ora t or y t ec hniques ar e indir ec t and n o t
a lways spe cific .Sc ient is t sar e in c ont inuous s e a r c h
fo r spe cific, s ens it iv e and s im ple diagn o s t i c
technioues for identification of diseases.
DNA, b ein g t he genet ic m at er ial of t he l i v i n g
organisms,contains the information that contributes
to various characteristic features of the specific
organism. Thus, fhe presence of a disease-causing
pathogen can be detected by identifing a gene or
ACG TTAG CA
ACG TTAG CA
lllllll'
_ I-TG CAATCG T-
ComPlementary
Pairing
Fig. 14.1 : Hybridization of target DNA with DNA
probe (with radioactive isotope label).
a set of genes of the organism. Likewise an
inherited genetic defect can be diagnosed by
identifying the alterations in the gene. In the
m o d e r n l a b o r a t o r y d i a g n o s t i c s ,D N A a n a l y s i s i s a
very useful and sensitivetool
The basic principles underlying the DNA
diagnosticsystems,and their use in the diagnosisof
certalnpathogenicand geneticdiseasesare described.
Besides these, the various approaches for DNA
f i n g e r p r i n t i n g( o r D N A p r o f i l i n g )a r e a l s o d i s c u s s e d .
The specific identification of the DNA sequence
is absolutely essentialin the laboratory diagnostics.
This can be achieved by employing the following
principles/tools.
NUCLEIC ACID HYBBIDIZATION
Hybridization of nucleic acids (particularly
DNA) is the basis for reliable DNA analysrs.
Hybridization is based on the principle that a
single-stranded DNA molecule recognizes and
s p e c i f i c a l l yb i n d s t o a c o m p l e m e n t a r y D N A s t r a n d
amid a mixture of other DNA strands. This rs
c o m p a r a b l et o a s p e c i f i c k e y a n d l o c k r e l a t i o n s h i p .
The general procedure adopted for nucleic acid
hybridization is as follows (Fig. laJ)
The single-strandedtarget DNA is bound to a
membrane support. Now the DNA probe (sinSle-
173
174
stranded and labeled with a detector substance)is
added. Under appropriate conditions (temperature,
ionic strength), the DNA probe pairs with the
c om plem ent ar y t ar get DNA. T h e u n b o u n d D N A
probe is removeci.Sequence of nucleotides in the
target DNA can be identified from the known
s equenc e of DNA pr obe.
There are two types of DNA hybridization-
r adioac t iv e and non- r adioac ti v er e s p e c t i v e l yu s i n g
DNA probes labeled with isotopes and non-
isotopes (discussedbelow) as detectors.
DNA PRO BES
A DNA probe or a gene probe is a synthetic,
single-stranded DNA molecule that can recognize
and specifically bind to a target DNA (oy
c om plem ent ar y bas e pair ing ) i n a m i x t u r e o f
.l
biom olec ules . DNA pr obes ar e e i t h e r l o n g ( > 0 0
nuc leot ides )or s hor t ( < 50 nu c l e o t i d e s ) ,a n d m a y
bind to the total or a small portion of the target
DNA. Ther e is a wide v ar iat io n i n t h e s i z e o f D N A
probes used (may range from 10 bases to 10,000
bas es ) . The m os t im por t ant r e q u i r e m e n t i s t h e r r
s pec if ic and s t able binding wi t h t a r g e t D N A s .
Methods employed to obtain DNA
probes
A great majority of DNA probes are chemically
synthesized in the laboratory.There are, however,
many other ways of obtaining them-isolation of
selected regions of genes, cloning of intact genes,
pr oduc ing ir om m RNAs .
lsolation of selected regions of genes : The
DNA from an organism (say a pathogen)can be cut
by us ing r es t r ic t ion endonucl e a s e s .T h e s e D N A
fragments are cloned in vectors and the DNA
probes can be selected by screening.
Synthesis of DNA probes from mRNA : The
m RNA m olec ules s pec if ic t o a p a r t i c u l a r D N A
s equenc e ( enc oding a pr ot ei n ) a r e i s o l a t e d . B y
us ing t he enz y m e r eve r s e t r a n s c r i p t a s e ,
c om plem ent ar y DNA ( c DN A ) m o l e c u l e s a r e
synthesized.This cDNA can be used as a probe to
detect the target DNA.
fulechanism of action of DNA probes
The bas ic pr inc iple of DNA p r o b e s i s b a s e d o n
the denaturation and renaturation (hybridization)
of DNA. W hen a double- s t r a n d e dD N A m o l e c u r e phosphatase,chromogenic substrateswill be used,
is subjectedto physical (temperature> 95'C or pH
BIOTECHNOLOCY
< 1 0 . 5 ) o r c h e m i c a l ( a d d i t i o n o f u r e a o r fo r m a r -
dehyde) changes, the hydrogen bonds break and
the complementary strands get separated. This
process is called denaturation. Under suitable
conditions (i.e., temperature, pH, salt concen-
tration), the two separatedsingle DNA strandscan
reassemble to form the original double-stranded
DNA. and this phenomenon is referred to as
r e n a t u r a t i o no r h y b r i d i z a t i o n .
Radioactive detection system
The DNA probe is usually tagged with a
radioactive isotope (commonly phosphorus-32).
The target DNA is purified and denatured, and
mixed with DNA probe. The isotope labeled DNA
m o l e c u l e s s p e c i f i c a l l y h y b r i d i z e s w i t h th e ta r g e t
D N A ( F i g . 1 4 . 1 ) . f h e n o n - h v b r i d i z e dp r ob e D N A i s
washed away. The presenceof radioactivity in the
hybridized DNA can be detected by
autoradiography.This reveals the presence of any
b o u n d ( h y b r i d i z e d ) p r o b e m o l e c u l e s a n d th u s th e
complementhry DNA sequencesin the target DNA.
Non-radioactive detection system
The disadvantage with the use of radioactrve
label is that the isotooes have short half-lives ancr
i n v o l v e r i s k s i n h a n d l i n g , b e s i d e sr e q u i ri n g sp e ci a l
laboratory equipment. So, non-radioactive
detection systems (e.g., biotinylation) have also
been developed. Biotin-labeled (biotinylated)
nucleotides arc incorporated into the DNA probe.
The detection system is based on the enzymatic
conversion of a chromogenic (colour producing) or
c h e m i l u m i n e s c e n t( l i g h t e m i t t i n g ) s u b s t r a te s.
The procedure commonly adopted for
chemiluminescent detection of target DNA is
depicted in Fig. 14.2. A biotin labeled DNA probe
is hybridized to the target DNA. The egg white
protein avidin or its bacterial analog streptavidin is
added to bind to biotin. Now a biotin labeled
enzyme, such as alkaline phosphataseis added
w h i c h a t t a c h e s t o a v i d i n o r s t r e p t a v i d i n .- fh e se
p r o t e i n s h a v e f o u r s e p a r a t e b i o t i n - b i n d i n g si te s.
T h u s , a s i n g l e m o l e c u l e ( a v i d i n o r s t r e p ta vi d i n )ca n
b i n d t o b i o t i n - l a b e l e dD N A p r o b e a s . v e ll a s b i o ti n -
labeled enzyme. On the addition of a
c h e m i l u m i n e s c e n t s u b s t r a t e ,t h e e n z v me a l ka l i n e
phosphataseacts and converts it to a light emitting
product which can be measured.(Note : When the
e n z y m e p e r o x i d a s ei s e m p l o y e d i n p l a c e o f a l ka l r n e
and the colour of the product formed is measurecr;.
lnaoter 14 : DNA D'ACNosts4\Dl'!FP-lgL-FsEltlrcj.-.-__
: rN- DT'EASE
-":E:____'----

{- DNA probe

Substrate
Light-emitting
product

Fig.1 4'2 :Non- r adioac t uedet ec t ions y s t em o f t a r g e t D N A b y u s i n g b i o t i n - l a bthe

e l eenzyme
dDNAprobe
(Note : Chemiluminescentdetectionis .iltustrated; :!.'::"?::ic,^detection'
-'?'
oeroxidaseand a cnromogenicsubstrateare used)
176, B IOTECHNO LO CY

Adv ant ages : The biot in- l a b e l e d D N A i s q u r t e Target DNA fragments with complementary
stable at room temperaturefor about one year. The sequences bind to DNA probes. The remaining
det ec t ion dev is es us ing c he m i l u m i n e s c e n c e a r e DNA fragmentsare washed away. The target DNA
preferred,since they are as sensitiveas radioisotope p i e c e s c a n b e i d e n t i f i e d b y t h e i r f l u o r e sce n ce
det ec t ion, and m or e s ens i t i v e t h a n t h e u s e o f e m i s s i o n b y p a s s i n g a l a s e r b e a m . A co m p u te r i s
chromogenic detection systems. used to record the pattern of fluoresenceemission
and DNA identification.
PCR in the use of DNA probes
T h e t e c h n i q u e o f e m p l o y i n g D N A ch i p s i s ve r y
DNA pr obes c an be s uc c e s s f u l l yu s e d f o r t h e
r a p i d , b e s i d e s b e i n g s e n s i t i v ea n d s p e ci fi c fo r th e
ident if ic at ion of t ar get D N A s f r o m v a r i o u s several DNA fr a g m e n ts
identification of
s am ples- blood, ur ine, f e c e s , t i s s u e s , t h r o a t
s i m u l t a n e o u s l y . S c i e n t i s t s a r e t r y i n g to d e ve l o p
was hingswit hout m uc h pur ifi c a t i o n .T h e d e t e c t i o n
C e n e c h i p s f o r t h e e n t i r e g e n o m e o f a n o r g a n i sm .
of target sequence becomes quite difficult if the
quant it y of DNA is v er y lor v . I n s u c h a c a s e , t h e
Applications of DNA chip
poly m er as ec hain r eac t ion ( P C R ) i s f i r s t e m p l o y e d
to amplify the minute quantities of target DNA T h e p r e s e n c em u t a t i o n s i n a D N A s e q u e n ceca n
( Chapt er E) , and ident if ied b y a D N A p r o b e . b e c o n v e n i e n t l y i d e n t i f i e d . I n f a c t , G e n ech i p p r o b e
array has been successfullyused for the detection
DNA probes and signal amplification o f m u t a t i o n s i n t h e p 5 3 a n d B R C A I ge n e s. Bo th
Signal am plif ic at ion is an a l t e r a t i v et o P C R f o r t h e s e g e n e s a r e i n v o l v e d i n c a n c e r ( d e ta i l s g i ve n
t he ident if ic at ion of m inut e q u a n t i t i e s o f D N A b y later).
us ing DNA pr obes . I n c as e o f P C R , t a r g e t D N A i s
am plif ied, while in s ignal a m p l i f i c a t i o n i t i s t h e
target DNA bound to DNA probe that is amplified
There are two general methods to achieve signal
am olif ic at ion
1 . Separate the DNA target-DNA probe
T h e u s e o f D N A a n a l y s i s ( b y e m p l o yi n g D N A
c om plex f r om t he r es t of t he D N A m o l e c u l e s , a n d probes) is a novel and revolutionary approach for
t hen am plif y it . specifically identifying the d i s ea se - ca u si n g
2. Amplify the DNA probe (bound to target pathogenic organisms This is in contrast to the
DNA) by us ing a s ec on d p r o b e . T h e R N A t r a d i t i o n a l m e t h o d s o f d i s e a s e d i a g n o si s b y
complementary to the DNA probe can serve as the detection of enzymes, antibodies etc., besides the
s ec ond pr obe. The RNA- DN A - D N A c o m p l e x c a n m i c r o s c o p i ce x a m i n a t i o no f p a t h o g e n s .Al th o u g h a t
be separated and amplified. The enzyme O-beta p r e s e n t n o t i n w i d e s p r e a d u s e , D N A a n a l ysi s m a y
r eplic as e whic h c at aly s es R N A r e p l i c a t i o n i s soon take over the traditional diagnostictests in the
c om m only us ed. years to come. Diagnosis of selected diseases by
genetically engineered techniques or DNA probes
THE DNA CHIP.MICROARRAY OF or direct DNA analysis is briefly described.
G ENE PRO BES
The DNA chip or Genechip contains thousands T U B E R C U L O S I S
of DNA probes (4000,000 or even more) arranged
T u b e r c u l o s i s i s c a u s e d b y t h e b a cte r i u m
on a s m all glas s s lide of t h e s i z e o f a p o s t a g e
Mycobacterium tuberculosis. The commonly used
s t am p. By t his r ec ent and a d v a n c e d a p p r o a c h ,
diagnostic tests for this disease are very slow and
thousandsof target DNA molecules can be scanned
sometimesmay take severalweeks. This is because
s im ult aneous ly .
M. tuberculoslsmultiplies very slowly (takes about
Technique for use of DNA chip 2 4 h r s . t o d o u b l e ; E . c o l l t a k e s j u s t 2 0 m i n u te s to
double).
The unk nown DNA m o l e c u l e s a r e c u t i n t o
fragmentsby restrictionendonucleases.Fluorescent A novel diagnostic test for tuberculosis was
markersare attachedto these DNA fragments.They d e v e l o p e d b y g e n e t i c e n g i n e e r i n g ,a n d is i l l u str a te d
are allowed to react to the probes of the DNA chip. in Fig. 14.3. A gene from firefly, encoding the
 14 : DNA IN DI SEASEDI ACNO SI S AND M E D I C A L F O R E N S I C S
ChA OICT
Phage
genome
Viral
genome
Luciferase
gene
Fig. 14.3 : Diagnosis of tuberculosis by using a
genetically engineered bacteriophage (phage).
enzyme luciferase is introduced into the
bacteriophage specific {or M. tuberculosis.
The bacteriophage is a bacterial virus, frequently
referred to as luciferase repofter phage or
mycophage. The genetically engineered phage
is added to the culture of M. tuberculosis. The
phage attachesto the bacteriai cell wall, penetrates
insicie, and inserts its gene (along with luciferase
gene) into the M. tuberculosis chromosome. The
enzyme luciferase is produced by the bacterium
When luciferin and ATP are added to the culture
medium, lu cife rasecleav es luc if er in. This r eac t ion
is accompanied by a flash of light which can be
detected by a luminometer. This diagnostic test is
quite sensitivefor the confirmation of tuberculosis.
The flash of light is specific for the
identification of M. tuberculosis in the culture. For
other bacteria, the genetically engineered phage
cannot attach and enter in, hence no flash of light
rvould be detected.
MALARIA
Malaria, mainly caused by Plasmodium
ialciparum and P. vivax, affects about one-third of
t he world 's p op ula t ion. The c om m only us ed
labora toryte stsfo r the diagnos isof m alar ia inc lude
-4otechnology [12]
177
microscopic examination of blood smears, and
d e t e c t i o n o f a n t i b o d i e si n t h e c i r c u l a t i o n .Wh i l e t h e
former is time consuming and frequently gives
false-negativetests, the latter cannot distinguish
between the past and present infections.
A specific DNA diagnostic test for identification
of the current infection ol P. falciparum has been
developed. This is carried out by using a DNA
probe that can bind and hybridize with a DNA
fragment of P. falciparum genonte and not with
other speciesof Plasmodium. lt is reported that this
DNA probe can detect as little as 1 ng of
P. falciparum in blood or 10 pg of its purified DNA.
CHAGAS' DISEASE
The protozoan parasite Trypanosoma cruzi
causes Chagas' disease. This disease is
characterizedby destructionof severaltissues(liver,
spleen, brain, lymph nodes) by the invading
p a r a s i t e . C h a g a s 'd i s e a s e i s d i a g n o s e d b y t h e
microscopic examination of the fresh blood
samples. lmmunological tests, although available,
are not commonly used, since they frequentlygive
false-positiveresults.
Scientistshave identified a DNA fragment with
1B8-base pair length present in T. cruzi genome.
This is however, not found in any other related
parasite.A PCR technique is employed to amplify
the lBB bp DNA fragment. This can be detected by
u s i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s .T h u s ,
PCR-basedamplification can be effectively used for
t h e d i a g n o s i so f C h a g a s ' d i s e a s e .
ACQUIRED IMMUNODEFICIENCY
S Y N D R OME (A rD S l
AIDS is caused bv the virus. human
immunodeficiencyvirus(HIV. fhe commonly used
laboratory testfor detectionof AIDSis the detection
of HIV antibodies.However,it might take several
weeks for the body to respond and produce
suffi ci ent H IV anti bodi es. C onseqqentl y,the
antibodies testmay be negative(i.e.,false-negative),
al thoughH IV i s presenti n the body. D uri ngthi s
period,being a carrier,helshecan transmitHIV to
others.
DNA probes,with radioisotopelabel, for HIV
DNA are now available.By using PCRand DNA
probes, AIDS can be specifically diagnosedin the
laboratory.During the courseof infectioncycre,
178
HIV existsas a segment of DNA integratedinto the by certain bacteria.At leastthree distinct speciesof
T-lyrnphocytesof tlre host. The T-lymphocytesof a
suspectedAIDS patient are isolated and disrupted developed for their detection. Early diagnosis of
to releaseDNA. The so obtained DNA is amplifieo p e r i o d o n t a l d i s e a s e w i l l h e l p t h e tr e a tm e n t
by PCR, and to this DNA probes are added. lf the modalities to prevent the tooth decay.
HI V DNA is pr es ent , it h y b r i d i z e s w i t h t h e
complementary sequence of the labeled DNA
probe which can be detected by its radioactivity.
The advantage of DNA probe is that it can detect
the virus when there are no detectable antibodres
in t he c ir c ulat ion.
HIV diagnosis in the raewborep
Detection of antibodies is of no use in tne organisms-bacteria, viruses, parasites.
newborn to ascertain whether AIDS has been
transmitted from the mother. This is because the
antibodies might have come from the mother
but not from the virus. This problem can be solved
by using DNA probes to detect HIV DNA in the
newborn.
HUMAhI PAPILLONfi& VEffiAJS
Human papilloma virus (HPV) causes genital
warts. HPV is also associated with the cervical
cancer in women. The DNA probe (trade name
Virapap detection kit) that specifically detects HPV
has been developed. The tissue samples obtained
from woman's cervix are used. HPV DNA, when
present hybridizes with DNA probe by
complementary base pairing, and this is the positive
test.
LYME DISEASE
Lyme disease is caused by the bacterium,
Borrelia burgdorferi. This disease is characterized
by fever, skin rash, arthritis and neurological
manifestations.
The diagnosisof Lyme disease is rather difficult,
since it is not possible to see B. burgdorferi under
microscope and the antibody detection testsare not
v er y r eliable.
Some workers have used PCR to amplify the
DNA of B. burgdorferi. By using appropriate DNA
probes,the bacterium causing Lyme diseasecan be
soecificallv detected.
PERI O DO NTAL DI SEASE
Periodontal disease is characterized by the
degenerativeinfection of gums that may ultimately
lead to tooth decay and loss.This diseaseis caused
B IOTE CHNO LO CY
bacteria have been identified and DNA pr,obes
MNA PF3@BES FGR OTHER DISEASES
In principle, almost all the pathogenic
organisms can be detected by DNA probes. Several
DNA probes (more than 100) have been developecl
and many more are in the experimental stages.The
ultimate aim of the researchersis to have a stock of
probes for the detection of various pathogenic
The other important DNA probes in recent years
include for the detection of bacterial infections
caused by E. coli (gastroenteritis) Salmonella typhi
(food poisoning), Campylobacter hyoitestinalis
(gastritis).
ffifragarosis of tropical diseases
Malaria, f i l a r i a s i s , t u b e r c u l o s i s, l e p r o sy,
s c h i s t o s o m i a s i sl,e i s h m a n i a s i sa n d t r y p an o so m i a si s
are the tropical diseasesaffectingmillions of people
throughout the world. As already described (See
p. 177), for the diagnosis of malaria caused by
P. falciparum, a DNA probe has been developed.
A novel diagnostic test, by genetic manipulations,
has been devised for the diagnosis of tuberculosis
.l
(See p. 76). Scientistsare continuously working to
develop better diagnostic techniques for other
tropical diseases.
Traditional laboratory tests for the diagnosis of
genetic diseasesare mostly based on the estimation
of metabolitesand/or enzymes.This is usually done
after the onset of symptoms.
The laboratorytests based on DNA analysiscan
specifically diagnose the inherited diseasesat the
genetic level. DNA-based tests are useful to
d i s c o v e r ,w e l l i n a d v a n c e , w h e t h e r t h e i n d i vi d u a l s
or their offspringsare at risk for any genetic disease:
Further, such tests can also be employed for the
prenatal diagnosis of hereditary disorders, besides
identifying the carriers of genetic diseases.
C hapt er14 : DN A l N D ISE ASD
E IAGN O S IS
AN D ME D IC A LFOR E N S IC S 179

By knowing the genetic basisof the diseases,the B-Globingene

ind ivid ua ls ca n be adv is ed on how t o lim it t h e
transmission of the disease to their offsprings. lt
may also be po ss ible,in due c our s e of t im e, t o t r e a t
genetic diseasesby appropriate gene therapies.

Theoretically,it is possibleto develop screening Mstll

tests for all single-gene diseases. Some of the
important genetic diseasesf<lr which DNA analysis
Y
is used for dia gn os isar e br ief ly des c r ibed.
Pro Glu Glu
GAG GAG F Base sequence
CYSTIG FIBROSIS in normalgene
567 FAmino acidnumber
Cystic fib rosis ( CF) is a c om m on and f a t a l
CCT GTG GAG tsBasesequencein
hereditary disease.The patients produce thick and
Pro Val Glu sickle-cellgene
sticky mucus that clogs lungs and respiratorytract (p-globingene)
(For some more details on CF, refer Chapter 13).
Cystic fibrosis is due to a defect in cftr gene that Fig. 14.4 : Single-base change resulting in sickle-cell
encodes cystic fibrosis transmembrane regulator anemia. (Note : A small and relevant DNA fragment
protein cftr gene is located on chromosome 7 rn of p-globin gene magnified and shown with
humans, and a DNA probe has been developed to encoded amino acids).
ide ntify this g en e.

The genetic diseasecystic fibrosis is inherited by

DNA fragments in and around B-globin gene,
a recessivepattern, i.e., the diseasedevelops when
followed by the electrophoreticpattern of the DNA
two recessivegenes are present. lt is now possible
fragments formed. The change in the base from A
to detect CF genes in duplicate in the fetal cells
to T in the'B-globin gene destroys the recognition
o bta ine d fro m sa m olesof am niot ic f luid. As t he t e s t
site (CCTCACC) for Mstll (Fig. 1a.4. Consequently,
can be done months before birth. it is possible to
the DNA fragments formed from a sickle-cell
know whether the offspring will-be a victim of CF.
anemia patient for B-globin gene differ from that of
One group of researchershave reported that CF
a normal person.Thus. sickle-cellanemia can be
gene can be detected in the eight-celled embryo
detected by digesting mutant and normal B-globin
obtained through in vitro fertilization.
gene by restriction enzyme and performing
a hybridization with a cloned p-globin DNA
SICKL E.CELL ANEM I A
probe.
Sickle-cell a nem ia. is a genet ic dis ea s e
characterizedby the irregular sickle (crescentlike) Single.nucleotide polymorphisms
shape of the erythrocytes. Biochemically, this The srhgle base changes that occur in some of
disease results in severe anemia and progressive t h e g e n e t i c d i s e a s e s( e . g . , s i c k l e - c e l l a n e m i a ) a r e
damage to major organs in the body (heart, brain, collectively referred to as single-nucleotide
lun gs, jo ints). polymorphisms (SNPs, pronounced snips). lt is
estimated that the frequency of SNPs is about one
Sickle-cellan em ia oc c ur s due t o a s ingle am in o
in every 1000 bases. Sometimes SNPs are
acid cha ng e in t he p- c hain of hem oglob i n .
associatedwith amino acid change in tlre protern
Specifically,the amino acid glutamate at the 6th
that is encoded A point mutation in a,-antitrypsrn
position of B-chain is replaced by valine. At the
gene is also a good example of SNPs, besides
m ole cu lar le ve l, s ic k le- c ell anem ia is due t o a
sickle-cellanemia.
s ing le-n ucleo tidec hange ( . A- + Tl in t he B- glob r n
gene of coding (or antisense)strand. ln the normal
DUCHENNE'S MUSCULAR DYSTROPHY
B-globin gene the DNA sequenceis CCTCACCAC,
wh ile in sickle- c ell anem ia, t he s equenc e i s D u c h e n n e 's m u s c u l a r d y s t r o p h y ( D M D ) i s a
CCTCTCCAC. This s ingle- bas em ut at ion c an b e genetic abnormality characterized by progressive
detected by using restriction enzyme Mstll Io cuI w a s t i n g o f l e g a n d p e l v i c m u s c l e s .l t i s a s e x - l i n k e d
180 B IOTECHNO LO CY

recessive disease that appears between 3 and 5 clinical manifestationsof this Ciseaseare observed
years of age. The affected chilcirenare unsteadyon after middle age, and by then the person might
their feet as they lose the strength and control of have already passed on the defective gene to his/
t heir m us c les . By t he age o f t e n , t h e v i c t i m s o f her offsprings.
DMD are confined to wheel chair and often die
before reaching 20 years age. FRAGILE X SYNDROME

The patients of DMD lack the muscle protein, F r a g i l e X s y n d r o m e , a s t h e n a m e in d i ca te s, i s

namely dystrophin which gives strength to the clue to a genetic defect in X chromosome (a sex
m us c les . Thus , DM D is due t o t h e a b s e n c e o f a chromosome) and affects both males and females.
gene enc oding dy s t r ophin.Fo r s p e c i f i c d i a g n o s i so f T h e v i c t i m s o f t h i s d i s e a s e a r e c h a r acte r i ze d b y
Duchenne's muscular dystrophy, a DNA probe to mental retardation.
identify a segment of Dt''lA that lies close to Researchershave found that sufferersof fragile X
defective gene (for dystrophin) is used This DNA syndrome have the three nucleotide bases (CGG)
segrnent, referred to as restriction fragment length repeated again and again. lt is beiieved that these
polymorphism (RFLP),serves as a marker and can trinucleotide repeats block the transcription
detect DMD with 95% certainity. process resulting in a protein deficiency. This
I n t he DNA diagnos t ict es t u s i n g R F L Pf o r D M D , protein is involved in the normal function of the
DNA samples must be obtained from as many n e r v e c e l l s , a n d i t s d e f i c i e n c y r e s u l ts i n m e n ta l
blood r elat iv es ( par ent s , g r a n d - p a r e n t s , u n c i e s , retardation.
aunt s et c . ) as pos s ible. T h e R F L P p a t t e r n s , A DNA probe has been developed for the
constructed for the entire fanrily are thoroughly detection of fragile X syndrome in the laboratory.
checked for the affected and unaffected reiatives
This is r eouir ed s inc e t her e i s a w i d e v a r i a t i o n r r r OTHER TRIPLE REPEAT DISEASES
RFLPsf r om f ar nily t o f am ily . T h u s , t h e r e i s n o s i n g l e
identifying test for the diagnosisof genetic diseases Excessiverepetition ol triplet bases in DNA are
bas ed on RFLPsanaly s is n o w k n o w n t o r e s u l t i n s e v e r a ld i s e a s e sw h i ch a r e
collectively referred to as triple repeat diseases.
$*WruTfrNGTON'$ DISEASE B e s i d e s H u n t i n g t o n 's d i s e a s e a n d fr a g i l e X
syndrome (discussed above), some more triple
Hunt ingt on' sdis eas eis a g e n e t i cd i s e a s e( c a u s e d repeatsare given below.
by a dominant gene) characterized by progressrve
deteriorationof the nervous system,particularly the Friedreich's ataxia
des t r uc t ionof br ain c ells .The v i c t i m s o f t h i s d i s e a s e
The trinucleotide GAA repeats200 to 900 times
( us ual11, abov e 50 y ear s of a g e ) e x h i b i t t h r a s h i n g
o n c h r o m o s o m e 9 i n F r i e d r e i c h 's a ta xi a . Th i s
(jerky) movements and then insanity [older name
disease is associated with degradation of spinal
was Huntington's chorea; chorea (Greek) means to
cord. Spinocerebellar ataxia is another triplet
dancel. Huntington's disease is invariably fatal.
disease, characterized by neuromuscular disorder,
The m olec ular bas isof Hu n t i n g t o n 'sd i s e a s eh a s and is due to trinucleotide repeatsof CAC by 40 to
been ident if iec i. The gene r e s p o n s i b l e f o r t h i s B 0 t i m e s o n c h r o m o s o m e 6 .
dis eas e lies on c hr om os om e n u m b e r 4 , a n d i s
There are a fer,vtriple repeat diseasesin which
chai-acterized by excessive repetition of the base
the repeatstend to increase with each generation
triplet CAG. The victims of Huntington's ciisease
and the diseases become more severe. This also
have CAC triplet repeated42-66 times, againstthe
results in the onset of clinical manifestations at
normal 11-34 times. The triplet CAG encodes for
early ages. Kennedy's disease, also called
the arnino acid glutamine. lt is believed that the
spinobulbar muscular atropy (CAG repeafl and
abnormal grofein (with very high content of
myotonic dystrophy (CTA are good examples.
glutamine) causes the death of cells in the basal
ganglia (the part of the brain responsiblefor motor Are triple repeat diseases confined to humans?
f unc t ion) .
Triple repeat diseases have so far not been
Huntington's disease can be detected by the detected in any other organisms (bacteria, fruit
analy s isof RFLPsin blood r e l a t e d i n d i v i d u a l s .T h e f l i e s , o t h e r m a m m a l s ) e x c e p t i n h u m a n s. M o r e
 -I4 181
Ch APICT : DNA I N DI SEASEDI AG NO SI S AND M E D I C A L F O R E N S I C S

studies however, may be needed to confirm this.
The occurrence of triple repeat diseasesindicates
that the structure of DNA may be rather unstable
and d yn amic. This is in c c nt r as tt o what m olec uiar
biolog ists ha ve be en t hink ing all along.
ALZHEIMER'S DISEASE
Alzheimer's disease is characterized by loss of
memo ry a nd im pair ed int ellec t ual f unc t ion
(de men tia). Th e vic t im s of t his dis eas e c annot
properly attend to their basic needs, besides being
unable to speak and walk.
The patients of Alzheimer's diseasewere founo
to have a specific protein, namely amyloid in the
plaqu es (o r. clump s ) of dead ner v e f iber s in
their brains. A group of researchershave identified
a specific gene on chromosome 21 that is
believed ro be responsible for familial Alzheimer's
disease.
gerre sodf . The deleterious effects of free radicals
can be reduced by administering certain
compounds such as vitamins C and E
Another group of workers have reportedthat the
defective superoxide dismutase cannot control a
transporter protein responsible for the removal of
t h e a m i n o a c i d g l u t a m a t ef r o m t h e n e r v e c e l l s . A s
a result, large qr-rantities of glutamateaccumulate in
t h e n e r v o u st i s s u e l e a d i n g t o d e g e n e r a t i v ec h a n g e s .
CANCERS
It is nov., agreed that there is some degree of
genetic predisposition for the occurrence of
cancers, although the influence of environmental
factors cannot be underestirnated.In fact, cancer
susceptible genes have been identified in some
f a n r i l i e s e . 9 . , g e n e s f o r m e l a n o m a s u s c e p t i b i l i t yi n
humans are located on chromosomes 1 and 9
p53 gene

A DNA probe has been developed to locate the The gene p53 encodes for a protein with a
genetic marker for Alzheimer's disease.l'he present m o l e c u l a r w e i g h t 5 3 k i l o d a l t o n s ( h e n c e t h e n a m e ) .
belief is that many environmental factors, and a It is believed that the protein produced by this gene
virus may also b e res pons iblef or t he dev elopm ent h e l p s D N A repair and suppresses cancer
of this d ise ase. lt m ay be pos s ible t hat in t he development. Certain damages that occur in DNA
individuals with genetic predisposition,the outside m a y l e a d t o u n l i m i t e d r e p l i c a t i o na n d u n c o n t r o l l e d
factors nray be stimulatory for the onset of the multiplication of cells. In such a situation, the
d isease. protein encoded by ps3 gene binds to DNA and
blocks replication. Further, it facilitates the faulty
A]I'IYOTROPHIC LATERAL SCLEROSIS DNA to get repaired. The result is that the
cancerous cells are not allowed to establishano
Amyotrophic lateral sclerosis (ALS) is multiply. Thus,
f3 is a cancer-suppressorgene and
characterized by degenerativechanges in the motor acts as a guardian of cellular DNA.
neurons of brain and spinal cord. A gene to explain
Any nrutation in the gene fi is likely to alter its
the inherited pattern of ALS was discovered.
tumor suppressor function that lead to cancer
The gene, known as sodl, encoding for the development And in fact, the altered forms of
enzyme superoxide dismutase is located on ps3 recovered from the various tunror cells (breast,
chromosome 21. T.his gene was found to be b o n e , b r a i n , c o l o n . b l a d d e r ,s k i n , I u n g ) c o n f i r m t h e
defective in families suffering from amyotrophic protective function of ps3 gene against cancers.
laterai sclerosis.In fact, certain point mutations in It is believed that the environrnentalfactors mav
the sodl resulting in single amino acid charrgesin
cause mutations in ps3 gene which may ultinrately
superoxide dismutase have been identified. lead to cancer. Some of the mutations of p53
Sup-^roxide ciismutase is a key enzyme in g e n e m a y b e i n h e r i t e c i ,w h i c h p r o b a b l y e x p l a i n s
elim in atin g th e hig hly t ox ic f r ee r adic als t hat t h e o c c u r r e n c e o f c e r t a i n c a n c e r s i t t s o m e l 'a m i l i e s .
damage the cells (free radicals have been
implicated in aging and severaldiseasee.B. cancer. Genes of breast cancer
cataract,'Parkinson'sdisease,Alzheimer's disease). Two genes, namely BRCN and BRCNI,
On the basis of the function of superoxide implicated ir-r certain hereditary forms of breast
dismutase, it is presumed that ALS occLtrs as a cancer in rvomerr, have beerr identified. lt is
'fre e estimated that abolrt Bj'k of irrherited breast
result o f ra di c al ac c um ulat ion due t o a
defective enzyme (as a consequence of mutatecl c a n c e 1 5a r e d u e t o m u t a t i o n s i n e i t h e r o n e o f t h e s e
182 B IOTECHNO LO CY

t wo ger r es- BRCAI or BRCA I I . I n a d d i t i o n , t h e r e l s Gene of retinoblastoma

a high r is k f or ov ar ian c anc e r d u e t o m u t a i i o n s i n
R e t i n o b l a s t o m ai s a r a r e c a n c e r o f th e e ye . l f
BRCAI.
detected early, it can be cured by radiation therapy
I t is s ugges t edt hat t he no r m a l g e n e s B R C A I a n d a n d l a s e r s u r g e r y o r e l s e t h e e y e b a l l h a s to b e
BRCAI I enc ode pr ot eins ( w i t h 1 8 6 3 a n d 3 4 1 8 removeo.
am ino ac ids r es pec t iv ely t)ha t f u n c t i o r r i n a m a n n e r
S c i e n t i s t sh a v e i d e n t i f i e da m i s s i n gor a d e fe cti ve
c om par able t o gene p53 p r o t e i n ( a s d e s c r i b e d
( m u t a t e d )g e n e o n c h r o m o s o m e n u m b e r 1 3 , b e i n g
abov e) . As s uc h, BRCAI a n d B R C A I I a r e D N A -
r e s p o n s i b l e f o r r e t i n o b l a s t o m a .T h e n o r m a l Se n e
r epair and t um or - s uppr e s s o r g e n e s . S o m e
w h e n o r e s e n to n c h r o m o s o m e 1 3 i s a nti ca n ce ra n d
researchersbelieve tl-ratthese tno proteirrs act as
gene reguiators. d o e s n o t a l l o w r e t i n o b l a s t o m at o d e v e l o p .

Diagnos t ic t es t s f or t he a n a l y s i s o f t h e g e n e s D I A B E T E S
BRCAI and BRCAII were developed. Unfortunately,
t heir ut ilit y is v er y lim it ed, s i n c e t h e r e c o u l d b e D i a b e t e s m e l i i t u s i s a c l i n i c al co n d i ti o n
hundr eds of v ar iat ions in t h e b a s e s e q u e n c e o f characterized by increased blood glucose level
t hes e genes . : (hyperglycemia) due to insufficient or inefficient
( i n c o m p e t e n t ) i n s u l i n . I n o t h e r w o r d s , i n d i vi d u a l s
Genes ef colon cancer with diabetes cannot utilize glucose properly in
their body.
The oc c ur r enc e of c olon c a n c e r a p p e a r s t o b e
genet ic ally link ed s ir r c e it r u n s i n s o m e f a m i l i e s . A rare form of type ll diabetes (i.e., non-insulrn
Some researchershave identified a gerre lir.rked d e p e n d e r r td i a b e t e s n r e l l i t u s , N I D D M ) i s m a tu r i ty
with hereditary nonpolyposis colon cancer or onset diabetes of the young (MODY) MODY
HNPCC (some times called lynch syndrome). fhis o c c u r r i n g i n a d o l e s c e n t sa n d t e e n a g e r s,i s fo u n d to
gene enc oded a pr ot ein t hat a c t s a s a g u a r d i a n a n d have a genetic basis A gene, synthesizing the
br ings about DNA r epair w h e n e v e r t h e r e i s a enzyme glucokinase, located on chromosome 7, is
danrage to it. However, as and when there is a found to be defective in MODY patients
mutation to this protective gene, an altered protetn
C l u c o k i n a s e i s a k e y e n z y m e i n g l u co se
r s pr oduc ed whic h c annot u n d o t h e d a m a g e d o n e
m e t a b o l i s m . B e s i d e s i t s i n v o l v e m e n t i n th e
t o DNA. This leads t o HNP C C . l t i s e s t i m a t e dt h a t
n r e t a b o l i s m , g l u c o k i n a s e i n t h e p a n cr e a ti c ce l l s
t he oc c ur r enc e of t his alt er e d g e n e i s o n e i n e v e r y
serves as a detector for glucose concentration in
200 people in gener al popu l a t i o n .
t h e b l o o d T h i s d e t e c t i o n s t i m u l a t e sB - ce l l s o f th e
p a n c r e a s t o s e c r e t e i n s u l i n . A g e n e m o d i fi ca ti o n
Micresatellite marker genes
t h a t r e s u l t si n a d e f e c t i v eo r a n a l t e r e d g l u co ki n a se
Microsatellites refer to the short repetitive h a m p e r s p a n c r e a t i c i n s u l i n s e c r e t i o n. L a te r r n ,o r k
s equenc es of DNA t hat c a n b e e m p l o y e d a s h a s s h o w n t h a t g l u c o k i n a s eg e n e i s d e fe cti ve i n th e
nr ar k er sf or t he ic lent if ic at i o no f c e r t a i n g e n e s . F o r common form of type ll diabetes.
c olon c anc er , nr ic r os at eil i t e m a r k e r g e n e s h a v e
been ident if iedon c lr r om oso m e2 i n h u m a n s .T h e r e DNA probes for type ll diabetes
is a lot of v ar iabilit y i n t h e s e q u e n c e o f
T h e g l u c o k i n a s eg e n e s f r o m n o r m al a n ci typ e l l
nr ic r os at ellit es( as is t he c as e w i t h R F L P sd e s c r i b e o
d i a b e t e s p a t i e n t s w e r e c l o n e d a n d s ca n n e d w i th
on p 185) .
DNA probes. lt was found that a single base
Ear ly det ec t ion of t he r is k f o r c o l o n c a n c e r b y mutation of the gene led to a defective glucokinase
DNA analy s isis a boon f or t h e w o u l d b e v i c t i m s o { production that is largely responsible for MODY,
t his dis eas e The s us pec t e d i n d i v i d u a l s c a n b e a n d a l s o a m a j o r i t y o f i n d i v i d u a l s w i th typ e l l
per iodic ally m onit or ed f or t h e s i g n s a n d t r e a t e d diabetes.Later,some workers reported a possibility
appr opr iat ely Unlik e m an y o t h e r c a n c e r s , t h e o f a t l e a s ta d o z e n m u t a t i o n s( t h o u g h I essi m p o r ta n t
c hanc es of c ur e f or c oion c a n c e r a r e r e a s o n a b l y t h a n t h e o n e d i s c u s s e d )i n B l u c o k i na seg e n e fo r
good. type ll diabetes.
C hapt er14 : DN A l N D ISE ASD
E IAC N OS IS
AN D ME D i C A LFOR E N S IC S
Genes responsible for type I diabetes
Type I d iab etes or ins ulin- dependentdiabet e s
mellitu s (IDDM) m ainly oc c ur s in c hildhood ,
.l
particularly between 2-1 5 years of age. IDDM rs
chara cte rize dby a lm os t t ot al def ic ienc y of ins ulin .
Researchershave identified at least 18 Cifferent
chromosome regions linked with type I diabetes.
These DNA seouences are located on
.l.l
chromosomes 6, and 18.
OBESITY
Obesity is an abnormal increase in the body
weight due to fat deposition. Men and women are
con sid ere d ob es e if t heir weight due t o f a t ,
respectively exceeds more than 2Oo/oand 25"h of
the body weight. Obesity increasesthe risk of high
blood pressure,diabetes, atherosclerosisand other
I ife-th rea ten
ing co ndit ions .
Although many believed that obesity could be
gen etically inh eri t ed, t he m olec ular bas is was no t
known for long. lt was in 1994, a group of workers
identified a mutated gene that caused obesity rn
mice. La ter, a sim ilar Sene was f ound in hum an s
atso .
The gene designatedob (Ior obese) is located on
chro mosome 6 in m ous e. The DNA of ob gen e
conta ins 6 50 kb a nd enc odes a pr ot ein wit h 16 7
amino a cid s in adipos e t is s ue. This pr ot ein i s
responsibleto keep the weight of the animals under
control. The genetically obese mice have mutated
ob gene and thereforethe weight-control protein is
not p rod uced . lt is believ ed t hat t his pr ot ei n
functions like a hormone, acts on the
hyp oth ala mus,a nd c ont r ols t he s it e of hunger and
energy metabolism (thesetwo factors are intimatel,/
linked with ob esit y ) .
With the discovery of ob gene, the treatment for
inherited obesity may soon become a reality. In
fact, one multinational biotechnology company has
started producing ob protein that can be used for
weight reduction in experimental mice.
Besides the ob gene, a few other genes (faf
gene, tub gene) that might be associated with
obesity have also been discovered.
DNA ANALYSIS FOR OT}IER
HUMAN DISEASES
There is a continuous search for the
identification of more and more genes that are
responsiblefor human diseases.Such an approach
183
will ultimately help in the specific diagnosis of
these diseases before their actr-raloccurrence. In
a d d i t i o n t o h u m a n d i s e a s e sd e s c r i b e da b o v e , s o m e
more are given below.
Deafness
T h e d e a f n e s s ,i n h e r i t e d i n s o m e f a m i l i e s , h a s
genetic basis. A team of workers have iclentifieda
gene on chromcsome 5, encoding a protein tl-rat
facilitatesthe assemblyof actin (protein) rrolecules
i n t h e c o c h l e a o f i n n e r e a r . T h e a s s o c i a t i o no f a c t i n
is very essentialfor the detection of sound waves
by the ear. A mutation of the gene on chromosome
5 results in a defective rrrotein svnthesisand non-
a s s e m b l yo f a c t i n m o l e c r - r l ew
s hich causedeafness.
Some other genes, besidesthe one described here,
have also been found to be associated with
deafness.
Glaucoma
C l a u c o m a i s a d i s e a s eo f t h e e v e t h a t m a v o f t e n
l e a d t o b l i n d n e s s .l t o c c u r s a s a r e s u l to f d a m a g e t o
t h e o p t i c n e r v e d u e t o p r e s s u r et h a t b u i l d s u p i n t h e
e y e . A g e n e r e s p o n s i b l ef o r t h e h e r e d i t a r yg l a u c o m a
in teenagershas been detected on chromosome 1
Another group of researchershave found a gene on
c h r o m o s o m e - ?w h i c h i s l i n k e d w i t h t h e a d u l t - r t n s e t
Sraucoma.
Baldness
There is an inherited fcrm of balciness,called
alopecia universalis. This is found to be associated
with a gene locatecjon chromosome 12.
Parkinson's disease
P a r k i n s o n 'sd i s e a s e i s a c o m m o n d i s o r d e r i n
many elderly people, with about 1% of the
population above 60 years being affected. lt rs
characterized by muscular regidity, tremors,
expressionless face, lethargy, involuntary
movements etc. In the victims of Parkinson's
disease, there is degeneration of brain cells,
besidesa low concentration of dopamine (a neuro-
transmitter). Researchers have identified that a
gene-encoded protein namely u-synuclein plays a
s i g n i f i c a n t r o l e i n t h e d e v e l o p m e n t o f P a r k i n s o n 's
disease.An altered form of cx,-synuclein (due to a
m u t a t i o n i n t h e g e n e ) a c c u m u l a t e si n t h e b r a i n a s
Lewy bodies This is responsible for nerve cells
d e g e n e r a t i o n a n d t h e i r d e a t h i n t h e P a r k i n s o n 's
d isease.
18,4 B IOTECHNO LO CY

Hemochromatosis

Hem oc hr om at os isis an ir o n - o v e r l o a dd i s e a s ei n
whic h ir on is dir ec t ly depos i t e d i n t h e t i s s u e s( l i r r e r ,
s pleen, hear t , panc r eas and s k i n ) A n a b n o i " m a l
gene on c hr onr os om e 6 i s l i n k e d w i t h h e m o -
cl.rromatosis.The amino acid tyrosine, in the
normal protein encoded by this gene is replaced by
cysteine. This abnormal protein is responsiblefor
excessiveiron absorption irom the irrtestinewhich
ar : c um ulat esin t he v ar ious ti s s u e sl e a d i n g t o t h e r r
dam age and r nalf r r nc t ion.
E n v i r o n m e n t a lp o l l u t i o n ( p a r t i c u l a r l yw a te r a n cl
foods) by path<-rgenic nricroorganismsand viruses is
a c o m m o n o c c u r r e n c e . r n t h e t r a d i t i o n a l a p p r o a ch ,
t h e p a t h o g e n sa r e i d e n t i f i e d b y c u l t i v a ti n g th e m tn
t h e l a b o r a t o r y I n r e c e n t y e a r s , t e c h n i q u e s h a ve
been developed for their detection by DNA
a n al y s i s .

WATER QUALITY TESTING

Menkets disease
The quality c.rfpotable water can be tested by
tVlenke'sclisease,a copper deficiency disorder,is
detecting the indicator bacteria such as Escherichia
c har ac t er iz ed by dec r eas e d c o p p e r i n p l a s m a ,
coli. For the DNA anzilysis,water is filtered and the
depigm ent at ion of hair , de g e n e r a t i o n o f n e r v e
bacteria trapped on the filter are broken to release
c ells and m ent al r et ar dat io n . A g e r r e l c . c a t e d o n
D N A B y u s e o f D N A p r o b e s t o d e te ct sp e ci fi c
X- c hr or nos onr e,enc oding a t r a n s p o r t p r o t e i n , i s
genes, ihe presence ol E coli can L-e specifically
link ed wit h M enk e' s dis eas e A d e f e c t i n t h e g e n e ,
d e t e c t e d . I h e D N A a n a l y s i s t e c h n i q u e i s ve r y
c ons equent ly in t he pr ot e i n , i m p a i r s c o p p e r
s e n s i t i v e a n d i s c a p a b l e o f i d e n t i f y i n g a si n g l e
absorotion from the intestine .l
E. coli cell in 00 ml of water. lt is also possibleto
d e t e c t o t h e r p a t h o g e n i c o r g a n i s ms su ch a s
G ENE BANKS- A NO VE L C O N C E P T
Salntonella, Vibrio ancl Shigella in due course of
As t he s ear c h c ont inues b y s c i e n t i s t s f o r t l . r e ilme.
ident if ic at ion of m or e and m o r e g e n e s r e s p o n s i b l e
D N A a n a l y s i s c a t - r a l s o b e e m p l oye d fo r th e
f or v ar ious dis eas es , t he e n l i g h t e n e d p u b l i c
specific detection of viruses and protozoal
( par t ic ular ly in t he dev elop e d c o u n t r i e s ) , i s v e r y
pathogens.
k een t o eniov t he f r uit s of t h i s r e s e a r c ho u t c o m e .
As of now. DNA or obes a r e a v a i l a b l e f c r r n e
deiec t ion a lim it ed nu m b e r o f diseases.
Res ear c her sc ont inue t o dev e l o p D N A p r o b e s f o r
a lar g, e nur nber of gen e t i c a l l y p r e d i s p o s e d
d is or der s .
DNA fingerorinting is the present day genetic
Gcne banks are the centres for the storage of
detective in the practice of modern medical
individual's DNAs for future use to diagnose
forensics. The underlying principles of DNA
drcease.s. For this purpose,the DNA isolatedfrom a
f i n g e r p r i n t i n ga r e b r i e f l y d e s c r i b e d .
per s on' sc elis ( us ually whit e b l o o d c e l l s ) i s s t o r e d .
As and when a DNA probe for the detection of a The structureof each person'sgenome is unique.
s pec if icdis eas eis av ailable,th e s t o r e d D N A c a n b e T h e o n l y e x c e p t i o n b e i n g m o n o z y g oti c i d e n ti ca l
us ed t br t he diagnos isor r is k a s s e s s m e not i t h e s a i d t w i n s ( t w i n s d e v e l o p e d f r o m a s i n g l e fe r ti l i ze d
genet ic dis eas e. ovum). The uniqr-re nature of genome structure
provides a good opportunity for the specific
I n f ac t , s om e ins t it ut ionsh a v e e s t a b l i s h e dg e n e
i d e n t i f i c a t i o no f a n i n d i v i d u a l .
bank s They s t ( ) r e t he D N A s a m p l e s 0 1 t h e
interesteclcLrstonrers at a fee (one firm lvas charging I t m a y b e r e m e m b e r e d h e r e t h a t i n th e
$ 200) for a sper:ifierJperiod (say around 20-2-5 t r a d i t i o n a l f i n g e r p r i n t t e c h n i q u e , t h e in d i vi d u a l i s
y ear s ) For t he r is k as s es s m e not f a n y d i s e a s e ,i t i s i d e n t i f i e d b y p r e p a r i n g a n i n k i m p r essi o n o f th e
adv is ablet o hav e t ir e DNAs f r o m c l o s e r e l a t i v e so f s k i n f o l d s a t t h e t i p o f t h e p e r s o n 'sf i ng e r . Th i s i s
at I eas t 2- 3 eener at ions . based on the fact that the nature of these skin folds
Chaot er14 : DN A l N D ISE ASD AN D ME D IC A LFOR E N S IC S
E IAGN O S IS 185

is genet ic ally
de te rmi n e da,n d th u s th e fi n g e rp ri nt R1 R2 R3
. c o n tra s t,th e D N A
is uniquef or an i n d i v i d u a l In
fingerprint is an analysisof the nitrogenous base DNA 1
sequencein the DNA of an individual.

History and terminology

T he or iginalD N A fi n g e rp ri n ti nteg c h n i q u ewas
dev elopedby A l e c i
J a ffre y s n 1 9 8 5 .A l th o u g ht he 4 fragments
DNA fingerprintingis commonly used, a more
generalterm DNA profiling is preferred.This is due R1 R3
to the fact that a wide rangeof testscan be carried
out by DNA sequencing with improvedtechnology. DNA 2

Applications of DNA fingerprinting

The amount of DNA required for DNA
f inger pr int is re m a rk a b l y s m a l l . T h e m i n ute
quantitiesof DNA from blood strains,body fluids, 3 fragments
hair fiber or skin fragmentsare enough.Polymerase
chain reaction is used to amplify this DNA for
Fig. 14.5 : An outline of the restriction fragment
. N A p ro fi l i n g h a s w i de
us e in f inger p ri n ti n gD
length polymorphism (RFLP) (R,, Rz, R3 represent
range of applications-mostof them related to the sites for the action of restriction endonucleases).
medicalforensics.Someimportantones are listed
below.
The general aspectsof the above DNA markers
o ldent if ic at ioonf c ri mi n a l sra . i e v e se tc
, p i s tsth
are described along with their utility in disease
o Settlement
of paternitydisputes. d i a g n o s i sa n d D N A f i n g e r p r i n t i n g .
. Use in immigrationtest casesand disputes.
RESTRICTION FRAGMENT LENGTH
In general,the fingerprinting techniqueis carried
POLYMORPH,SMS (RFLPSI
out by collectingthe DNA from a suspect(or a
per s onin a pate rn i tyo r i m m i g ra ti o nd i s p u te and
) A RFLP representsa stretch of DNA that serves
matchingit with that of a referencesample(from as a marker for mapping a specified gene. RFLPs
the victim of a crime,or a close relativein a civil are located randomly throughout a person's
c as e) . chromosomes and have no apparent function.

DNA MARKERS IN DISEASE DIAGNOSIS A DNA molecule can be cut into different
A ND F I NG E RP R IN T IN G fragments by a group of enzymes called restriction
endonucleases(For details, Refer Chapter 6). These
The DNA markers are highly useful for genetic fragments are called polymorphisms (literally
mapping of genomes.There are four types of DNA means many forms).
seouenceswhich can be usedas markers.
A n o u t l i n e o f R F L Pi s d e p i c t e d i n F i g . 1 4 '5 . T h e
fra g m e n tl e n g th p o l y m o rp h i sms
1. Res t r ic t i o n DNA molecule t has three restriction sites (Bi, R,
(RFLFs,
pronouncedas rif-lips).
Rr), and when cleaved by restrictionendonucleases
s r v a ri a b l e n u m b e r ta n d em forms 4 fragments. Let us now consider DNA 2
2. M inis at e l l i te o
(VNIRs,
repeats pronounced as vinters). with an inherited mutation (or a genetic change)
that has altered some base pairs. As a result, the
3. Microsatellitesor simple tandem repeats site (Rr) f or the recognition by restriction
(5fRs).
e n d o n u c l e a s ei s l o s t . T h i s D N A m o l e c u l e 2 w h e n
4. Single nucleotide polymorphisms(SNPs, cut by restriction endonuclease forms only 3
p r onounc ed
as s n i p s ). fragrnents(instead of 4 in DNA 1).
186 B IOTE CHNO LO CY

Rr Re Ra
t-
- DNAwith
(A) - restriction(R) sites
DNAprobe One band

Two bands

Nylonmembrane

( B) Suspectedsite One band

I I
+ PCRfollowed
R1 R2 R3 Hybridization
--# by treatmentwith
endonucleases,
restriction
EE and electrophcresis
+^
Y
PCR primers Two bands
map
DNAwithrestriction

Fig. 14.6 : Two common methods used for scoring restriction fragment length polymorphism (RFLP)
(A) BFLP by Southern hybridization (B) RFLP by polymerase chain reaction (PCH)'

As is ev ident f r om t he ab o v e d e s c r i o t i o n . a m e m b r a n e . A D N A p r o b e t h a t s p a n st h e su sp e cte d
stretch of DNA exists in fragments of various r e s t r i c t i o n s i t e i s n o w a d d e d , a n d t h e h vb r i d i ze o
lengths (polymorphisrns), derived by the action of bands are detected by autoradiograph. lf the
restriction enzymes, hence the name restriction r e s t r i c t i o n s i t e i s a b s e n t , t h e n o n l y a si n g l e
f r agm ent lengt h poly m or phis m s . restrictionfragment is detected.lf the site is present,
then two fragments are detected (Fig. 1a.6A).
RFLPs in the diagnosis of diseases
2. Polymerase chain reaction : RFLPscan also
lf t he RFLPlies wit hin or ev e n c l o s e t o t h e l o c u s b e s c o r e d b y P C R . F o r t h i s p u r p o s e , P C R p r i m e r s
of a gene t hat c aus es a par t i c u l a r d i s e a s e , i t i s t h a t c a n a n n e a l o n e i t h e r s i d e o f t h e su sp e cte d
pos s iblet o t r ac e t he def ec t iv eg e n e b y t h e a n a l v s i s r e s t r i c t i o ns i t e a r e u s e d .A f t e r a m p l i f i c a t i o n b y PC R ,
of RFLP in DNA. The oer s o n 's c e l l u l a r D N A i s t h e D N A m o l e c u l e s a r e t r e a t e d w i t h r e str i cti o n
isolated and treated with restriction enzymes The e n z y m e a n d t h e n a n a l y s e d b y a g a r o se Be l
DNA fragn.rentsso obtained are separated by e l e c t r o p h o r e s i sl.f t h e r e s t r i c t i o ns i t e i s a bse n t o n l y
electrophoresis.The RFLP patterns of the disease o n e b a n d i s s e e n w h i l e t w o b a n d s a r e f ou n d i f tn e
s us pec t edindiv idualsc an be c o m p a r e d w i t h t h a t o f site is found (Fig. 1a.68\.
nor m al people ( pr ef er ablywit h t h e r e l a t i v e si n t h e
Applications of RFLPs: The approach by RFLP
s am e f am ily ) By t his appr o a c h , i t i s p o s s i b l e
is very powerful and has helped many genes to be
t o det er m ine whet her t he i n d i v i d u a l h a s t h e
m a p p e d o n t h e c h r o m o s o m e s e . g . si ckl e -
m ar k er RFLP and t he dis eas e g e n e . Wi t h 9 5 %
cell anemia (chromosome 11), cystic fibrosis
certainity, RFLPs can detect single gene-based (chromosome
7), Huntington'sdesease(chromosome
diseases.
4 ) , r e t i n o b l a s t o m a( c h r o m o s o m e 1 3 ) , Al zh e i m e r 's
Methods of RFLPscoring : Two methods are in d i s e a s e( c h r o m o s o m e2 1) .
common use for the detection of RFLPs(Fig. 1 .5)
VAR'ABLE NUMBEN TANDEM BEPEATS
1. Sout her n hy br idiz at ion : T h e D N A r s
(VNTRS)
diges t ed wit h appr opr iat e r es t r i c t i o n e n z y m e , a n d
separated by agarose gel electrophoresis.The so V N T R s , a l s o k n o v v na s m i n i s a t e l l i t e s li , ke R FL Ps,
obtained DNA fragmentsare transferredto a nylon a r e D N A f r a g m e n t s o f d i f f e r e n t l e n g t h Th e m a r n
ChA P I CT
14 : DN A IN D IS EA SE
D IA C N O S IS
A ND ME D IC A LFOR E N S IC S 147

Rl R2 Use of RFLPs and VNTRS in genetic

---ffiDNA1 fingerprinting
| 4 bands I
(VNTRS) R F L P sc a u s e d b y v a r i a t i o n s i n t h e n u m b e r o f
VNTRs between two restriction sites can De
detected (Fig. 14.8). The DNAs from three
individuals with different VNTRs are cut by the
DNA 2 s p e c i fi c r e s t r i c t i o n e n d o n u c l e a s e . T h e D N A
R1 R2 fragments are separated by electrophoresis, and
identified after hybrid ization with a probe
Fig. 14.7 : A diagrammatic representation of variable complementary to a specific sequence on the
number tandem repeats (VNTRS) Each band (or fragments.
copy) represents a repeating sequence in the DNA
(e g. 100 base pairs each). R, and R, indicate the
slfes cul by a restriction enzyme-

difference is that RFLPs develop from random

mutations at the site of restriction enzyme activity
while VNTRs are formed due to different number of
base sequences between two points of a DNA
m o lecule . In g ener al, VNTRs ar e m ade up o f
tandem repeats of short base sequences (.10-1 00
b ase pa irs). Th e num ber of elem ent s in a give n
region may vary, hence they are known as variable
number tandem reoeats.

An in dividu al' s genom e has m any dif f er e n t

VNTRs an d RFLPs whic h ar e unioue t o t n e
individual. fhe pattern of VNTRs and RFLPs
forms the basis of DNA fingerprinting or DNA
p rofilin g

ln the Fig. 14.7, Iwo different DNA molecules

with different number of copies (bands) of VNTRs
are shown. When these molecules are subjected to (B)
restrictionendonucleaseaction (at two sites R, and
Rr), the VNTR sequences are released, and they
can be detected due to variability in repeat
sequencecopies. These can be used in mapping of
ge no mes, b es ides t heir ut ilit y in DN A
fi ngerprinting.

VNTRs are useful for the detection of certarn

genetic diseasesassociatedwith alterations in the
Fig. 14.8 : Use of restriction fragment length
degree of repetition of rnicrosatellites e.g.
polymorphisms (RFLPI) caused by variable number
Hu ntin gto n's cho r ea is a dis or der whic h is f oun d
tandem repeats (VNTRs) in genetic fingerprinting
rvhen the VNTRs exceed 40 reoeat units. (A) An illustration of DNA structure from three
individuals (B) Hybridized pattern of DNA fragment
Limitations of VNTRs : The maior drawback of
with a probe complementary to the sequence shown
\/NTRs is that they are not evenly distributed
in black circles (1, 2 and 3 represent the individuals;
throughout the genome. VNTRs tend to be
R, and R, indicate restriction sites; coloured squares
localized in the t elom er ic r es ions at t he ends of t n e
are the number ot VNTR9)
chro mosome s.
18 8 B IOTE C H NO LO CY

DNA 1 (A)
-cAGCTGTCGAT-

GGCGAGAGAGAGATC -CAGoTCTCGAT
(5 repeatingunitsof GA)
(B)
I
?"/'
\--
v --S N P
- OAGCTGTCGAT - TagetDNA
--- DNA2
-..-.' lllllllllll
GGCGAGAGAGAGAGAGAGAGAGATCT...-.. - GTCGACAGCTA- oligonucleotide
(10 repeatingunitsof GA) +L Matchedbase
Fig. 14.9 : Two alleles of DNA molecules representing Completearid stable
5 and 10 dimer repeating units. hybridizationof base pairs

I
rSNP
Y
MICBOSATELLITES -CAGCTGTCGAT - TagetDNA
(stMPLE TANDEM REPEATS)
-GTCGAGAGCTA - oligonucleotide
M ic r os at ellit es ar e s hor t r epe a t u n i t s ( 1 0 - 3 0 fL-
Mismatchedbase
co pies ) us ually c om pos ed of d i n u c l e o t i d e o r
Hybridizationnot formed
tetr anuc leot ideunit s . Thes e s im ple t a n d e m r e p e a t s due to mismatchbase pair
) e m or e popular t han m inis a t e l l i t e s( V N T R s )
(S TRs ar
as DNA markers for two reasons. Fig. 14.10 : (A) An illustration of single nucleotide
polymorphism (SNP) (B) Oligonucleotide hybridization
1 M ic r os at ellit es ar e ev en l y distributed to detect SNP.
thr oughout t he genom e

2. PCR can be effectively and conveniently

u s ed t o ident if y t he lengt h of poly m o r p h i s m .
Two v ar iant s ( alleles )of DNA m o l e c u l e s w i t h 5
a nd 10 r epeat ingunit s of a dim er n u c l e o t i d e s( C A )
are depicted in Fig. 14.9.
By us e of PCR, t he r egion s u r r o u n d i n g t h e
microsatellites is amplified, separated by agarose
g el elec t r ophor es isand ident if ied .
S'ATCIE NUCLEOTIDE POLYMORPHISMS
fSIVPS/
A n o l i g o n u c l e o t i d e l s a s h o r t s i n g l e - s tr a n d e d
D N A m o l e c u l e s y n t h e s i z e di n t h e l a b o r a t o r yw i th a
l e n g t h n o t u s u a l l y e x c e e d i n g5 0 n u c l e o t i d e s .U n d e r
a p p r o p r i a t e c o n d i t i o n s , t h i s n u c l e o t i d e s e qu e n ce
will hybridize rvith a target DNA strand if both
have completely base paired structure. Even a
s i n g l e m i s m a t c h i n b a s e p a i r w i l l n o t a l l ow th e
hybridization to occur.
DNA chip technology is most cornrnonly used
to screen SNPs hybridization with oligonucleotide
(Refer Chapter 7).

SNPs r epr es ent t he pos it ions i n t h e g e n o m e

CURRENT TECHNOLOGY OF DNA
rvher e s or ne indiv iduals hav e one n u c l e o t i d e ( e . 9 .
FINGEBPRINTING
C) while others have a different nucleotide (e.9. C).
'fher e In the forensic analysis of DNA, the original
ar e lar ge num ber s of SNPs i n g e n o m e s . l t i s
estimatedthat the human genome contains at least techniques based on RFLPs and VNTRs are now
3 m illion SNPs . Som e of t f r es eSN P s m a y g i v e r i s e largely replaced by microsatellites (short tandem
to RFLPs . r e p e a t s ) . T h e b a s i c p r i n c i p l e i n v o l v e s th e
a m p l i f i c a t i o n o f m i c r o s a t e l l i t e s b y p o l y m e r a se
SNPs ar e highly us ef ul as DNA m a r k e r s s i n c e
chain reaction followed by their detection.
there is no need fclr gel electrophoresisand this
saves a lot of time and labrour.The detection of It is now possible to generate a DNA profile by
SN Ps is bas ed on t he oligonuc leoti d e h y b r i d i z a t i o n automated DNA detection system (comparable to
analysis (Fig. 1a.l0). t h e D N A s e q u e n c i n ge q u i p m e n t ) .
 A l'e w de cd de s dgo, il w. t s r ealis ( ' ( lt lr . r t t er l, ti r t I . Human proteirrreplac-ernents
A p rote ins co ulc l be us ed as ; r har m ac eut i c a l
- ) I h e r a p e u l i c a g e n t sl o r h r r m a n d i s e a s c s .
agents for the treatnrent of humart dise.rses e g
3 Vaccines.
r nsulin for d iab et es m ellit us , int er f er on f or v ir a l
disea se s. Howe v er , t he av ailabilit y of s uc h Some authors do not make such categorization
therapeutic/pharrn;rceutical products was very and consicler all of them together as pharma-
linrite d d r-reto cos t ly and c um ber s om e pr oc edLt r e s ceutically important products of biotechnology
Fur t hel t he i r
invo lve d in the ir is olat ion/ pr odr - r c t ion
r rse in hu man s was as s oc iat ed wit h s ev e r a l
co mplicatio ns. F or ins t anc e, adm inis t r at ion of p i g lllr
insulin to d iabet ic pat ier r t s r es ult s in t h e
develo pme nt o f a r r t ibodr c s .
l-he synthesis of the cellular proteins rs
Th e ad rrcn t ot r ec om binani DNA t ec hnolo g y u l t i m a r t e l yu n d e r t h e c o n t r o l o f g e n e s .A n y d e f e c t r n
h era lde da ne w c hapt erf or t he pr oduc t ionof a wid e i r g e n e p r o d u c e sa n i n c o r r e c t p r o t e i n o r n o p r o t e i n
ran 1leof the rap eut r cagent s in s uf f ic ient quant it i e s a t a l l S o m e t i n r e s ,t h e o c c u r r e n c e o f a d e f e c t i v e
f or hu man use. The c om m er c ial ex ploit at ion o f (i e functionally ineffective)or deficient protein
recombinant DNA (rDNA) technology began in late m a y c a u s e a d i s e a s e T h u s , g e n e d e f e c t sw i l l r e s u l t
1 c)7 0s by a few biot ec hnologic al c om panies t o i n i r . r h e r i t e do r g e n e t i c a l l y l i n k e d d i s e a s e s .
pro du ce p rote ins Ther e ar e at leas t 400 dif f er e n t
pro tein s l-.ein g pr oduc ed ( by DNA t ec hnolog y ) l d e n t r f i c a t i o no f d e f e c t i v e o r d e f i c i e n t p r o t e i n s
r vh ich n ray se rve as t her apeut icagent sf or hum an s in the causation of inherited diseases is very
A ie lected list of s om e im por t ant hum an pr ot ei n s important. fhe recombinant DNA technology can
proteins
pro du ce d b y r ec om binant DNA t ec hnoio g y be fruitfully employed to produce human
po ten tial fo r th e t r eat m ent of hun' r ar rdis or der s r s that can be used for the treatment of genetically
given in Table 15.1 As of now, only a selectedfew linked diseases. This is referredto as human protern
o f th em (a rou nd 30) hav e been appr ov ed f or hum a n replacement strategy in bictechnology
use , an d tl-remost im por t ani am ong t hes e ar e giv e r r
in Table 15.2 INSULIN

The p ha rmaceut ic al pr oduc t s of r ec om bina n t The hormone insulin is produced by the p-cells
DNA techn olo gv ar e br oac lly div ided int o t h e o f i s l e t so f L a n g e r h ; i n so f p a n c r e a s .H u m a n i n s u l i n
follo wing th ree c at egor ies and br ief ly dis c us s e d c o n tains 51 amino acids, arranged in two
with impo rtan t ex am ples . polypeptide chains. The chain A has 21 amino

189
190 B IOTE CHNO LO CY

acids while B has 30 amino acids. Both are held

Trsrr.15.I A selectedlist of ftumanproteins together by disulfide bonds.
producedby reconbinantDNAtechnology for
Diabetes mellitus

Disorder Recombinantprotein(s) Diabetes mellitus affects about 2-3'h of the

g e n e r a lp o p u l a t i o n . l t i s a g e n e t i c a l l yl i n k e d d i se a se
Anemia Hemoglobin, erythropoietin
characterized by increased blood glucose
Asthma Interleukin-l
receptor
concentration (hyperglycemia) The occurrence of
Atherosclerosis growth
Platelet-derived d i a b e t e s i s d u e t o i n s u f f i c i e n t o r i n e ffi ci e n t
faclor ( i n c o m p e t e n t )i n s u l i n I n s u l i n f a c i l i t a t e st h e ce l l u l a r
Delivery Relaxin u p t a k e a n d u t i l i z a t i o n o f g l u c o s e f o r t h e r e l e a seo f
Bloodclots Tissue plasminogen e n e r g y I n t h e a b s e n c e o f i n s u l i n , g l u co se
activator,
urokinase a c c u m u l a t e s i n t h e b l o o C s t r e a n r at h i g h e r
B u rn s Epidermal growth factor c o n c e n t r a t i o n , u s u a l l y w h e n t h e b l o o d g l u co se
c o n c e n t r a t i o ne x c e e d sa b o u t 1 8 0 m g / d l , g l u co se r s
Cancer Intederons,lumornecrosis
e x c r e t e d i n t o u r i n e T h e p a t i e n t s o f d i a b e te s a r e
factor,
colony stimulating
weak and tired since the production of energy (i e
factors,interleukins,
ATP) is very much depressed.
lymphotoxin,
macrophage-
qvilvquilu I^^r^"
^^+;,,^+i^^ rautul

Diabetes Insulin, growth

insulin-like
factor
Tlsrr 15.2 A selected list of reconblnant
Emphysema a, -Antitrypsin proteins that have been approved for hunan
Femaleinfertility Chorionicgonadotropin use by U.S. Food and Drug Administration
Freeradical
damage Superoxide
dismutase
(minimizing) Recombinantprotein DisorderG) treated

Growth
defects growth
Growlhhormcne, factorVlll
Coagulatlon Hemophilia
A
hormone'releasing
factor, factorlX
Coagulation Christmas
disease
somatomedin-C DNaseI fibrosis
Cystic
Heartattacks Prourokinase
Erythropoietin Anemia,
kidney
disease
HemophiliaA Factor
Vlll
Glucocerebrosidase Gaucher's
disease
HemophiliaB FactorlX
Growth
hormone Growth in children
defects
Hepatilis
B F.lonatitic R rrennino
Granulocyte
colony Cancer
Hypoalbuminemia Serumalbumin
factor
stimulating
lmmune disorders Interleukins,
B-cellgrolvth
factors Hepatitis
B surface Hepatitis
B
(vaccine)
antigen
Kidneydisorders Erythropoietin
l nsul i n Diabetes
mellitus
LowGehrig's disease Brain-derived
neurotropic
(amytrophiclateralsclerosis)factor Interferon
a Leukemia,kaposisarcoma
genitalwarts,hepatitis
B
Multiplesclerosis Interferons
(u, 0, y)
Nervedamage Nervegrowthfactor interferon
B Multiple
sclerosis
Osteomalacia Calcitonin y
Interferon granulomatous
Chronic
drsease
Pa i n Endorphins and
enkephalins Interleukin-2 Renalcellcarcinoma
Rheumatic disease Adrenocorticotropic Interi euki n-10 Thrombocytopenia
n0rm0ne Somatotropin Growthdefects
Ulcers Urogastrone Tissueplasminogen Aculemyocardial
infarction,
Viralinfections (u, 0, y)
Interferons activator pulmonary
embolism
 Ch a pt er15 : P HA R MA C E U T IC AL
P R OD U C fSO F DN A TE C H N OLOC Y 191

The more serious complications of uncontrolled

diabetesinclude kidney damage (nephropathy),eye for ,,/--Gene for
.-Gene

) nu*" I/ B'"ilfi
damage (retinopathy),nerve diseases(neuropathy)and
circulatory diseases(atherosclerosis,stroke). In fact,
diabetes is the third leading cause of death (afterheart
diseaseand cancer) in many developed countries.
Plasmid plasmid
In the early years, insulin isolated and purified
from the pancreasesof pigs and cows was used fcr
| ,r"n"rorminto I
t he trea tmen t o f d iabet ic s . Ther e is a s light E.coti
difference (by one to three amino acids) in the
J J
st ructure o f a nima l ins ulin c om par ed t o hum an
insu lin. Th is re su lted in aller gy in s om e of t he
diabe tics whe n a nim al ins ulin was adm inis t er eo.
A nothe r pro ble m wi t h anim al ins ulin is t hat lar ge
numb er of a nima ls hav e t o be s ac r if ic ed f or
extracting insulin from their pancreases. For
instance, about 7O pigs (giving about 5 kS
E. coli E. coli
pancre atictissue )ha v e t o be k illed t o get ins ulin f or
treating a single diabetic patient iust for one year! Culturecellsin
a nutrientmedium
containinglactose.
Production of recombinant insulin lsolateand purify
A and B chains
Attemps to produce insulin by recombinant
DNA technology started in late 1970s. fhe basic
technique consisted of inserting human insulin -/--.--l.t-'-'t-'
B chain
gene and the promoter gene of lac operon on to
the plasmids of E. coli. By this method human
insulin wa s pro du c ed. lt was in J uly 1980, Joiningof chains
seventeen human volunteers were, for the first and
reconstitution
t ime , a dmin iste red r ec om binant ins ulin f or
treatment of diabetes at Cuy's Hospital, London.
And in fact, insulin was the f irst ever
pharmaceu tica l p roduc t of r ec om binant DNA
t ech no log y ad minist er edt o hum ans . Rec om binant Humaninsulin
insulin worked well, and this gave hope to scientists
that DNA technology cbuld be successfully Fig. 15,1 : The production of recombinant insulin in
employed to produce substancesof medical and E, coli (l-lnducer gene, P-Promoter gene,
commercial importance. An approval, by the
concerned authorities, for using recombinant
insulin for the treatment of diabetes mellitus was
given in 1982. And in 1986, Eli Lilly company
received approval to market human insulin under F i g . 1 5 . 1. T h e g e n e s f o r i n s u l i n A c h a i n a n d B
the trade name Humulin. chain are separatelyinsertedto the plasmids of two
different E. coli cultures. The /ac operon system
Technique for recombinant insulin production :
(consisting of inducer gene, promoter gene,
The orginal technique (described briefly above) of
operator gene and structural gene Z lor
insulin synthesis in E. coli has undergone several
change s, fo r impro v ing t he y ield. e. g. addit ion of B-galactosidase)is used for expressionof both the
genes. The presence of lactose in the culture
signal pe ptid e, synt hes is of A and B c hains
m e d i u m i n d u c e s t h e s y n t h e s i so f i n s u l i n A a n d B
seDaratelvetc.
c h a i n s i n s e p a r a t ec u l t u r e s . T h e s o f o r m e d i n s u l i n
The procedure employed for the synthesis of chains can be isolated,purified and joined together
t wo insulin cha ins A and B is illus t r at ed in t o g i v e a f u l l - p l e d g e d h u m a n i n s u l i n .
192
Second generation
recombinant insulins
A f t er injec t ing t he ins ulin, t h e p l a s m a
con c ent r at ion of ins ulin r is es s lowl y . A n d f o r t h i s
.l
reason, insulin injection has to be done at last 5
rrinutes before a meal. Furthet; decrease in the
in su lin lev el is als o s low, ex pos ing t h e p a t i e n t st o a
da nger of hy per ins ulinem ia.All t hi s i s d u e t o t h e
exis t enc e of t her apeut ic ins ulin as a h e x a m e r ( s t x
mol ec ules as s oc iat ed) whic
, h dis s o c i a t e ss l o w l y t o
the biologic ally ac t iv e dim er or m o n o m e r .
Aftempts have been made in recent years to
produce second generation insulins by site-
directed mutagenesis and protein engineering. (For
mor e det ailson t hes e t ec hniques ,r e f e r C h a p t e r 1 0 ) .
The second generation recombinant proteins are
terrned as muteins (See p. 198). A large nurnber of
in su iin m ut eins hav e been c ons t r u c t e d w i t h a n
objective of faster dissociation of hexamers to
biologically active forms. Among these is insulin
lispro, with modified amino acid residues at
position 29 and 30 of the B-chain of insulin
lnsulin lis pr o c an be injec t ed im m ed i a t e l y b e f o r e a
me al as it at t ains t he phar m ac olo g i c a l l y e f f i c i e n t
levels very fast.
Ghemically altered porcin insulin
A s alr eady s t at ed, por c in ( pig) i n s u l i n d i f f e r s
fro m hum an ins ulin jus t by one am i n o a c i d - a l a n i n e
in plac e of t hr eonine at t he C- t e r m i n a l a n d o f
B-chain of hum an ins ulin. Biot ec h n o l o g i s t sh a v e
develooed methods to alter the chemical structure
of oor c in ins ulin t o m ak e it i d e n t i c a l t o
h um an ins ulin.And t lr is c hem ic ally m o d i f i e d p o r c i n
insulin can also be ernploved for the treatmeni
dia bet es r nellit us .
HU M AN G RO W TH HO RM O NE
Crowth hormone is produced by the pituitary
gland. lt regulaies the growth anci development.
Crowth hormone stimulatesoverall body growth by
in cr eas ingt he c ellular upt ak e of am i n o a c i d s , a n d
protein synthesis,and promoting the use of fat as
body fuel.
I ns uf f ic ient hum an gr owt h hor m o n e ( h C H ) r n
yo ung c hildr en r es ult sin r et ar dedgr o w t h , c l i n i c a l l y
referred to as pituitary dwarfism. The child usually
is le s st han f our f eet in height , and h a s c h u b b y f a c e
a nd abundant f at ar ound t he wais t .
B IOTE C H N O LO CY
Traditionaltreatment for dwarfism : The children
of pituitary dwarfism were treated with regular
injections of growth hormone extracted from the
brains of deceased humans. lt may be noted that
o n l y h u m a n g r o w t h h o r m o n e i s e f f e c t i v e fo r
t r e a t m e n to f d w a r f i s m ( T h i si s i n c o n t r a s t o d i a b e te s
w h e r e a n i r n a l i n s u l i n sa r e e m p l o y e d ) .A t l e a s te i g h t
pituitary glands from cadaversmust be extractedto
g e t h C H a d e q u a t ef o r t r e a t i n ga d w a r f c h i l d j u st fo r
o n e y e a r ! A n d s u c h t r e a t m e n th a s t o b e c o n t i n u e d
f o r B - 10 y e a r s ! !F u r t h e r ,a d m i n i s t r a t i o nh C H i s o l a te d
fron'r h'uman brains exposesthe children to a Sreat
r i s k o f t r a n s m i t i i n g i h e c a d a v e r b r a i n d i se a se s
( t h r o u g h v i r u s o r v i r a i - l i k e a g e n t s )e . g . C r e u z fe l d t-
J a c o b ( C J )s y n d r o m e c h a r a c t e r i z e db y c o n v u l s i o n s,
wasting of muscle etc.
Production of recombinant hGH
B i o t e c h n o l o g i s t sc a n n o w p r o d u c e h C t l b y
g e n e t i c e n g i n e e r i n g .T h e t e c h n i q u e a d o p t e d i s q u i te
c o m p a r a b l e w i t h t h a t o f i n s u l i n p r o d u c t i o n . Th e
procedure essentially consists of inserting hCH
gene into E. coli plasmid, culturing the cells and
i s o l a t i o no f t h e h G H f r o m t h e e x t r a c e l l u l a rn r e di u m .
Limitation in hGH production : The hCH is a
p r o t e i n c o m p r i s e d o f 1 9 1 a m i n o a c i d s . D u r i n g th e
c o u r s e o f i t s n a t u r a l s y n t h e s i si n t h e b o d y , h CH i s
t a g g e d w i t h a s i n g l e p e p t i d e ( w i t h 2 6 a m i n o a ci d s)
T h e s i g n a l p e p t i d e i s r e m o v e d d u r i n g s e c r e t io n to
r e l e a s et h e a c t i v e h C H f o r b i o l o g i c a l f u n c t i o n s.Th e
errtire process of hCH synthesisgoes on in an
orderly fashion in the body. However, signal
peptide interrupts hGH production by
recombi nant technology. The complementary D NA
( c D N A ) s y n t h e s i z e df r o m t h e n r R N A e n c o d i n g h C H
i s i n s e r t e di n t o t h e p l a s m i d .T h e p l a s m i d c o n t a i n i n g
E . c o l i w h e n c u l t u r e d , p r o d u c e s f u l l l e n g t h h Gl l
along with signal peptide. But E. coli cannot
r e m o v e t h e s i g n a l p e p t i d e . F u r t h e r ,i t i s a l s o q u i te
d i f f i c u l t t o g e t r i d o f s i g n a l p e p t i d e b y v a r i o u s o th e r
m e a n s . T h e o r e t i c a l l v , c D N A e n c o d i n g si g n a l
p e p t i d e c a n b e c u t t o s o l v e t h e s e p r o b l e m s.
U n f o r t u n a t e l y ,t h e r e i s n o r e s t r i c t i o ne n d o n u ci e a se
to do this job, hence this is not possible.
A novel approach for hGH production : Bio-
technologists have resolved the problem of
s i g n a l p e p t i d e i n t e r r u p t i o n b y a n o v e l a p p ro a ch
( F i g . 1 5 . 2 \ . T h e b a s e s e q u e n c e i n c D N A e n c od i n g
s i g n a l p e p t i d e ( 2 6 a m i n o a c i d s ) p l u s th e
n e i g h b o u r i n g2 4 a m i n o a c i d s ( i . e . a t o t a l 5 0 a m i n o
 - : ac r er l5 : P H A R MA C E U T IC AL
PR O D U C T S
O F D N A TE C H N OLOC Y 193

cDNAfor
hGH
Sequencefor Sequencefor Sequencefor 167
signalpeptide 24 amino acids aminoacidsof hGH
(26 aminoacids) of hGH I
I ECoRI
Jcreavage
:
Deletionof sequencefor 50
amiho acidd(26 + 24) I
+

Sequencefor 167aminoacids
- of hGH

ffi ---l
Ligation
cDNAencodingfor \
24 amino acids I
of hGH +
cDNAfor hGH alono
with expression-
veclor genome

I tnsertion
intoa
vector
J

E. coli

Fig. 15.2 : The production of recombinant human growth hormone

pDNA-Complemenw DNA,hGH-tluman grawfh hormone)

acids) is cut by restriction endonuclease ECoRl.
Now a gene (cDNA) for 24 amino acid sequenceof
hCH (that has been deleted) is freshly synthesized
an d liga ted to th e r em aining hCH c DNA. The s o
constitued cDNA, attached to a vector, is inserted
into a bacterium such as E. coli for culture ano
pro du ctio n o f hGF l. I n t his m anner ,t he biologic all y
f unction al hGH c an be pr oduc ed bv DNA
technology.
Recombinant hGH was approved for human
use in 1985. lt is marketedas Frotropin by Cenetech
company and Humatrope by Eri.Lilly company.
Gontroversy over the use of hGH
Recombinant hCH can be administered to
children of very short stature. lt has to be given
daily for many years with an annual cost of about
$ 20,000. Some workers have reported substantial
increase in the height of growth retarded children.
One group of workers observed that the normal
growth pattern in children was not restoredon hCH
administration,although there was an initial spurt.
Another big question raised by the opponents of
hCH therapy is whether it is necessaryto consider
short stature as a disorder at all for treatment!

Biotechnology [i3]
r94 B IOTE CHNO LO CY

Tmr.r 15.3 A selected list of rDilA-derived therapeutic agents

{approvedby FDA)with trade namesand their applicationsin humans

rDNA product Trade name(s) Applications/uses

In s u l i n H umul i n Diabetes
Growth hormone Protropin/Humatrope Pituitary
dwarfism
q,-lnterferon Intron
A Hairycellleukemia
Hepatitis
B vaccine Recombinax B
HB/Engerix HepatitisB
plasminogen
Tissue activalor Activase Myocardial infarction
Factor
Vlll nate
Kogenate/Recombi H emophi i i a
DNase Pulmozyme Cystic{ibrosis
Erythropoietin Epogen/Procrit Severe anemia withkidney
damage

Use of recombinant growth i s r e s p o n s i b l ef o r t h e s y n t h e s i so f f a c t o r Vl l l Th i s

horqnone for farm animals m R N A c o n t a i n s 9 , 0 0 0 b a s e s a n d s y n t he si ze sth e
p r o t e i n , f a c t o r V l l l . I a c t o r V l l l c o n t a i n s2 3 3 2 a m i n o
Rec om binantCH is now a v a i l a b l e f o r a d m i n i s -
a c i d s , w i t h c a r b o h y d r a t e m o l e c u l e s a tta ch e d a t
tration to farm aninralsto promote early growth ancJ
least at 25 sites.
dev elopm ent .Suc h f ar m anim p l s y i e l d l i n e a r m e a t ,
bes idesinc r eas edm ilk pr oduc ti o n . H o w e v e r , u s e o f DNA technologists s y n t h e s i ze d th e
CH in f ar m anim als is a c ont r o v e r s i a li s s u e . complementaryDNA (cDNA) for mature mRNA of
f a c t o r V l l l . T h i s c D N A c a n b e i n s er te d i n to
CLO TTI NG FACTO R VI I I m a m m a l i a n c e l l s o r h a m s t e r k i d n e y c e l l s fo r th e
production of recombinantfactor Vlll
The clotting factor Vlll is required for proper
blood c lot t ing pr oc es s . A ge n e t i c d e f e c t i n t h e S i n c e 1 9 9 2 , f a c t o r V l l l i s a v a i l a b l e i n th e
s y nt hes is of f ac t or Vlll r es u l t s i n t h e d i s o r d e r m a r k e t . l t i s p r o d u c e d b y G e n e t i c s I n sti tu te i n
hemophilia A This is a sex-linked disease C a m b r i d g e , Massachusetts and so l d as
( inc idenc e 1 in 10, 000 m a l e s ) t r a n s m i t t e d b y Recombinate while Miles Laboratoriessell under
femalesaffectingmales.The victims have prolonged the trade name Kogenate
clotting time and suffer from internal bleeding.

Traditional treatment for hemophilia A

Clot t ing f ac t or Vlll was is ol a t e df r o m t h e w h o l e

blood and adm inis t er edt o t he p a t i e n t so f h e m o o h i l i a
A. This appr oac h r equir eslar ge q u a n t i t i e so f b l o o d .
Anot her pr oblem is t he r is k of t r a n s m i s s i o no f c e r t a i n
dis eas esI ik e AI DS t o t he hem o o h i l i a c s
Production of recombinant factor Vlll
The gene for the formation of factor Vlll rs
loc at ed on X c hr om os om e lt i s a c o m p l e x g e n e o f
186 k b ( i. e. , 186, 000 bas e pai r s ) i n s i z e , o r g a n i z e d
into 26 exons of varying length. In between the
exons, many introns are present. The introns vary
in t heir s iz e, s t ar t ingf r om 200 b a s e p a i r s t o a s h i g h
as 32. 000 bas e oair s .
Biotechnologists were able to isolate mature
m RNA ( c ont ainingonly ex ons a n d n o i n t r o n s )t h a t
B i o t e c h n o l o g y i s v e r y u s e f u l f o r t h e p r o d u cti o n
of several therapeutic products for treating human
d i s e a s e s . A s e l e c t e d l i s t o f r D N A- d e r i ve o
t h e r a p e u t i c a g e n t s a l o n g w i t h t r a d e n a m e s a n cl
t h e i r u s e s i n h u m a n s a r e q i v e n i n T a b l e 15 .3 . So n r e
o f t h e s e a r e d e s c r i b e da b o v e ( u n d e r h u m a n p r o te r n
r e p l a c e m e n t s )w h i l e t h e r e m a i n i n g a r e d i scu sse cJ
below
TISSUE PLASMINOGEN A C T I V A T OR
T i s s u ep l a s n r i n o g e na c t i v a t o r ( t P A )i s a n a tu r a l l r
occurring protease enzyme that helps to dissolt'e
blood clots. tPA is a boon for patients suffering
from thrombosis
C hapt er15 : P HA R MA C E L J T ICPAL
R OD U C T S
OF D N A TE C H N OLOC Y 195

Tissue Rec<;mbinant tPAhasbeenin usesince1987for

Plasminogen
activator treatment of patients with acute myocardial
infarction or stroke. Cenetech was the first to
II rnarkettPA with a trade name Activase.
I
*
Plasminogen | Plasmin

cDNA for tPA

Degraded Soluble
products
Synthetic
plasmid
Fig. 15.3 : Role of tissue plasminogen activator in
dissolving blood clots.

The majority of naturaldeathsworldwide are

due to a blockadeof cerebralor coronaryarteryby
a blood c lot , t ec h n i c a l l yc a l l e da s th ro m b u sT. fr e
phenomenon of thrombus blockage of blood
vesselsis referredto as thrombosis.

Chemically,thrombusconsistsof a networkof
fibrin, formed from the fibrinogen.In the normal
circumstances,plasmin degrades fibrin and
d is s olv esblood c l o ts . T h i s p l a s mi n i s a c tu a l l y
producedby activationof plasminogenby tissue
plasminogenactivator (Fig. 15.3). The natural
biological systemsis however,not that efficientto
remove the blood clots through this machinery.
Tissueplasminogenactivatoris very useful as a
therapeutic agentin dissolving blood clots(thrombi)
by activating plasminogen.By removing the
arterial,thrombi, the possibledamagecausedby
them on heartand brain could be reduced.

Production of recombinant tPA

DNA technologists synthesized the

y N A (c D N A) m o l e c u l efo r ti s s u e
co m plem ent arD
plasminogen activator. This cDNA was then
attachedto a syntheticplasnridand introducedinto
mammaliancells (Fig. 15.4).They were cultui'ed
and tPA-producingcells were selectedby using J
methotrexate to the medium.tPA-producing cells lsotaretPA
were transferredto an industrialtank (fermenter). from culture
medium
tPA, secretedinto the culture medium, is isolated
for therapeuticpurpose.It may be noted heretlrat
tPA was the first pharmaceutical product to be
produced by mammalian cetl culture.
 196 B IOTECHNO LO C\

Alteplase and Reteplase: These are the second

generation recombinant tPAs. They have increased
in vivo half-lives and are functionally more
efficient. The general aspectsof second generation
r ec om binant pr ot eins ar e g i v e n e l s e w h e r e ( S e e
o. 198) .

Antibody.plasminogen
activator coniugates
An ant ibody agains t f ibr in ( a n t i f i b r i n a n t i b o d y )
c an be c onjugat ed wit h t i s s u e p l a s m i n o g e n
ac t iv at or t his c onjugat e is a p p r o p r i a t e l y r e g a r d e d
as im m unot l. r er apeut ic t hr o m b o l y t i c a g e n t . l t
quic k ly and s pec if ic ally bind s i o f i b r i n c l o t s a n d
loc ally inc r eas est he c c nv er s i o n o f p l a s m i n o g e nt o
plasmin to dissolve fibrin (Fig. lS.S) In fact,
ant if ibr in m onoc lonal ant i b o d i e s h a v e b e e n
synthesized, conjugated with tPA and tried for
s olubilis ing blood c lot s

Advantages of tPA as
thrombolytic agent
lis s ue plas m inogenac t iv a t o ra c t s o n b l o o d c l o t s
( s olubiliz es by degr adat ion)w i t h o u t r e d u c i n g t n e
blood c lot t ing c apabilit y el s e w h e r e l - h i s i s i n
contrast to the action of urokinase or streptokinase
whic h ar e m or e gener alis edin t h e i r a c t i o n . F u r t h e r ,
t PA c an be adm inis t er ed i n t r a v e n o u s l y w h i l e
ur ok inas e and s t r ept ok in a s e h a v e t o oe
adm inis t er eddir ec t ly t o t he b l o c k e d b l o o d v e s s e r .
Another merit of using tPA is tlrat its action is mucn
faster than other thrombolytic agents with much
reduced side effects.
I NTERFERO NS
Interferon is an antiviral substance, and is the
first line of defense against viral attacks. -fhe term
interferon has originated from the interference of
t his m olec ule on v ir us r eplic a t i o n . l t w a s o r i g i n a n y
dis c ov er ed in 1957 by Ali c k l s a a c s a n d J e a n
Lindem ann and was c ons id e r e d t o b e a s i n g l e
s ubs t anc e.I t is now k nown t h a t i n t e r f e r o na c t u a l l y
consistsof a gi'oup of more than twenty substances
with molecular weights between 20,000-30,000
dalt ons . All t he inier f er onsar e p r o t e i n s i n n a t u r e
and m any of t hem ar e gly c o p r o t e i n s . T h e y a r e
broadly categorized into three groups based on
t heir s t r uc t ur eand f unc t r on
Interferon-cr(l FN-cx)
I nt er ier on- p( lFN- p)
Interferon-y(lFN-y)
Fig. 15.5 : Action of antifibrin antibody-tPA
conjugate in dissalving blood clot
(tPA-tissue plasminogen activator).
Mechanism of action of interferons
I n t e r f e r o n sa r e p r o d u c e d b y m a m m a l i a n ce tt:
when infected by viruses. As the virus releasesi:'
n u c l e i c a c i d i n t o c e l l u l a r c y t o p l a s m , i t sti m u l a te .
the host DNA to produce interferons. The:e
interferons, secreted by the cells, bind to the
a d j a c e n t c e l l s . H e r e , t h e y s t i m u l a t e t h e ce l l u l a '
D N A t o p r o d u c e a s e r i e so f a n t i v i r a l e n z ym e s.Th e
s o f o r m e d p r o t e i n s i n h i b i t v i r a l r e p l i ca ti o n a n .
protect the cells (Fig. 15.6). lt is believed that the
p r o t e c t i v e ( e n z y m e s ) p r o t e i n s b i n d t o m R N A c'
v i r u s e sa n d b l o c k t h e i r p r o t e i n s y n t h e s i s.Th e a cti o ^
of interferonsappears to be species specific. Thu.
h u m a n i n t e r f e r o n so p e r a t ei n h u m a n s .O th e r a n i m "
(dog, mouse) interferonsare ineffective in man.
lsolation of interferons in
the early years
B l o o d w a s t h e o n l y s o u r c e o f i n t e r f e r o n se a r l i e -
T h e p r o c e d u r ew a s v e r y t e d i o u s a n d t h e q u a n ti t\ ,:-
i n t e r f e r o n si s o l a t e d w a s v e r y l i t t l e . T h u s, a s m u c-
a s 5 0 , 0 0 0 l i t r e so f h u m a n b l o o d w a s r e q ui r e dto e=-
.l
just 00 mg of interferons!Therefore, it was re-
d i f f i c u l t t o c o n d u c t r e s e a r c ho r u s e i n t e r fe r o n s .-
therapeuticpurposes.
Chapt er15 : P H A R MA C E U T IC AL
PR O D U C T S
OF D N A TE C H N OI-OC Y 197

Production of hvbrid interferons : Severar

attempts have been rnade to prcduce hybrici
interferons.This is advantageoussince clifierent
interferons with different antiviral activities can be
combined to produce a more efficient interferon.
Further,the glycosylationstep can be bypasseu,
Viral nucleicacid and bacteria can be used to produce hybrid
interferons.The hybridinterferons are morereacttve
lnterferons i n performi ngthei r functi on.
The creationof hybrid genesfrorn the genesof
Host ceil IFN-u, and IFN-c,,is illusti-ated
in Fig. 15.7.1-hese
AA genesare digested by restrrction
endonucleases. The
A AA
A resultingfragmentsare ligatedto generatehybrid
genes.The appropriate hybridgenescan be selected
and usedfol producinghybridinterferons. As already
stated,E. coli.can be employedfor this purpose.

Therapeutic applications of interferons

Antiviralenzymes
lnterferons-cr,-B and -y were respectively
approvedfor therapeuticuse in humans in the

64:
Adjacentcell
Blockviral reolication
years1986,1993and 1990.A S w i ssbi otechnol ogy
firm was the first to market infurteron-a with a
trade name lntron. Interferonsare used for tne
treatmentof a largenumberof viral diseasesand
cancers.The cancersi ncl ude l eukemi a.kaoosi s
Fig. 15.6 : The mechanism of action of interferons. sarcoma,bladdercancer,head and neck cancer,
renal cel l carci noma,ski n cancer and mul ti pl e
myeloma.The other diseases employinginterferon
Production of recombinant inter{erons therapyare AIDS,multiplesclerosis, genitalwans,
hepatitisC, herpeszosteretc.
The complementary DNA (cDNA) was synthe-
sized from the mRNA of a soecific interferon.This is
insertedto a vector (say plasmid)which is introduced
into E. coli or-other cells. The interferon can be RE 1 RE2
is ola tedfro m the c ult ur e. m edium .This is t he bas r c \P \P
m e ch an ismo f p roduc ing r ec om binantint er f er ons. r--- -------A------A-- IFN-cqgene

The production of interferonsis relati'relyless in RE1 RE2

bacterial hosts, although E. coli Was the first to be \PV
used. This is mainly because most interferonsare IFN-cbgene
glycoproteins in nature and bacteria do not possess
the machinery for glycosylation of proteins.
Production interferons by yeasts : The yeast
Saccharomyces cerevisiae is more suitable for the
production of recombinantinterferons.This is mainly Hybrid
because the yeast possessthe mechanism to carry genes
out glycosylationof proteins,similar to that occurs in
m a mmalia n cells . The DNA s equenc e c oding f o r
soecific human interferon can be attached to the
yeast alcohol dehydrogenasegene in a plasmid and
Fig. 15.7 : Creation af hybrid inierleron genes
introduced into 4 yeastcells. The yield of interferons (RE-Restriction endonuclease, IFN-lnterteron).
is severalfold higher compared to E. coli.
r-
r98
Interferonsare also employed in the treatmentof
c om m on c olds and inf luenz a . F o r t h i s p u r p o s e ,
interferonscan be used as nasal sprays.
The basic mechanism of action of interferons
against viruses has already been described.
lnterferonsare found to causethe death of cancerous
c ells .This is br oughtout by s t im u l a t i n gt h e a c t i o n o f
natural killer (NK) cells, a specialised form of
lymphocytesthat can destroy cancerouscells.
Des pit e t he wides pr eadt her a p e u t i ca p p l i c a t i o n s
of interfei-ons,they are noi within the reach of a
lar ge num ber of c om m on peo p l e d u e t o t h e c o s t
factor (the cost of production being very high).
ERYTHROPOIETIN
Erythropoietinis a hormone synthesizedby the
kidneys. lt stimulatesthe stem cells of bone marrow
to produce nrature erythrocytes. Biotechnologists
wef e s uc c es s f ul in pr odu c i n g r e c o m b i n a n t
erythropoietin. An approval for its therapeutic use
in hunr answas obt ained in t he y e a r . l 9 8 9 . A n r g e n
lnc. first marked erythropoietin with a trade name
Epagen. lt is useful in treating ihe oatients with
s ev er e aner nia t hat ac c om oan i e s l <i d n e v d i s e a s e
Another firrn Ortho-Biotech cornpany produced
Procrit, a gerreticallyengirreerederythropoietin in
1997 Pr oc r it ac t s lik e t he na t u r a l h o r m o n e a n o
stimulatesthe production of erythrocytes.lt is used
in anen- : icpat ient s under g, oin gn o n - c a r d i a c , n o n -
vascular surgery. Procrit administration before
surgeryservesas an alternativeto blood transfusion.
However, therapeutic use of procrit is quite
ex pens iv e,henc e not widely u s e d .
DEO XYRI BO NUCLEASE | (DNase ll
The enz y m e DNas e I hy d i 'o l y s i s I o n g D N A
c hains int o s hor t er olig o n u c l e o t i d e s . T h e
bioiec hnology f ir m Cenente c i r i s o l a t e d a n d
expressed the gene to produce i"ecombinant
DNas e I . This enz y m e is ve r y u s e f u l i n t h e
treatrnent olc common hereditary disease cysfic
fibrosis. as exolained hereunoer.
Cy s t ic f ibr c s is ( CF) is one o f t h e m o s t c o m m o n
( f r equenc y1 : 25, 000) genet ic d i s e a s e s .P a t i e n t so f
CF ar e highly s us c ept ible t o I u n g i n f e c t i o n s b y
bacteria. The oresence of live or dead bacteria
leads t o t he ac c um ulat ion of t h i c k m u c u s i n t h e
I ungs m ak ing t he br eat hingv er y d i f f i c u l t . T h e m a ; o r
c ons t it uent of t his m uc us is t h e b a c t e r i a l D N A
B IOTE C HNO LO C\
(released on bacterial lysis). Adnrrnistration of the
enzyme DNase I to the lungs of CF patients
decreases the viscosity of the mucus, and the
breathing is made easier. lt must be remembered
that DNase I cannot cure cystic fibrosis. lt can onlr
relieve the severe symptoms of the disease in mosi
oatients.
ALGINATE LYASE
Alginate lyase acts on a polysaccharidepolymer
n a m e l y a l g i n a t e . A l g i n a t e i s f o u n d i n so i l a n d
m a r i n e b a c t e r i a . T h e o c c u r r e n c e o f m u c u s i n th e
Iungs of cystic fibrosis patients is partly due to
alginate, produced by the bacterium Pseudomonas
aeruginosa Therefore, administration of alginate
I y a s e i n s t e a do f o r i n a d d i t i o n t o D N a s e I h e l p s to
clear lungs of CF patients.
Alginate lyase gene has been isolated from a
Cram-negative soil bacterium, Flavobacterium sp
T h i s g e n e w a s u s e d t o p r o d u c e r e co m b i n a n t
alginate lyase in E. coli. Trails are being conducted
for therapeutic use of this enzyme in CF patients
SECOND GENERATION T H E R A P E UTIC
PROTETNS (MUTEINSI
By employin g site-directed mutagenesis, the
a m i n o a c i d s e q u e n c eo f a r e c o m b i n a n t p r o te i n ca n
b e s u i t a b l y m o d i f i e d a s d e s i r e d , b y a te ch n i q u e
referred to as protein engineering (For details Refer
Chapter 10). The mutated proteins are collectivelr
referred to as muteins. Protein engineering is a
rational approach to modify a protein with regarcr
to its stability, solubility, specificity, substrate
affinity, pharmacokineticsetc.
The muteins obtained by protein engineerine
technique are considered as Second generation of
therapeutic proteins. Selected examples of such
proteins (e.g. insulin lispro, Alteplase) are alreadr
described(Seep. 192 and P. 196).
Vaccines are another important Sroup oI
pharmaceuti calproducts of recombinantDNA
technology.They are separatelydealt with rr'
.l
Chapter 6. However,the readermust treat ano
learnvaccinesalso as a part of this chapter.
\ /a ct ina tion is t he phenom enon of
pr ev ent iv e
V immu nizatio n. ln t he m oder n c onc er l t ,
vaccination involves the administrafion (iniection
or oral) of an antigen to elicit an antibody response
that will protect the organism against future
infections.
Vaccines, althclugh in a vei-y crude form, were
us ed in a pe cu liar m anner by Chines e in as ear ly a s
eleventh century. They used to rernove the dried
sca bso f sma llpo x pat ient sand gr ound t hem . The so
prepared powder was sprayed in the noses of
healthy people lt was observed that these people
we re less su scep t iblet o s m allpox when t her e wa s
an o utb rea k o f e pidem ic .
Sma llpo x is a v ir ulent dis eas ewit h a high deat h
ra te. Even the s ur v iv or s bec om e v ic t im s o f
p erma ne nt disfigur em ent , blindnes s and m ent a l
re tard atio n. Edwa r d J enner , an Englis h Phy s ic ia n ,
observed th at the f ar m er s and m ilk m aids wor k in g
with co ws d eveloped a m ild f or m of s m allpo x
ca lled co wpo x or v ac c inia. And t he c owpo x
infection could protect these people from the
infe ctio n o f smallpox . lt was in 1796, J enn e r
e xp erime nta lly t es t ed t his obs er v at ion. H e
inocr-rlatedan B-year old boy with exudate from a
cowpox lesion, and he repeated it twice with a gap
o f some we eks. H e t hen inoc ulat ed m or e hum a n
volu nte ers. Je nn er not ed t hat s m allpox did no t
d evelo o in th ese v olunt eer s . This was t he f ir s t
disco ve ry of th e p r inc iple of v ac c inat ion.
It took nearly hundred years for the scientiststo
clea rly u nd erstan dt he bas isof s m allpox im m unit y .
We n r r w k n o r v t h a t c o w p o x v i r u s e s ( r 'a <, c . i n i a j
rnocr-rlated by Jenner,stimr,rlatetlre bodlr's imnrune
s y s t e m t o p r o c l u c ea n t i b o d i e s w h i c h n e u t r a l i s et l r e
c ( ) w p o x a s w e l l a s s m a l l p o x v i r i r s e s .T h e p r e s e n t
day vaccines which are more rr:fined lvork in a
- s i m i l a rf a s h i o n
Vaccinesare mainly of three typer
1 . Dead bacteria or inactivated viru-ces
*
2. Live non-virulent or weakened (attenuated) t
bacteria/or viruses.
3. Viral fragments or bacterial moleatles
(subunit vaccines)
A v a c c i n e t r i g g e r st h e b o d y 's i m m u n e s y s t e mt o
p r o d r . r c ea n t i b o d i e s a g a i n s t a s p e c i f i c d i s e a s e -
causing organism (virus, bacterium or other
p a r a s i t e ) T h i s p r c ; v i d e ss u r v e i l l a n c e a g a i n s t f u t u r e
exposureto such an organism and thus protectsthe
body. Many ccmmunicable diseases (smallpox,
c h o l e r a , t y p h o i d , t u b e r c u l o s i s ,p o l i o m y e l i t i s ) h a v e
been brought under control through vaccination.
However, as on today, for several diseases,there
a r e n o v a c c i n e s e . g . , A I D S , l e p r o s y ,f i l a r i a s i s .
TRADITIONAL VACCINES
The disease producing organisms (infectious
a g e n t s )a r e g r o w n i n c u l t u r e . T h e y a r e t h e n p u r i f i e d
a n d e i t l r e r k i l l e d ( i n a c t i v a t e d )o r n r a d e n o n - v i r u l e n t
( a t t e n u a t e d )T. h i s h a s t o b e c a r e f u l l y d o n e w i t h o u t
t h e l o s s o f t h e o r g a n i s m 'sa b i l i t y t o e v o k e i m m u n e
! 'e s p o n s ea g a i n s ta v i r u l e n t f o r m o f d i s e a s e - c a u s i n g
o r g a nr s m .
199
200 BIOTECHNOLOCY

The traditional production of vaccines has

several drawbacks.
It is not possible to develop vaccines for the
organisms not grown ir-rculture.
or being developed
a The yield of vaccines is very low.
a Cell c ult ur es ar e c os t ly t o m a i n t a i n . Disease Pathogenic organism
a Ther e is a danger of non- v i r u l e n t o r g a n i s m s Viral diseases
getting converted to virulent ones. Vaccinations
Accuteinfantile Rotavirus
by s uc h or ganis m sm ay c aus e t h e d i s e a s e i t s e l f
gastroenteritis
It is not possible to prevent all the diseasesby
Acuterespiratory A andB viruses
Influenza
us e of t r adit ional v ac c ines e. g . , A I D S .
diseases
Purified antigen vaccines A ID S Human immunodef virus
iciency
Some improvementsin traditional vaccines have pox
Chicken virus
Viricella-zoster
been made by isolating the antigens frorn the
Encephalitis Japanese virus
encephalilis
pathogenic organisms. The antigens of bacterial
cell walls (e.9., Streptococcuspneumoniae causing Genital
ulcers Herpes virus
simplex type-2
pneurnonia),and the endotoxins ai'egood examples Hemonhagic
fever virus
Dengue
of purified vaccines. The endotoxins that do not
Liverdamage A virus
Hepatitis
possess toxicity but retain immunogenecity are
referred to as toxoids e.g. toxods of tetanus, Liverdamage B virus
Hepatitis
diohtheria etc. Upperandlower Yellow
levervirus
tractlesions
respiratory
RECOMBINANT VACCINES-GENERAL
Recombinant DNA technology in recent years, Bacterialdiseases
has become a boon to produce new generation Cholera Vibriocholerae
vaccines.By this approach, some of the limitations
(listed above) of traditional vaccine production Dianhea E. coli
could be overcome. In addition, several new Dysentery Shigeilasltain
strategies,invclving gene manipulation are being Gonorrhea Neissriagonorroheae
tried to create novel recombinant vaccines.
Lepr0sy Mycobacterium leprae
The list of diseases for which recombinant
vaccines are developed or being developed is given Meningitis ria meningitidis
Neisse
in Table 16.1. lt may be stated here that due to very Pneumonia s pneumoniae
Streptococcu
stringentregulatoryrequirementsto use in humans, fever
Rheumatic StreptococcusgroupA
the new generation vaccines are first tried in
animals, and it may take some more years before Tetanus Clostridiumtetani
most of them are approved for use in humans. Tuberculosis Mycobacterium tuberculo
sis
Typhoid Salmonella
typhi
Types of recombinant vaccines
Urogenital
tracl groupB
Streptococcus
The recombinant vaccines mav be broadly
infection
categorized into three Broups :
1 . Subunit recombinant vaccines : These are Parasiticdiseases
the components of the pathogenic organisms.
Filariasis Wuchereria
bancrofti
Subunit v ac c ines inc lude pr ot e i n s , p e p t i d e s a n d
DNA. Malaria Plasnodium
sp
2. Attenuated recombinant vaccines : These Riverblindness volvulus
Onchocerca
are the genetically modified pathogenic organisms Schistosomiasis mansoni
Schistosoma
(bacteria or viruses)that are made non-pathogenic
sickness
Sleeping Trypanosoma
sp
and used as vaccines.
Chapt er16 : R E C OMB IN A NVTAC C IN ES 2(J1

3. Vector recombinant vaccines : These are the

genetically modified viral vectors that can be used
as vaccines against certain pathogens.
Some of the developments made in the DNA
(A)
production of recombinant vaccines againstcertain HBsAg
diseasesare briefly described.

HepatitisB virus

As already stated,subunit recombinant vaccines

are the camponents (proteins, peptides, DNAs) of
the pathogenic organisms. The adrrantages of these
(B) 'n
ADH
vaccin es include t heir pur it y in pr epar a t i o n ,
pMA56 promoter
sta bility an d s af e us e. The dis adv ant agesar e - (yeastvecto0
high cost factor and possible alteration in native
conformation. Scientistscarefully evaluate the pros
and cons of subunit vaccines {or each disease,and
oroceed on the considered merits.

HEPATITIS B zA\
tl f- \
He pa titis B is a wides pr ead dis eas e in m a n ' l t \\ ) l+-- HBsAssene
primarily affects liver causing chronic hepatitis,
cirrhosis and liver cancer. Hepatitis B virus is a 42
nm particle, called Dane particle. lt consists of a
core containing a viral genome (DNA) surrounded
by a ph osph olipid env elope c ar r y ing s ur f a c e

de@&
antigens (Fig, 16,1A). Infection with hepatitis B
virus produced Dane particles and 22 nm sized
oarticles.The latter contain surface antigenswhich
are more immunogenic. lt is however, very difficult
to gro w h ep atit isB v ir us in m am m alian c ell c u l t u r e Grow in tryptophan-free
and produce surface antigens. mediumand selectcells
The gene encodipg for hepatitiS B surface
antigen HBsAg) has been identified. Recombinant
hepatitis B vaccine as a subunit vaccine, is
produced by cloning HbsAg Sene in yeast cells'
@36/
Saccharomyces cerevisiae, a harmless baking and
Culturecells
brewing yeast, is used for this purpose (Fig- 16.18)' I
The gene for: HBsAg is inserted (pMA 56) which is
+
Lyseyeast cells
linked to the alcohol dehydrogenase promoter'
These plasmids are then transferred and cultured'
The cells grown in tryptophan, free medium are +
I
selected and cloned. The yeast cells are cultured'
PurifyHBsAg
1-heHBsAg gene is expressedto produce 2nm sized (as 22 nm particles)
particles similar to those found in patients infected
with hepatitis B. (These particles are
Fig. 16.1 : (A) Hepatitis B virus-Dane particle (42 nm
immu no rea ctiv e wit h ant i- HBs Ag ant ibod i e s ) '
Th e su bu nit HBs Ag as 22 nm par t ic les c a n b e
isola ted a nd us ed t o im m uniz e indiv iduals ag a i n s t
heoatitis B.
2|J2 B IOTE C H N OLO CY

Hepatitis B vaccine.the first

synthetic vaccine
Shortpeptides
ln 1987,the recombinant vaccinefor hepatitisB
(i.e. HBsAg)becamethe first syntheticvaccinefor
ptrblic use. lt was marketed by trade names
Recombivaxand Engerix-0.HepatitisB vaccine is
safeto use,very effectiveand producesno allergic
reactions.For these reasons,this recombinant
v ac c i n e h a s b e e n i n u s e s i n ce 1987. The
individualsmust be administered three dosesover
a p e ri o d o f s i x mo n th s . l mmu ni zati onagai nst
hepatitisB is strongly recommendedto anyone Fig. 16.2 : A general structure of a synthetic
coming in c<-rntact with blood or body secretions. peptide vaccine.
All the healthprofessionals-physicians, surgeons,
medical laboratorytechnicians,nurses,dentists,
besidespolice officers,firefightersetc., must get
protein1 was usedto vaccinatearrimals.However,
vaccinatedagainsthepatitisB.
VPI vaccinationwas foundto be lesseffectivethan
that of the whole virus in protectingFMD. Further,
Hepatitis B vaccine in India
studiesare beinBpursuedto improvethe efficiency
lndia is the fourth country (after USA, France of subuni tvacci ne.
and Belgium)in the world to developan indigenous
hepatitisB vaccine.lt was launchedin 1997, and The concept of peptide vaccines
is now being used.
Theoretically,it is expectedthat only srnall
portions of a given protein (i.e., domains) are
Hepatitis B vaccine tomato?
immunogenicand bind to antibodies.Logically,it
Biotechnologistshave been successful in is possible to use short peptides that are
insertinghepatitisB gene into the cells of the immunogenicas vaccines.Theseare referredto as
tomato plant. Thesegeneticallyengineredplants peptidevaccines.
producehepatitisB antigens.The day may not be
far otf to get immunizedagainsthepatitisB by Peptide vaccines for foot and
havinga tomaio with lunch! mouth disease
Somedetailson the FMD are describedabove.
FOOT AND MOUTH DISEASE
The domainsof viral proteinI (VPl)of FMDV were
Foot and mouth disease(FMD) is a highly' chemicallysynthesized. Fromthe C-terminalend of
corrtagiousdiseaseaffectingcattle and pigs. A V P l , ami noaci ds14i to 160, 15i to 160 an d 200
forrnalin killed foot and mouth disease virus to 213,and from N-terminalend, aminoacids9 to
(FMDV)was previouslyused to vaccinateagainst 24, 17 to 32 and 25 to 41 were synthesized. Each
t his d i s e a s e . one of these shorf peptides (domains) was bound
to the surface of a carrier protein (Fig. 16.2 and
The genomeof FMDV is composedof a single-
usedas a vaccine.Among the peptidesused, the
strandedRNA,coveredby four viral proteins(VPl,
one correspondi ng to ami noaci ds141 to 16 0 was
VP2, VP3 and VP4). Among these, VPI is
found to be effectivein immunizingguinea pigs
immunogenic.The nucleotidesequenceencoding
againstFMD. ln addition,when two peptideswere
VPI was identifiedin the FMDV genome.A double- joined together (amino acids 141 to 158, and
strandedcomplementaryDNA (cDNA) from the
amino acids 200 to 213), they servedas more
single-stranded vira.l RNA (genome) was
efficientrecombinantvaccines.
synthesized. This cDNA was then digestedwith
restrictionenzymesand the fragments were cloned The successso far to use recombinantpeptides
by using plasmid pBR322 in E. coli. The as vaccineshas been very limited.This is mainly
recombinant vaccinefor FMDV in the form of virar becausea shortpeptideusuallyis not enoughto be
Ch AOICT
1 6 : RECO M BI NANT VACCI NES
HSV glycoproteinD
Membrane
Transmembrane
portion
cooH
I Bacillus
NHr
t-
cooH
ModifiedHSV glycoprotein
Fig. 16.3 : Modification of HSV glycoprotein D by
deleting transmembrane portion.
(HSV-Herpes simplex virus)
sufficienty immunogenic, since it may not have the
same con for m at ion as t hat of t he or igina l v i r a l
particle. However. scientistscontinue their search
for specific, inexpensiveand safe synthetic peptide
vaccines for various diseases.
HERPES S I M PLEX VI RUS
Herp es sim plex v ir us ( HSV) is an onc o g e n i c
(can ce r-ca us ing)v ir us . I n addit ion, it als o c a u s e s
se xu ally trans m it t ed dis eas es , enc ephalit i s a n d
severe eye infections.Attempts have been made to
produce subunit vaccines against HSV.
An envelope glycoprotein D (gD) of HSV that
can elicit antibody production has been identified.
This is a mem br ane bound or ot ein. and dif f ic u l t t o
isolate and purify. The glycoprotein D was
modified by deleting the transmembrane portion
of the protein (Fig. 16.3) and the gene was
mo difie d. This gene f or gD was c loned i n a
ma mmalia n v ec t or and ex or es s ed in Ch i n e s e
hamster ovary (CHO). The advantage here is that
the protein can get glycosylated (unlike in E. coli
system). In the experimental trials, the modified
form of gD was found to be effective against HSV.
203
TUBERCULOSIS
Tuberculosis is caused by the bacterium
Mycobacterium tuberculosls. lt is often fatal, and as
p e r s o m e e s t i m a t e sn e a r l y 3 m i l l i o n d e a t h s o c c u r
every year due to this highly infectious disease.
Antibiotics are used to treat tuberculosis.However,
drug-resistant M. tuberculosrs strains have been
developed making the drug therapy some tinres
ineffective.Vaccination for tuberculosisis therefore.
advocated.
Calmette.Guerin (BGGf vaccine
In some countries, particularly the developing
ones, BCC vaccine is widely used to protect against
tuberculosis However, countries like United States
have not approved BCC vaccination for various
r e a s o n s B C C v a c c i n e i t s e l f c a u s e s t u b e r c u l o s i sr n
s o r n e i n d i v i d u a l s( A I D S v i c t i m s ) a n d t h e v a c c i n a t e d
people respond positively for laboratory diagnosis
of tuberculosis.
Subunit vaccines
The secretory (extracellular proteins of M.
tuberculosis have been purified and used for
i m m u n o p r o t e c t i o n a g a i n s t t u b e r c u l o s i s .O f a b o u t
100 such proteins, six were found to be useful
( e i t h e r i n d i v i d u a l l y o r i n c o m b i n a t i o n )t o i m m u n r z e
Surneaprgs.
Attempts are underway to develop recombinant
s u b u n i t v a c c i n e a g a i n s tt u b e r c u l o s i s .
MENINGITIS
Croup B strain of meningococci, narnely
Neisseria meningitidis causes meningitis In
adolescents and young adults. Meningitis is
characterized b), inflammation of the membranes
covering brain and spinal cord. The symptoms
include headache, photophobia, irritability, and
neck stiffness.
Pizza et al (2OOO)made a novel approach to
develop a vaccine against meningitis. They
identified 350 proteins (potential protecttve
antigens) and the entire sequence of genome
coding for these proteins in N. meningitidls.All the
350 candidate antigens were expressedin E. coli,
purified and used to immunize mice. A good
bactericidal antibody response was observed rn
these mice.
20,4 B IOTE C H N OLO G Y

(A)

Cell surface

Bindingof gp41
Attachmentof virus to host cell for
to hostcellby 9p120 virus entry

(B)

F-CD+ receptor

Anti-9p120antibody . Anti-qp41
-/ anttbodY
V-t
t/
-\*
()-so+t
)-
_/
Anti-9p120
antibody Anti-9p41antibody
preventsbindingof bindsto gp41 and blocks
9p120to CD4 receptor fusionof HIV to host cell

Fig. 16.4 : Subunit recombinant vaccines against AIDS. (A) The functions of 9p120 and gp41 (B) The

A|DS (ACOUTRED TMMUNODEFTCTENCY continuously developed and field tested, although

SYNDROMEI there has been no suci:essso far.

AIDS is a retroviraldiseasecausedby human

Subunit vaccines
immunodeficiency virus (HfI4. This disease is
characterizedby immunosuppression, neoplasma The developrnent of two subunit vaccines,
and neu ro l o g i c aml a n i fe s ta ti o nAsID S i s i nvari abl y s p e c i f i c a l l y t h e g l y c o p r o t e i n s o f H I V e n v e l o p e is
fatal,sinceas of now thereis no cure.Development d e s c r i b e dh e r e . T h e f u n c t i o n so f g p 1 2 0 a n d g p 4 1 o f
t S i s a to p p ri o ri tyby D N A
of a v acc i n ea g a i n sAID HIV are illustrated in Fig. 16.4A. The glycoprotein
technologists worldover.In fact,vaccinesare being gp120 projects out of the HIV envelope while the
Chapt er1 6 : R E C OMB IN A NVTAC C IN ES 20'5

ot her gl),c o p ro te i ng p 4 1 l i e s b e n e a t h gp120. the DNA vaccine gene, origin of replication, a

On enteringthe body, the HIV binds to the host selectable marker sequence (e.g. a gene for
cells (T-lvmphocytes) by attachinggp.l20 to the a m p i c i l l i n r e s i s t a n c e )a n d a t e r m i n a t o r s e q u e n c e
CDo receptor sites on the cell surface. This (a poly-A tail).
at t ac hme nut n c o v e rsg p 4 1 mo l e c u l e a
s n d the vi ral
DNA vaccine-olasmids can be administeredto
envelope.Now gp4.l bindsto the hostcell surface
the animals by one of the following delivery
and opensa passage for the entryof the virus into
methods.
t he c ell,
. Nasal spray
Biotechnologists have isolatedthe genes for
o I n t r a m u s c u l a ri n l e c t i o n
9p120 and gp41 and inserted them into the
bacteriumE. coli. These bacterialcells oroduce o lntravenous iniection
gpl20 an d g p 4 1 th a t c a n b e u s e da s re c ombi nant
o I n t r a d e r m a li n j e c t i o n
v ac c inesa g a i n s tAID S.T h e a c ti o n o f g p120 and
gp41in im m u n i z i n gh o s tT -l y mo h o c y tei ssdepi cted . C e n e g u n o r b i o l i s t i c d e l i v e r y ( i n v o l v e s p r e s s u r e
in Fig. 16.48.The gp120 r-nolecules stimulatethe delivery of DNA-coated gold beads).
hos t im m u n e s y s te m to p ro d u c e a n ti - gp120
antibodies.These antibodiesbind to gp.l20 and DNA VACCINE AND IMMUNITY
preventits attachmentto CDo In a comparable An illustration of a DNA vaccine and the
m anner , g p 4 1 m o l e c u l e s a l s o re s u lt i n the m e c h a n i s m o f i t s a c t i o n i n d e v e l o p i n g i m m u n i t y
pr oduc t i o n o f a n ti -g p 4 1 a n ti b o d i es. These i s g i v e r r i n F i g . 1 6 . 5 . T h e p l a s n r i d v a c c i n e c a r r y i n g
ant ibodie a s l s o b i n d to g p 4 1 a n d b l o c k the vi rus- the DNA (gene) for antigenic protein enters the
hostcell union.The net resultof usinggpt20 and n u c l e u s o f t h e i n o c u l a t e d t a r g e t c e l l o f t h e h o s t .
gp41 vaccines is that the entry of HIV into the T h i s D N A p r o d u c e s R N A , a n d i n t u r n t h e s p e c i f i c
host cells is prevented. antigenic protein The antigen can act directly
for developing humoral immunity or as fragments
Vaccine against AIDS- in association with major histocompatability
not yet a reality class (MHC) molecules for developing cellular
The descriptionof vaccinedevelopmentagainst i m m u n i t y .
AIDS(givenabove),which appearsattractiveis not
s o s im ple .T h e mo s t i m p o n ta nlti mi ta ti o ns
are that Humoral immunity
the HIV hashigh frequency of mutations. Therefore As the antigen molecules bind to B-
the vaccines developed cannot hind to the new lymphocytes, they trigger the production of
virus (i.e., mutatedone). In addition,9p120 and antibodies which can destroy the pathogens.
gp41 are very pqor stimulators of immunesystem. Some of the B-lymphocytes become memory
Despitethese Iimitations,scientistshave not lost cells that can protect the host against future
hope, and continue their researchto develop infections.
vaccinesagainstAIDS.
Gellular immunity
Theproteinfragments
of the antigenboundto
MHC molecules can activate the cytotoxic
T-lymphocytes.They are capable of destroying the
infected pathogenic cells. Some of the activated
T-lymphocytes become memory cells which can
Ce ne tic im m ur r iz at ion by us ing DNA v a c c i n e s
kill the future infecting pathogens.
is a novel approach that came into being in 1990.
fhe immune response of the body is stimulated by
Gomplementary DNA vaccines
a DNA molecule. A DNA vaccine consists of a
gene encoding an antigenic protein, insertedonto a F o r g e n e t i c i m m u n i z a t i o n , c o m p l e m e n t a r yD N A
pla smid , a nd t hen inc or por t ed int o t he c e l l s i n a (cDNA) vaccines can also be used. Some workers
ta rge t an im al. The plas m id c ar r y ing DNA v a c c i n e have successfully used cDNA as vaccines e.g.
normally contains a promoter site, cloning site for immunization of mice ieainst influenza.
 N
o
o

MemorvB-lvmPhocvte

YYY (protecisagairistfu[ureinfection)

YYY Antibodies

Antigen

HUMORALIMMUNITY

CELLULARIMMUNITY

Fragments
of antigen

Killpathogenic
ceils

T-lymphocyte
boundio antigen Activatedcytotoxic
T-lymPhocYtes
"B[1"' Nucteus ---l
m
rf
lnoculated T
cell Z

--
(MHC-Maior histocompatabilitycomplex molecule) -o
Fig. 16.s : DNA vaccne and mechanism of its action in devetoping immunity
Chaot er' 16 : R E C OMB IN A NVA
T C C IN E S 2lJ7

DNA vaccines for production of 5. The deliverymethodsto the hostare simpler

antigens and antibodies for DNA vaccines.
A novelapproachfor the productionof antigens
as well as antibodies by DNA vaccine was Disadvantages of DNA vaccines
devefopedin 1997. In the experiments conducted 1. The fateof the DNA vaccinein the hostcells
in mice, researchersinjected plasmids containing is not yet clear.Thereis a possibilityof this DNA
the genes for malarial parasite and also the getting integratedinto the host genomeand this
genesfor the antibodiesagainst malarial parasite.
may interruptthe normalfunctions.
The B-lymphocytesof these mice performeda
double duty, and produced antigens for, and 2. Therealso existsa dangerof cancerdue to
antibodiesagainstmalarialparasite.The antigens D N A vacci nes.
stimulateto oroduce more and more antibodies.
The antibodiesso produced react with malarial 3. The oost-translationalmodificationof the
parasite.
The generationof antigensand antibodies gene (DNA vaccine)productin hostcells may not
by usinga DNA vaccineis a recentdevelopmentin be the sameas that found in the nativeantigen.
immunology, and is referred to as antigenic
antibody approach of DNA vaccine. Present status of DNA vaccines
Since 1990, severalgroupsof workersworld-
Screening of pathogenic Aenome for
over have been trying to develop DNA vaccines
selecting DNA vaccines
againstvariousdiseasesin experimentalanimals.
The ultimategoal of scientistsis to choosethe C eneti c i mmuni zati onhas been done agai nsta
right DNA fragmentfrom the patirogento serveas number of pathogeni corgani sms. Thesei ncl ude
immuneresponse
a vaccinefor the strongest against i nfl uenzaA vi rus, rabi esvi rus, hepati ti sB vi rus,
the invading pathogen. For this purpose, the bovine herpesvirus, HIV type l, and Plasmodium
pathogen's DNA can be broken into fragmentsand species(malarialparasite).
a large number of vaccine DNA-plasmids can be
prepared. The immune responsefor each one of It must be noted that DNA vaccineshave not
the DNA vaccinescan be studiedby injectingthe been tried in humans for obvious reasons.Tne
pathogen.By screeningthe DNA fragmentsof the most important being rhe unknown risks of these
pathogenicBenome,it is possibleto chooseone or foreignDNAs in human subjects.
few DNA vaccinesthat can offer maximalimmune
orotection. R N A V A C C IN E S

Advantages of DNA vaccines Severalworkersaretryingto useRNA molecules

as vaccinesTheseRNAscan readilysynthesize the
There are severaladvantagesof using DNA antigenic proteins and offer immunity. But
v ac c inesin im mu n i z a ti o rr. unfortunately,RNAs are iess stable than DNAs.
1. The tedious and costly procedures of Thi s poses a bi g probl em for R N A vacci ne
purifyingantigensor creatingrecombinant vaccines manufacture and distribution. Therefore, the
are not necessary. progressin the developmentof RNA vaccineshas
been rather slow comparedto DNA vaccines.
2. DNA vaccinesare very specificin producing
the targetproteins (antigens Thus,
or antibodies).
they trigger immune responseonly againstthe
specificpathogen.
, N A v a c c i n e se l i c i tm u c h h i gher
3. I n gene ra lD
immune responsecompared to other kinds of
vaccrnes. Plants serve as a cheap and safe production
4. DNA vaccines are more stable for systemsfor subunit vaccines. The edible vaccines
temperaturevariations (low or high) than the can be easily ingested by eating plants. This
conventional vaccines. Thus, the storage and eliminates the processing and purification
transpoftproblemsassociatedwith vaccinesare procedures that are otherwise needed. Transgenic
m inim al. planfs (tomato, potato) have been developed for
20,4 B IOTE C H N OLO CY

a c t a s a n i r n m u n i z i n g a g e n t . T h i s t y p e o f a p p r o a ch
is almost outdated now.
edible subunit vaccines
lt is now possible to genetically engineer the
Antigen Host plant organisms (bacteria or viruses) and use them as
Iive vaccines, and such vaccines are referred to as
glycoprotein
Habies Tomato attenuated recombinant vaccines. The genetic
virus(VPl)
Footandmouth Arabidopsis m a n i p u l a t i o n sf o r t h e p r o d u c t i o n o f t h e s e v a cci n e s
are broadly of two types
Herpes
virusB surface
antigen Tobacco
1 . Deletion or modification of viruience genes
toxinB subunit
Cholera Potato
of pathogenic organisms.
Human glycoprotein
cytomegalovii'us B Tobacco
2 . C e n e t i c m a n i p u l a t i o n o f n o n - p a t h og e n i c
organisms to carry and express antigen
d e l e r m i n a n t sf r o m p a t h o g e n i c o r g a n i s m s .
expressing antigens derived from animal viruses
(ra bies v ir us , her pes v ir us ) . A s e l e c t e d l i s t o f l-he advantage with attenuated vaccines is that
re c om binant v ac c ines agains t a n i m a l v i r u s e s the native conformation of the immunogenic
prcduced in plants is given in l-able 16.2. determinants is preserved, hence the immune
responseis substantiallyhigh. This is in contrast to
Edible vaccine production and use purified antigens which otten elicit poor
in-rmunological response.
The production of vaccine potatoes is iilustrated
in Fig. 16.6. The bacterium, Agrobacterium Some of the imoortant attenuated vaccines
tumeiaciens is commonly used to deliver the DNA developed by genetic manipulatior.rsare briefly
(g enet ic m at er ial)f or bac t er ial or v i r a l a n t i g e n s .A described.
p las m id c ar r y ing t he ant igen gene a n d a n a n t i b i o t i c
CHOLERA
resistancegene are incorporated into the bacterial
cells (A. tumefaciens). The cut pieces of potato Cholera is an intestinaldiseasecharacterizedby
le av es ar e ex pos ed t o an ant ibiot ic w h i c h c a n k i l l d i a r r h e a ,d e h y d r a t i o n ,a b d o m i n a l p a i n a n d f e ve r . l t
th e c ells t hat lac k t he new genes . T h e s u r v i v i n g is caused by the bacterium, Vibrio cholerae. This
cel ls ( i. e. , gene alt er ed ones ) c an m u l t i p l y a n c if o r m p a t h o g e n i c o r g a n i s m i s t r a n s m i t t e d b y d r i n ki n g
a c allus ( c lum p of c ells ) . This c allu s i s a l l o w e d t o water contaminated with fecal matter. Cholera
sprout shoots and roots, which are grown in soil to epidemics are frequently seen in developing
form plants. In about three weeks, the plants bear countries where the water purification and sewage
potatoes with antigen vaccines. disposal systemsare not well developed.

The first clinical trials in humans, using a plant-
de riv ed v ac c ine wer e c onduc t ed i n 1 9 9 7 . f h i s
involved the ingestion of transgenic potatoes with
a toxin of E. coli causing diarrhea. Some success
was reported. For some more details on edible
vaccines, the reader nrust refer Chapter 51.
In the early years of vaccine research,attenuated
strains of some pathogenic organisms were
prepared by prolonged cuitivation - weeks,
months or even years.Although the reasonsare not
known, the infectious organism would lose its
ability to cause diseasebut retains its capability to
On entering the small intestine, V. cholerae
colonizes and starts producing large amounts of a
toxic protein, a hexameric enterotoxin. This
e n t e r o t o x i n s t i m u l a t e s t h e c e l l s l i n i n g i n t e sti n a l
w a l l s t o r e i e a s e s o d i u m , b i c a r b o n a t e a n d oth e r
i o n s . Wa t e r a c c o m p a n i e s t h e s e i o n s l e a d i ng to
severe diarrhea, dehydration, and even death.
The currently used cholera vaccine is composed
of ohenol-killed V. cholerae. The imrnuno-
protection, !astingfor 3-6 months is just moderate.
Attempis aie being made to develop better
vacctnes.
The DNA technologistshave identified the gene
encoding enterotoxin (toxic protein). Enterotoxin,
an hexamer, consists of one A subunit and five
i d e n t i c a l B s u b u n i t s . T h e A s u b u n i t h a s tw o
functional domains-theA, peptide which possesses
 Chaot er16 : RE C OMB IN A NVTAC C IN ES 249

Plasmid

Plantcell

Genetransfer
Potato

R*Leaf Cut pieces of leaves

Sprouts
with bacteria
Callus

Potato

: An illustration for edible

the toxic activity and A, peptide that joins A 2. The DNA sequence of A, peptide is
. y g e n e ti ce n g i n e e ri n g,
s ubunitt o B s u b u n i tsB it incorporatedinto a plasmid,cloned and digested
was possibleto deletethe DNA sequenceencoding with restrictionenzymes(ClaI and XbaD. In this
A. peptideand createa new strain of V. cholerae. manner,the A.' peptidecodingsequenceis deleted
This strain is non-pathogenic,since it cannot (the DNA encodingfor 183 of the 194 aminoacids
produceenterotoxin.The genetically engineeredV of the A, peptideis actuallyremoved).By usingTo
cholerae is a good candidate to serve as an D N A l i gase,the pl asmi d i s reci rcul ari zed.
Thi s
attenuated vaccine. plasmid containsa small portion of A., peptide
coding sequence.
Gieating a new strain ol V, cholerae
3. The pl asmi ci ,contai ni ngthe del eted A ,
The develooment of a new strain of Vibrio
peptidesequenceis transferred by conjugationinto
choleraethat can effectivelyserveas an attenuated
the V. cholerae strain carrying a tetracycline
recombinantvaccine is depicted in Fig. 16.7, anc)
resistance gene.
brieflydescribedbelow.
1. A tetracyclineresistance gene was inserted 4. Recombinationcan occur between the
into the A, peptide sequence of V. cholerae plasmid (containinga srnall portion of peptide
chromoSome.This destroysthe DNA sequence A 1 codi ng sequence)and the.chromosomeof
encodingfor Ar peptide,besidesmakingthe strain V. cholerae (carryingtetracyclineresistancegene).
resistant to tetracycline. Unfortunately, the The resultof this double crossoveris the formation
tetracycline gene
resistant is easily lost and the of V, cholerae containing a chromosomal DNA
enterotoxinactivityis restored.Because of this,the lacking A, peptide DNA sequence. As the
new strainof V. choleraeas suchcannot be usedas bacterium,V. choieraemultiplies,the plasnridsare
a v ac c t ne. lost in the next few generations.

Biotechnology
[14]
21'J B IOTE C H N OLO CY

5. The V. choleraecellsdefectivein A. peptide

are selected,based on tetracycline sensitivity.lt
mav be notedthat this new strainlackstetracycline
gene.
resistance
The geneticallyengineeredV. cholerae cells
with deleted,{1 pepticleDNA sequenceare quite
stable.They cannotproduceactiveenterotoxinbut
possessall other biochemicalfunctions of the Chromosomal
pathogen. This new strain of V. cholerae is
undergoingtrials for its efficiencyas a vaccine.
Preliminaryresults indicate that this attenuated
C)
vaccine can orotect about 907" of the volunteers J ,oo*o',nu."
againstcholera. But there are some side effects. Tetracycline
Scientists continuetheir work to developa better
v ac c in ea g a i n sct h o l e ra .
Vibrio
cholerae
resistance
gene c)",*"0o,
peptideDNA
sequence
Potato as a vehicle for
cholera vaccine
A group workershavedevelopeda genealtered
potato containing attenuatedcholera vaccine.
Thesepotatoeswhen fed to mice inducedimmunity
againstcholera.

SALMONELI.A SPECIES
The differentstrainsof Salmonellagenus are
responsiblefor causingtyphoid, entericfever,food
poisoning and infant deafh. Immunoprotection f**b-chromosomat DNA
againstSalmonellapathogensis really required. t-.--J
Someworkershave been successful in deleting
,'-l\
aro genesand pur genesin Salmonella.Aro genes ( )+- Plasmid
encode for the enzymes responsiblefor the \___--l

biosynthesisof aromatic compounds,while pur t /rA

genesencodefor enzymesof purine metabolism. l')\J
The new strainsof Salmonellacan be grown in I Plasmid
,,...,G
vitro on a complete medium. The doubly deleted /\
strainshave a very restrictedgrowth in vivo, while i 41peptide
I Deleted
\--nlEJ- DNA sequence
they can stimulate immunological response.The T-=--.<
genetically engineered attenuated vaccines of
Chromosomal DNA
Salmonellahave been shown to be effectiveas oral
vaccines in experimentalanirnals (mice, cattle,
sheep,chickens). Someworkersclaim that the new Fig. 16.7 : Developntent of a new strain of Y,
cholerae as an attenuated recombinant vaccine
strain of Salmonellaoffers immunoprotectionin
hum a n sa l s o .

LE'SHMAN'A SPECIES An attenuated strain of leishmania has been

Leishmaniaspeciesare flagellatedprotozoan
parasitesand are responsiblefor the disease
Ieishmaniasis.This disease is characterized by
cutaneous, visceral and mucosal leisons.
is transmittedby sandflies.
Leishmaniasis
created and successfullyused in mice to offer
immunaprotection against leishmaniasis. ln
Leishmaniamajor,the genesencodingdihydrofolate
synthase
reductase-thymidylate can be replacedby
the genesencodingresistance to antibioticsC-418
Chapt er16 : R EC O MB IN A NVA
T C C IN E S 211

and hygromycin. This new strain of L. major against pathogenic organisms Another advantage
invaria bly req ui r est hy m idine in t he m edium f or i t s of vaccinia virus is the possibility of vaccinating
g rowth a nd mu lt iplic at ion.The at t enuat eds t r ain o f individuals against different diseases
L. major can survive only a few days when simultaneously.fhis can be done by a recombinant
administeredto mice. This short period is enough to vaccinia viruses which carries genes encoding
ind uce immun it y in m ic e agains t t he les ions o f d ifferent antigens.
leishma nia .Ho wev er , m or e ex per im ent son anim a l s
Antigen genes for certain diseases have been
ha ve to be ca r r ied out bef or e t he leis hm a n i a
successfullv incorporated into vaccinia virus
a tten ua tedvacc ine qoes f or hum an t r ials .
genome and expressedT . h u s , v e c t o r v a c c i n e sh a v e
b e e n d e v e l o p e d a g a i n s th e p a t i t i s ,i n f l u e n z a , h e r p e s
s i m p l e x v i r u s , r a b i e s , a n g u l a r s t o m a t i t i sv i r u s a n d
malaria. However, none of these vaccineshas been
Iicensed for human use due to fear of safety lt is
Some of vectorscan be geneticallymodified a r g u e dt h a t r e c o m b i n a n tv a c c i n i a v i r u s m i g h t c r e a t e
and em ploy eda s v a c c i n e sa g a i n spt a th o g e n s . l i f e t h r e a t e n i n gc o m p l i c a t i o n s i n h u m a n s .

VACCINES AGAINST VIRUSES- Production of recombinant

vAcctNtavrRUs vaccinia viruses

Va ccinia viru s e is bas ic allyt he v ac c ine t hat w a s T h e d e v e l o p m e n to f r e c o m b i n a n tv a c c i n i a v i r u s

o rigin ally u se d by J enner f or t he er adic at ion o f is carried out by a two-step procedure (Fig. 16.8)
smallpox. The molecular biology of this virus has 1 . Assembly of plasmid insertion vector : Fresh
b ee n clea rly wor k ed out . Vac c inia v ir us c ont ain s a v a c c i n i a ( c o w p o x ) v i r u s e sa r e p r o c e s s e dt o r e l e a s e
doubie-strandedDNA (187 kb) that encodes about their DNAs. Now genes from hepatitis B virus,
20 0 d iffere ntpro t eins .The genom e of t his v ir us c a n h e r p e ss i m p l e x v i r u s a n d i n f l u e n z a v i r u s a r e a d d e d
accommodate stretchesof foreign DNA which can one after another and insertedinto vaccinia virus
be e xp resse dal ong wit h t he v ir al genes g e n o m e . T h e s e D N A c l u s t e r sa r e c l o n e d i n E . c o l i
f o r i n c r e a s i n gt h e i r n u m b e r a n d t o p r o d u c e p l a s m i d
The vaccin ia v ir us c an r eplic at e in t he hos t c e l l
i n s e r t i o n v e c t o r s .T h e p l a s m i d c o n t a i n s t h e f o r e i g n
cytoplasm (of the infected cells) rather than the
s u b u n i t g e n e s ,t h e n a t u r a l v a c c i n i a g e n e s ,i n c l u d i n g
nu cle us. Th is is pos s ible s inc e t he v ac c inia v i r u s
t h e p r o m o t e r g e n e s .T h e r e c o m b i n a n t p l a s m i d s a r e
p osse ssesthe m ac hiner y f or DNA r eplic at i o n ,
i s o l a t e da n d p u r i f i e d a n d s e r v e a s p l a s m i d i n s e r t i o n
tran scriptio n-D NA poly m er as e, RNA poly m er a s e
vectors.
etc. The foreign genes inserted into the vaccinia
viru s ca n also b e ex pr es s edalong wit h t he v i r a l 2 Production of recombinant vaccinia
g en ome .Thu s, the f or eign DNA is under t he c ont r o l v i r u s e s : T h e a n i m a l c e l l s a r e i n f e c t e dw i t h p l a s m i d
o f the virus, a nd is ex pr es s edindependent lyf r o m i n s e r t i o n v e c t o r s a n d n o r m a l v a c c i n i a v i r u s e s .A s
the h ost cell g enom e t h e v i r a l r e p l i c a t i o n o c c u r s , t h e p l a s m i d sa r e t a k e n
u p t o p r o d u c e r e c o m b i n a n t v a c c i n i a v i r u s e s .T h e
The vaccin ia v ir us es ar e gener ally har m le s s ,
p l a s m i d i n s e r t i o nv e c t o r i n c o r p o r a t e si t s g e n e s i n t o
relatively easy to cultivate and stable for years after
vaccinia virus genome at a place that encodesfor
lyo ph iliza tion ( f r eez e- dr y ing) .All t hes e f eat u r e s
the enzyme thymidine kinase (TK). Thus the
ma ke the va cc inia v ir ues s t r ong c andidat es f o r
recombinant viruses have Iost their ability to
vector vaccine. The cloned foreign genes (from a
produce TK. There are two advantagesof loss of TK
p ath og en ico rga nis m )c an be ins er t edint o v ac c i n i a
gene. One is that it is easy to select recombinecl
virus g ,en omefor enc oding ant igenswhic h in t u r n
v a c c i n i a v i r u s e st h a t l a c k T K g e n e a n d t h e s e c o n c |
p rod uces a ntib odies agains t t he s pec if ic dis ea s e -
is that these viruses are less infectious than the
causing agent. The advantage with vector vaccine
normal viruses.
is that it stimulates B-lymphocytes (to produce
antibodies)and TJymphocytes(ro kill virus infected T h e r e c o m b i n a n tv a c c i n i a v i r u s e s ,r e l e a s e df r o m
ce lls).Th is is in cont r as tt o a s ubunit v ac c ine whi c h t h e c u l t u r e d a n i m a l c e l l s , c a n b e s u c c e s s f u l l yu s e d
ca n stimula te o nly B- ly m phoc y t es .Thus , v ac c i n t a a s v a c c i n e s . T h e s e l i v e v i r a l v a c c i n e s h a v e s o m e
viru s ca n p rovide a high lev el of im m unbpr ot ec tt o n a d v a n t a e e so v e r t h e k i l l e d o r s u b u n i t v a c c i n e s .
212 B IOTE C H N OLO CY

Advantages
1. Authenticatedantigensthat closelyresemble
naturalantigenscan be produced.

2. fhe vi ruscan repl i catei n the hostcel l s .This

+ of the anti gensfor t heir
enabl esthe ampl i fi cati on
actionon B-lymphocytes and T-lymphocytes.
I A portionof genome
3. Therei s a possi bi l i ty sever al
of vacci nati ng
lr-17
disebseswith one recombinantvacciniavirus.
I He-patitisB
+ vrrusgene
Disadvantages
lr
--
I Herpessimplex 1. The most i mportantl i mi tati on i s thd yet
+ virusgene unknow nri sksof usi ngthesevacci nesi n hum ans.
2. Theremay be seriouscomplications of using
lr - @
I Influenza
virus recombinantviral vaccinesin immunosuppressed
I gene i ndi vi dual such
s as A ID S pati ents.
Y

=-r772--T-l
Vacciniavirus genornewith 3 insertedgenes Other viral recombinant vaccines

Most of the lvork on the developmentof lrve

vi ral vacci neshas been carri edout on vac cinia
virus. Other virusessuch as adenovirus,poliovirus
and varicella-zoster virus are also being tried as
recombinantvaccines.Scientistsare attractedto
devel op a recombi nantpol i ovi rusas i t can be
orally administered.lt mighttake many more years
for the recombinantviral vaccinesto become a
realityfor human use.

D E LIV E R Y OF A N TIGE N S B Y B A C TERI A

It is known that the antigenslocatedon the
surfaceof a bacterialcell are more immunogenic
than the antigensin the cytoplasm.Basedon this
observation, scientists to
havedevelopedstrategies
coat the surfacesof non-pathogenic with
organisms
antigensof pathogenicbacteria.
Flagellinis a protein presentin the fragella
(threadlike filaments)oI Salmonel/a.A synthetic
encodi ngthe epi topeof choler a
ol i gonucl eoti de
toxin B subunit was inserted into Salmonella
flagellingene.This epitopewas in fact found on
pdt-"qq
the flagellum surface.These flagella-engineered
t A/\4 4 bacteria, when administeredto mice, raised
"t"*a-l
anti bodi esagai nstthe chol era toxi n B subunit
Recombinant vacc Intavrrus
peptide.lt may be possiblein futureto incorporate
mul ti pl eepi topes(2 or 3) i nto the fl agel l i ngenet o
Fig. 16,8 : Production of recombinant vaccinia virus
createmultivalentbacterialvaccines.
 tii: ntibodies or immwnoglabulins are protein B-lylnphocytes is difficult,.rnd the synthesis of
rf "'. mo lecule s pro duc ed by a s pec r aliz edgr oup r . r t ' M A l r '" v a s s h o r t - l i v e d a n d v e r y l i n r i t e d .
cells calle d B-lym phoc y t es ( plas m a c ells ) in I t i s i n t e r e s t i n g t h a t i m n r r r r - t ; r lm o n o c l o n a l
mamn rals. Th e s t r uc t lr r es , c har ac t er is t ic s and a n t i b o d y p r o c l u c r r - rcge l l s c l o e x i s t i n n a t L r r r .T l r e y
variou s oth er aspe c t sof im m unoglobr - r lins ( lgs ) ar e
a r e i o u n d i n t h e p a i i . : n t s s L r f i e r i n g1 - r o m. r c l i s e a s e
describe delsewh er e( Ref erChapt er 68) . Ant ibodies callecl multiple myeloma (a cancer of lJ-
are a part of the defensesystenlto protect the body .l
lvnrphocytcs) lt was in 975. Ceorge l(oirler and
again st the in va ding f or eign s ubs t anc , esnam ely Ccsar Milstein ( N o b e l Prize, 1984) achieved large
antigens Each antigen has specific antigen s c a l e p r c r c l u c t i o no f M A b s T h e y c o u l c i s u c c e s s f u l l y
determinants (epitopes) located on it The hybridize antibody-producing B-iymphacytes
antib<.rdies have complementary determining with myeloma cells in vitro ancl creale a
regions (CDRs) which are mainly responsible for hybridoma The resr-rit is that the arlificially
t he a ntib od y spe ci f ic it y inrmortalized B-lynrphocytcs c;m nrultipll,
In re sp on seto an ant igen ( wit h s ev er aldif f er ent i n d e f i n i t e i l , i n v i t r o a n d p r o d L r c e M A b s f h e
epito pe s), B-lymph oc y t es gear up and pr oduc e h y b r i d o m a c e l l s p o s s e s st h e g r o w t h a r . r dm L r l t i p l y i r r g
ma ny diffe ren t an tibodies This t y pe of ant ibodies p r o p e r t i e so f m y e l o m a c e l l s b u t s e c r e t ea n t i b o d y o f
wh ich ca n re act wit h t he s am e ant igen ar e B-lyrrphocytes. The production of monoclonal
designatedas polyclonal antibodies Tlre polyclonal antibodies by the hy,brid cells is reierred to as
antib od y p rod uction is v ar iable and is deper r den t hybridoma technology
on factors such as epitopes, responseto in'rrr-iunity
et c Due to lack o{ s pec if ic it y and het er ogenr c :
natu re,th ere are sev er alI im it at ionson t he r - r t ilit yof
polyclon al a ntib od iesf or t her apeut icand c iiagnos t i c The rryelonra cells used in hybriciorr.ra
pu rposes technology must not be capable of syntfresizing
Monoclonal antibody (MAb) is a single type of t i - r c i ro n , r 'ra n t i i r o c i i e s I. h e s e l e c t i o n o i h y l r r i c l o r r r . .
that is directecl against a specific cells is basecl on inhibiting tlre nrrcleotide
antibady
( c o n s e c ] u . : n t l 1t hz e D N A ) s y n t h e s i z i n g n r a c h i n c r l ,
antigenic determinant (epitope). lt was a dleam of
T h r : m a n r r r a l i a n c e l l s c a n s y r r t h e s i z en t r c i e o t i ( j c s
scie ntiststo pro du c e M Abs f or dif f er entant igens .I n
lry trvo 1;athways-r/e novo sylrthesisanil salva5,;e
the ea riy ye ars, an im als wer e im m uniz ec lagains ta
pathrvay fFig. 17.1)
specific a rrtige n,B- ly m phoc y t eswer e is olat ed ancl
cultu red in vitro f or pr oduc ing M Abs . I his The de novo synthesis of nucieotiiles requires
appro ach was no t s
s uc c es s f ul inc e c ult r - t r it - nor
t gi r na l t e t r a h y c l r o f o l a t ew h i c h i s f o r m e t l f r o n r d i h y c l r L . r -

213
214 BIOTECHNOLOCY

Dihydrofolate PRODUCTION OF MONOCLONAL

I Aminopterin
ANTIBODIES

-
precursors Tetrahydrofolate
t The establishment of hybridomas and
productionof MAbs involvesthe following steps
(Fig. 17.2).

o e .rovo
^ ' ---
I 1. l mmuni zati on
|
^.Y,, - 2. C el l fusi on
-J"t4es;"
-l Nucreotides- - - ->DNA 3. Selectionof hybridomas
the products
4. Screening
5. Cloningand propagation
6. Characterization and storage.
Hypoxanthine 1. lmmunization : The very first step in
Thymidine hybridomatechnologyid to immunizean animal
(usuallya mouse),with appropriateantigen.The
Fig. 17,1 : Pathways for the synthesis of nucleotides
antigen, along with an adjuvant like Freund's
complete or incomplete adiuvant is injected
subcutaneously (adjuvants are non-specific
potentiatorsof . specific immune responses). The
folate. The formation of tetrahvdrofolate(and injectionsat multiple sites are repeatedseveral
therefore nucleotides) can be blocked by the ti mes. Thi s enabl es i ncreasedsti mulat ion of
inhibitor aminopterin. B-lymphocyteswhich are responding to the
anti gen.Threedayspri orto ki l l i ngof the anim al,a
The salvage pathway involves the direct final doseof antigenis intravenously administered.
conversionof purines and pyrimidinesinto the The immune-stimulatedcells for synthesisof
c o rre s p o n d i nngu c l e o ti d e s.
H ypoxanthi ne guani ne antibodieshavegrown maximallyby this approach.
phosphoribosyltransferase(HCPRT) is a key The concentrationof the desired antibodiesis
enzyme in the salvagepathway of purines. lt assayedin the serum of the animal at frequent
convertshypoxanthine and guaninerespectively to intervalsduring the courseof immunization.
i n o s i n e mo n o p h o s p h a t e and guanosi ne
When the serum concentration of the
monophosphate. Thymidinekinase(TK), involved
antibodiesis optimal,the animal is sacrificed. The
in the salvagepathway of pyrimidinesconverts
spleen is asepticallyremoved and disruptedby
th y m i d i n eto th y m i d i n emo n ophosphate (TMP ).A ny
mechanicalor enzymaticmethodsto releasethe
mutationin eitherone of the enzymes(HCPRTor
cells. The lymphocytesof the spleenare separated
TK) blocksthe salvagepathway.
from the rest of the cells by density gradient
When cells deficient(mutatedcells) in HCPRT centrifugation.
a re g ro w n i n a me d i u mc o n tai ni nghypoxanthi ne 2. Cell fusion : The thoroughly washed
a m i n o p te ri na n d fh y m i d i n e(H A T medi um),they lymphocytesare mixed with HGPRT defective
cannot survive due to inhibition ol de novo myelomacells.The mixtureof cells is exposedto
synthesisof purine nucleotides(Note : Salvage polyethylene glycol (PEC)for a shortperiod(a few
pathwayis not operativedue to lack of HCPRT). minutes),since it is toxic. PEC is removed by
T h u s ,c e l l sl a c k i n gH C P R I g row ni n H A T medi um washingund th" cells are kept in a freshmedium.
die. These cells are composed of a mixture of
The hybridoma cells possessthe ability of hybridomas(fusedcells),free myelomacells and
myeloma cells to grow in vitro with a functional free lymphocytes.
HCPRT gene obtained from lymphocytes(with 3. Selectionof hybridomas: When the cellsare
which myeloma cells are fused).Thus, only the culturedin HAT medium (the principledescribed
hybridomacells can proliferatein HAT medium, above),only the hybridomacells grow, while the
and this procedureis successfully used for their rest will slowly disappear.This happensin 7-.10
selection. days of culture.
17: M O N OC L O N AL A N T IB O D IES (HY B R ID OMA TE C H N OLOC Y )

lmmunizedanimal
Spinnerculture

Selectionof
hybridsin HATmedium

tleeze +-- Positive'Pots'

/I
l-
l<- | AssayantibodY
I
+
Freeze '71\".
'-+
l{------1
lRecloning
Characterizeclones
Selectvariants

Tumoursof cells
producingantibody
216 B IOTE CHNO LO CY

Selec t ion of a s ingle a n t i b o d y p r o d u c i n g characterizedfor their ability to withstand freezing,

hy br id c ells is v ei' y im por t ant .T h i s i s p o s s i b l ei f t h e a n d t h a v r i n g . T h e d e s i r e d c e l l l i n e s a r e fr o ze n i n
hv br idom as ar e is olat eC and g r o w n i n d i v i C u a l l y . liqr-rid nitrogen at several stages of cloning and
The s us oens ionof hv br idom a c e l l s i s s o d i l u t e d t h a t c u l t ur e .
t he indiv idual aliquot s c ont ain o n a n a v e r a g e o n e
c ell eac h Thes e c elis , r v het r g r o w n i n a r e g u l a r &AHGE 5GAIE PRO.DUCTION OF MAbs
c ult r r r e r nedium , pr oduc e t he d e s i r e d a n t i b o d y . The production MAbs in the culture bottles ls
4 Screening the products : The hybridomas rather low (5-10 pg/ml). The yield can be increased
nrust be s.:reetredfor tlre secretion of the antibody by growing the hybrid cells as ascites in the
of des ir ed s pec if ic it y . The c u l t u r e m e d i L t m f r o m peritoneal cavity of mice The ascitic fluid contains
eac h hv br idor na c ult ur e is pe r i o d i c a l l y t e s t e d f o r a b o u t 5 - 2 0 m g o f M A b / m l . T h i s i s f a r s u pe r i o rth a n
. l r e t w o t e c h n i q t r e s the in vitro cultivation techniques. But collection of
t he des ir edant ibody s pec if ic it y T
nam ely ELI SAand RI A ar e c om m o n l y u s e d f o r t h i s MAb fronr ascitic fluid is associatedwith the heavy
purllose. ln Lrothihe assays,the antibody binds to r i s k o f c o n t a m i n a t i o n b y p a t h o g e n i c o r g a n i sm so f
t he s pec if icant igen ( us uallyc oa t e d t o p l a s t i c p l a t e s ) t h e a n i m a l . I n a d d i t i o n , s e v e r a la n i m a l s h a ve to b e
and t he unbound ant ibodv an c i o t h e r c o m p o r r e n t s sacrificed to produce MAb. Hence, many workers
of tlre medium can be washed off. Thus, the prefer in vitro techniques rather than the use of
hy br idom a c ells pr oduc ing t h e d e s i r e d a n t i b o d y a n i m al s .
can be identified b), screeninB. The antibody
hyhridorna ce!ls for
secreted by the hyhrid cells is referred to as Encapsuelated
csffi?mercEai Broduction of MAbs
monoclonal antibody.
The yield of MAb production can be
5. Cloning and pr opagat io n : T h e s i n g l e h y b r i d
s u b s t a n t i a l l yi n c r e a s e db y i n c r e a s i n gt h e h yb r i d o m a
c ells pr oduc inp,t he des ir ed a n t i b o d y a r e i s o l a t e d
c e l l d e n s i t y i n s u s p e n s i o nc u l t u r e . T h i s c an b e d o n e
and c loned. T\ nio t ec hniqu e s a i 'e c o m m o n l y
by encapsulating the hybridomas in alginate gels
em ploy ed f or c loning hv br id c e l l s - l i m i t i n g d i l u t i o n
a n d u s i n g a c o a t i n g s o l u t i o n c o n t a i n i n g p o l y- l ysi n e
method and soft agar method.
(Fig. 17.3). These gels allow the nutrients to enter
Limiting dilution method : In this procedure, in and antibodies to come oLtt. By this approach, a
t he s us pens ionof hy br idom a c e l l s i s s e r i a l l yd i l u t e d m u c h h i g h e r c o n c e n t r a t i o n o f M A b p r o d u cti o n
and t he aliquot s of eac h di i u t i o n a r e p u t i n t o (10-1 00 pg/ml) can be achieved. Damon Biotech
m ic r oc ult ur e wells . The dilut io n s a r e s o m a d e t h a t company and Cell-Tech use encapsulated
eac h aliquot in a we! l c ont ains o n l v a s i n g l e h y b r i d hybridoma cells for large-scaleproduction of MAbs.
c ell This ens ur es t hat t he an t i b o d v p r o d u c e d i s They employ .l00-liter fernrentersto yield about
m onoc lonal. 1 O C go f M A b s i n a b o u t 2 w e e k s p e r i o d .
'
Soft agar method : In thiS technique, the
hy br idonr a c ells ar e c ult ur ed i n s o f t a g a r . l t i s }t U M A N M O N O C L O N A L A N T I B O D IES
pos s ible t o s im ult aneous ly g r o w m a n y c e l l s i n T h e m o n o c l o n a l a n t i b o d i e s p r o d u c e d b y u si n g
s em is olidm edium t o f or m c olc n i e s . T h e s e c o l o n r e s mice are quite suitable for in vitro use. However,
will be nr onoc lonal in nat ur e . t h e i r a d m i n i s t r a t i o n t o h u m a n s i s a s s o c i a te dw i th
ln actuai practice, both the above techniques i m m u n o l o g i c a l c o m p l i c a t i o n s , s i n c e th e y a r e
are combined and used for maximal production of foreign to human body. Production of human
MAbs. monoclonal antibodies is preferred. However, it is
difficult to produce human MAbs by conventional
6. Characterization and storage : The
h y b r i d o m a t e c h n o l o g y .T h e f o l l o w i n g a r e th e m a j o r
m onoc lonal ant ibody has t o b e s u b j e c t e d t o
limitations.
bioc her nic al and biophy s ic al c h a r a c t e r i z a t i o nf o r
t he des ir ed s pec if ic it y li is a l s o i r n p o r t a n t t o . For ethical reasons, humans cannot be
eluc idat eihe r \ ' lAbf or t he im m u n o g l o b u l i n c l a s s o r i m m u n i z e d a g a i n s ta n t i g e n s .
s ub- c las s ,t he epit opr : f or whi c h i t i s s p e c i f i c a n d . T h e f u s e d h u m a n l y m p h o c y t e - m o u se m ye l o m a
t he num ber ot bindir r g s it es it p o s s e s s e s . cells are very unstable.
The s t abilit y of t he c ell l i n e s a n d t h e M A b s . T h e r e a r e n o s u i t a b l e m y e l o m a c e l l s in h u m a n s
ar e im por t ant . The c ells ( a n d M A b s ) m u s t b e that can replace mouse myeloma cells.
 (H YB R ID OMA
17 : M O N O C L O N ALAN T IBOD IES
C hA P t Cr TE C H N OLOC Y ) 217

Transgenic .mouse for producing

human MAbs
Hybridcells' Attempts have been made in recent years to
(in sodiumalginate)
intraduce human immunoglobulin genes into the
mice Io develop transgenicmice. Such mice are
c a p a b l e o f s y n t h e s i z i n gh u m a n i m m u n o g l o b u l i n s
when immunized to a particular antigen. The
B-lynrphocytes isolated fi'om transgenic mice can

I be used to pr.oduce MAbs by the standard

hybridoma technology.
The above three approachesare quite laborious,
I i,,, i.',)fl-Encapsulated
and the yield of human MAbs is very low.
c ells
[ , ,,, J
\___- Consequently,researcherscontinue their searchfor
better alternatives.
L- Coatingsolution
[ (poly-lysine)
GENETIC ENGINEERING STRATEGIES
J FOR THE PRODUCTION OF HUMAN-
M O T 'S E M A b S
I t.i;' l..,,FHybrid cellsin
P o ro u s c a Ps u l e s
[ , , ' ,-: l -, ,l Wi t h t h e a d v a n c e s i n g e n e t i c e n g i n e e r i n g ,i t i s
\i- li'------l n o w p o s s i b l et o a d d c e r t a i n h u m a n s e g m e n t st o a
II mouse antibody. This is truely a hybridized
+ antibody and is referreclto irs humanized antibody
Antibociyproduction or chimeric antihody
and separation
Substitution of Fv region of
Fig. 17.3 : Production of monoclonal antibodies by human lg by mouse Fv
microencapsulation.
T h e D N A c o d i n g s e q u e n c e sf o r F v r e g i o n s o f
both L and H chainsof human immunoglobulinare
'
For the above reasons,alternative arrangements r e p l a c e db y F v D N A s e q u e n c e( f o r L a n d H c h a i n s )
are made to oroduce human MAbs. These are from a mouse monoclonal antibody (Fig. 17.4A).
briefly described below. The newly developed humanized MAb has Fc
r e g i o n o f l g b e i n g h u m a n . T h i s s t i m u l a t e sp r o p e r
Viral transformation of human
immunological response.
B.lymphocytes
The chimeric antibodies produced in this
B-Lymphocytes,actively synthesizing antibody,
manner were found to be effective for the
are treated with fluorescent-labeled antigen.
destruction of tumor cells in vitro.
The fluorescent-activated cells are separateo.
However, B-cells on their own, cannot grow in Substitution of human lg by
cu lture . This li m it at ion c an be ov er c om e b y mouse GDRs
transforming B-lymphocytes with Epsfein-Bar virus
(EBV).Some of the EBV-transformedcells can grow Cenetic engineers have been successful in
in cu lture a nd pr oduc e m onoc lonal ant ibodie s . d e v eloping human MAbs containing mouse
Unfortunately,the yield of MAb is very low by this c o m p l e m e n t a r yd e t e r m i n i n g r e g i o n s( C D R s ) .T h i s i s
ap pro ach. m a d e p o s s i b l e b y r e p l a c i n g C D R s g e n e s ( C D R ',
CDR2, CDR3) of humans by that of mouse. These
SGID rnotrse for producing human MAbs chinreric antibodies (Fig. 17.48) possessthe antigen
The mouse suffering from severe combined binding affinities of the mouse and they can serve
immu no de ficie nc y ( SCI D) dis eas e lac k s it s nat u r a l as effective therapeutic agents. So far, about 50
immu no log ica l s y s t em . Suc h m ous e c an b e monoclonal antibodies have been produced by this
c ha llen ge d with appr opr iat e ant igens t o pr odu c e a p p r o a t h . H o w e v e r , t h i s t e c h n i q u e i s c o s t l y a n d
h uma n MAb s (For det ails , Ref er Chapt er 17) . time consuming.
218
Fv mouse
(A)
(B)
Fig. 17,4 : Genetically engineered human-mouse
antibodies (A) Substitution of Fv region ol human lg
Bispecific monoclonal antibodies
The M Abs in whic h t he t wo a r m s o f F a b
(antigen-binding)have two different specificitiesfor
two different epitopes are referred to as bispecific
MAbs. They may be produced by fusing two
different hybridoma cell lines (Fig. 17.5) or oy
ge ne tic engineer ing. Bis pec if ic F a b M A b s
the ore t ic ally , ar e us ef ul f or a s im ult a n e o u s a n d
combined treatment of two different diseases.
PRODUCTION OF MAbs lN E. COL,
Th e hy br idom a t ec hnology is v er y l a b o r i o u s ,
expensiveand time consuming. To overcome these
limitation, researchers have been trying to
g en eti c ally engineer bac t er ia, plant s a n d a n i m a l s .
The objective is to develop biareactors for the large
sca le p r oduc t ion of m onoc lonal ant ibo d i e s .l t m a y
be no t ed t hat t he ant igen binding r e g i o n s o f
antibody (Fv or Fab fragments) are very crucrar,
while the Fc por t ion is dis pens able.
B IOTE C H N OLO CY
A schematic representation of the procedure
adopted for the production of functional antibody
fragments is shown in Fig. 17.6, and is brieflv
described.
The mRNA from isolated B-lymphocytes
of either human or mouse is converted to
c D N A . T h e H a n d L c h a i n s e o u e n c e so f t h i s c D N A
are amplified by PCR. The so produced cDNAs
are then cut by restrictionendonucleases.H and L
chain sequences are separately cloned in
bacteriophage vectors. These sequences are put
together and cloned in another bacteriophage
v e c t o r . T h e c o m b i n e d H a n d L c h a i n s ( f o r m i n g Fv
fragment) are screenedfor antigen binding activity.
The specific H and L chains fornring a part of the
plasmid are transformed in E. coli. These E. coli, n
turn, can be harvestedto produce Fv fragmentsto
bind to specific antigens.
SECOND GENERATION MONOCLONAL
ANTIBODIES
In the recent years, a number of improvements
have been made to produce more specific,sensitive
a n d d e s i r e d M A b s . T h i s h a s b e e n p o s s i b l e d u e to
the rapid advantes made in genetic engineering
techniques. For instances, by employing site-
directed mutagenesis, it is possible to introduce
cysteine residuesat the predeterminedpositions on
the MAb. These cysteine residues which facilitate
the isotope labeling may be more useful in
diagnostic imaging and radioimmunotherapy.
Specific cytotoxicity
Specific of T-cells
for tumor
Monoclonal Monoclonal
antibodyA antibodyB
Specificfor
cytotoxicity
of T-cells
Bisoecificmonoclonal
antibody
Fig. 17.5 : Production of a bispecific
 (HY B R ID OMA
Chapt er17 : MON O C L O N ALAN T IBOD IES TE C H N OLOC Y ) 219

mRNAfrom LIMITATIONS OF MONOCLOI{AL

B-lymphocytes ANTIBODIES
I
c DNA
Hybridoma technology is laborious and time
consuming. MAbs are produced against a single
I
PCR amplifies
antigenic determinant, therefore, they cannot
d i f f e r e n t i a t et h e m o l e c u l e a s a w h o l e . S o m e t i m e s ,
H and L chains
t h e y m a y b e i n c a p a b l e o f d i s t i n g u i s h i n gg r o u p s o f
I different moleculesalso
Cuttingby restriction
endonucleases The presence of retrovirusesas a part of the
mammalian chromosomes is a common
occurrence. Mice used in MAb production carry
several viruses (adenovirus, hepatic virus,
r e t r o v i r u s ,r e o v i r u s ,c y t o m e g a l o v i r u s ,t h y m i c v i r u s ) .
The presence of some of these viruses has been
H-chainDNA L-chainDNA detected in the hybridomas. This poses a
sequencecloned sequencectoneo great danger, since there is no guarantee that
in vector in vector MAb produced is totaily virus-free, despite the
p u r i f i c a t i o n . F o r t h i s ! 'e a s o n ,U S F o o d a n d D r u g
Administration insists thal MAb for human use
should be totally free from all pathogenic
organisms, including viruses.
H- and L-chainscombined
and clonedin vector

+
I
Screenfor
antigenbinding

+
I
M o n o c l o n a l a n t i b o d i e sw i t h s p e c i f i c i t ya n d h i g h
lncorporateH and L purity have a wide range of applications,which
sequencein a plasmid
can be broadly categorized as follows.
I
+ 1 Diagnosticapplications
E coiltransformed
with H-L DNA construct 2 Therapeutic uses

+
I 3. Protein purification
4 . M i s c e l l a n e o u sa p p l i c a t i o n s .
Productionof
Fv fragmentin E. coli A summary of the important applications of
MAbs is given in Table 17.1, and briefly discussed
Fig. 17.6 : Production of monoclonal antibodies
below.
in E. coli. ,
1. DIAGNOSTIC APPLICATIONS
ADVANTAGES OF MONOCLONAL M o n o c l o n a l a n t i b o d i e s h a v e r e v o l u t i o n i z e dt n e
ANTIBODIES l a b o r a t o r y d i a g n o s i s o f v a r i o u s d i s e a s e s .F o r t h i s
purpose, MAbs may be employed as diagnostic
Monoclonal antibodies truely represent a
reagents for biochemical analysis or as tools for
homogeneous sfafe of a single molecular species
diagnostic imaging of diseases.
Each MAb is s pec if ic t o a giv en ant ige n i c
d ete rmina nt.This is in c ont r as tt o t he c onv ent io n a r
a ntiseru mtha t c ont ains poly c lonal ant ibodies .T h e lAl MAbs IN BTOCHEMTCALANALYSTS
wide range of applications of MAbs are described Diagnostic tests based on the use o{ MAbs as
Iater. r e a g e n t sa r e r o u t i n e l y u s e d i n r a d i o i m m u n o a s s a y s
220 B IOTE C H N OLO CY

Tmrr 17.1 A sunmary of lmportant appllcatlons of monoclonal antlbodles

i applications
Oiagnostic
(A) Biochemical for the diagnosis
analysis of pregnancy,cancers, disorders,
hormonal diseases.
infectious
imaging(immunoscintigraphy)
(B) Diagnositic of
tor the detection myocardial deep
infarction, veinthrombosis,
cancers,
atherosclerosis, bacterialinfections.

applications
2. Therapeutic
(A) Directuseas therapeuticagents disease-causing
to destroy in thetreatment
organisrns, in the
of cancers,
oforgan
immunosuppression inthetreatment
transplantation, ofAIDS, andautoimmune diseases.
(B) As targetingagentsin thercpyas immunotoxins(forireatment
of cancers), fordissolving
in drugdelivery,
bloodclots, (of
in tadioimmunotherapy tumors).

; ;;i;i; ;;'ri.',i.' ov'"r.'.*''tt''.r,.iqr.'

agents(abzymes),
as catalytic
applications
4. Miscellaneous fingerorinting.
in autoantibody

(RlA) and enz y m e- link ed im m unos o r b e n t a s s a y s lBl MAbs IN DIACNOSTTC TMAG|NG

(ELISA)in the laboratory.These assaysmeasure thq
R a d i o l a b e l e d - M A b s a r e u s e d i n t h e d i a g r r o sti c
circulat ing c onc ent r at ions of hor m o n e s ( i n s u l i n ,
i m a g i n g o f d i s e a s e s ,a n d t h i s t e c h n i q u e i s r e f er r e d
hu man c hor ionic gonadot r opin, gr ow t h h o r m o n e ,
to as immunoscintigraphy. The i-adioisotopes
pro ge s t er one,t hy r ox ine, t r iiodot hy r o n i n e , t h y r o i d
commonly used for labeling MAb are iodine--l 31
stimu lat ing hor r none, gas t r in, r enin) , a t r d s e v e r a l
a n d t e c h n e t i u m - 9 9 . T h e M A b t a g g e d , wi th
o the r t is s ue and c ell pr oduc t s ( b l o o d E r o u p
radioisotope are injected intravenously into the
antigens, blood clotting factors, interferons, patients.
These MAbs localise at specific sites (say
in ter leuk ins , his t oc om pat ibilit y anti g e n s , t u t n o r
a tumor) which can be detected by imaging the
rnarkers).
radioactivity. In recent years, single photon
ln recent years, a number of diagnostic kits emission computed tomography (SPECT) cameras
u sin g M Abs hav e bec om e c om m er c i a l l y a v a i l a b l e . are used to give a more sensitivethree dimensional
For i ns t anc e, it is now pos s ible t o d o t h e e a r l y appearanceof the spots localized by radiolabeled-
dia gn os isof t he f ollowing c ondit ion s / d i s e a s e s . MAbs.
Pregnancy-by detecting the urinary levels of l m m u n o s c i n t i g r a p h yi s a b e t t e r d i a g n o s t i c to o l
h uma n c hor ionic gonadot r opin. t h a n t h e o t h e r i m a g i n g t e c h n i q u e ss u c h a s C T sca n ,
Fo r
Cancers----estimation of plasma carcino- u l t r a s o u n d s c a n a n d m a g n e t i c r e s o n a n c e .
immunoscintigraphy can differentiate
e mbr y onic ant igen in c olor ec t al c a n c e r , a n d instance,
growth,
prostatespecific antigen for prostatecancer. Besides between cancerous and non-cancerous
since radiolabeled-MAbs are tumor specific. This
diagnosis,estimationof tumor markersis also useful
i s n o t p o s s i b l e w i t h o t h e r i m a g i n g t e c h n i q u e s.
fo r the pr ognos isof c anc er s .That is a g r a d u a l f a l l
in a specific tumor ntarker is observed with a M o n o c l o n a l a n t i b o d i e s a r e s u c c e s s f u l l yu s ed i n
red uc t ion in t um or s iz e, f ollowing t r e a t m e n t . t h e d i a g n o s t i c i m a g i n g o f c a r d i o v a s c u l a rd i s e a se s,
Hormonal disorders-analysis of thyroxine, cancers and sites of bacterial infections.
triio dbt hy r onine and t hy r oid s t im ula t i n g h o r m o t r e
fo r thy r oid dis or der s . Cardiovascular diseases

lnfectious diseases-by detecting the circulatory M y o c a r d i a l i n f a r c t i o n : T h e c a r d i a c p r o t6 i n

levels of antigens specific to the infectious agent myosin gets exposed wherever myocardial necrosis
e.g., antigensof Neisseriagonorrhoeae and herpes ( d e a t h o f c a r d i a c c e l l s ) o c c u r s . A n t i m y o s i n M Ab
simp lex v ir us f or t he diagnos is o f s e x u a l l y l a b e l e d w i t h r a d i o i s o t o p ei n d i u m c h l o r i d e ( 1 1 1I n ) i s
transmitted diseases. used for detecting myosin and thus the site of
C h A P I CT (H YB RID OMA
17 : M O NO C L O N ALAN T IBOD IES TE C H N OLOC Y )
Tumor tnarker
Carcinoembryonic (CEA)
antigen
Alphafetoprotein
Human gonadotropin
chorionic
Prostatic
acidphosphatase
growthfactorreceptor
Epiderminal
Tumor--associated
cellsurface
antigens
myocard ial in farction. lm aging of r adiolabeled
MA b, is u su ally d one af t er 24- 48 hour s of
int raven ou sa dmin is t r at ion.This is c ar r ied out eit her
by p lan ne r ga mma c am er a or s ingle phot on
emission co mpu ted t om ogr aphy ( SPECT) . lt is
possible Io detect the location and the deg,ree of
damage to the heart by using radiolabeled
antimyosin MAb. fhus, this technique is useful for
the diagnosis of heart attacks.
Deep vein thrombosis (DVT) : DVT refersto the
f ormatio n o f b loo d c lot s ( t hr om bus ) wit hin t ne
blood vein s, prima rily in t he lower ex t r em it ies .For
the detection of DVI radioisotope labeled MAb
directed against fibrin or platelets can be used.
T he ima gin g is u s ually done af t er 4 hour s of
injectio n. Fibrin sp ec if ic M Abs ar e s uc c es s f ully
used fo r th e de tection of c lot s in t high, pelv is , c alf
anci knee regiorrs.
Ath ero sclero sis : Thic k ening and los s of
elasticity of arterial walls is referred to as
at hero sclero sis. Ath er os c ler ot ic plaques c aus e
dise ases o f co ron ar y and per ipher al ar t er ies .
A t hero sclero sis ha s been im olic at ed in t ne
development of heart diseases.MAb tagged with a
radiolabel directed against activated platelets can
be used to localize the atherosclerotic lesions by
imaging, technique.
Gancers
Mon oclon al an tibodies agains t m any t y pes of
huma n can ce rsare n ov v av ailable.A s elec t edlis t of
tumor markers (along rvith the associatedcancers)
t hat can be u se d for M Ab im aging is giv en in
Table 17.2. Tumors can be located in patientsusing
radioisotopelabeled MAbs specificto the protein(s),
particu larly o f me m br ane or igin. I t has been
possible to detect certain cancers at early stages
221
the associated.un."r, useAii MAbimaging
Assoaiated cancerG)
Cancers
of colon,siomach,pancreas
Cancers
of liver,andgermcellsof testes
Choriocarcinoma
Prostate
cancer
Melanoma
Various
cancers
(lung cancer/ breast cancer/ ovartran cancer/
m a l a n o m a , c o l o r e c t a lc a n c e r )b y e m p l o y i n g N l A b s
About B0 per cent specificity has been achieved for
detecting cancers by this approach
A n i o d i n e ( 1 3 1 1 )l a b e l e d m o n o c l o n a l a n t i b o o y
s p e c i f i ct o b r e a s tc a n c e r c e l l s w h e n a d m i n i s t e r e dr <t
the patients detects (by imaging) the spread of
cancer (metastasis)to other regions of the body.
This is not possibleby scanningtechniques
The imaging technique by using MAb can also
be used to monitor therapeutic responses of a
cancer.There are certain limitations in using MAb
i n c a n c e r d i a g n o s i s a n d p r o g n o s i s .T h e s e i n c l u d e
t h e d i f f i c u l t y i n t h e s e l e c t i o no f a s p e c i f i c M A b a n d
the access of MAb to the target site of the tumor
which may be lessvascularized.
MAbs in immunohistopathology of cancers :
The pathological changes of the canceroustissue
can be detected bv immunohistochemical
t e c h n i q u e s .T h i s c a n b e d o n e b y u s i n g M A b a g a i n s t
a specific antigen.
MAbs in hematopoietic malignancies :
Hematopoietic stem cells in bone marrow are
the precursors for different blood cells, B- and
T-lymphocytes which are produced in a stepwise
t r a n s f o r m a t i o n .D u r i n g m a l i g n a n c y ,t r a n s f o r m a t i o n
of lymphocytes stops at a particular stage of
maturation. This can be detected by using stage-
specific MAbs.
Bacterial infections
In recent years, attempts are made to detect the
sites of infections by using MAbs. This is made
possible by directing MAb against bacterial
antigens. Further, monoclonal antibodies against
inflammaton, leucocvtes which accumulate ar
222 B IOTE C H N OLO G Y

infectionsite are also usefulto specificallydetect bone marrow cells, due to non-availabilityof a
localizedinfections. sui tabl edonor).

2. THERAPEUTIC APPLICATIONS Limitations for direct use of

MAbs in cancer
Monoclonal antibodieshave a wide range of
therapeuticapplications.MAbs are used in the 1. The MA bs producedi n mi ce and di rect ly
treatmentof cancer,transplantation
of bone marrow used for therapeuticpurposesmay lead to the
and organs,autoimmunediseases,cardiovascular development of antimouse antibodies and
reactions.
diseasesand infectiousdiseases.The therapeutic hypersensitivity
applicationsof MAbs are broadlygroupedinto 2 2. All the cancercells may not carry the same
types. antigenfor which MAb has been produced.Thus,
(A)Direct use of MAbs as therapeuticagents MAbs may not be attachedto somecancercellsat
al l .
(B)MAbs as targetingagents.
3. The free antigens (of target cells) present in
lll MAbs AS DTRECT THERAPEUTTC the circulation may bind to MAbs and prevent them
AGENTS from their action on the target cells.

Monoclonalantibodiescan be directlyusedfor In the immunosuppression of

enchancing the immunefunctionof the host.Direct organ transplantation
use of MAbs causesminimal toxicity to the target
tissuesor the host. l n the normal medi cal practi ce, i mmun o-
suppressi vedrugs such as cycl ospori n an d
In destroying disease-causing prednisone are administeredto overcome the
organisms rejectiohof organtransplantation. ln recentyears,
MAbs specific to T-lymphocyte surface antigensare
MAbs promote efficient opsonization of
bei ng used for thi s purpose.The monocl on al
pathogenicorganisms(by coating with antibody)
antibody namely OKTt, was the firsf MAb to be
and enhance phagocytosis.In fact, MAbs were for
licensedby U.S.Food and Drug Adminstration
found to protect chimpanzesagainst certain viral
use as immunosuppressive agent alter organ
(hepatitis B-virus) and bacterial (E. coli
transpl antati oni n humans. OK T3 speci fi ci a lly
Haemophilus influenza, Streptococcus sp and
directedagainstCD, antigenof T-lymphocytes is
Pseudomonas sp) infections.
successfullyused in renal and bone marrow
transplantations.In the normalcourse,CD, antigen
ln the treatment of cancer
activatesT-lymphocytes and plays a key role in
MAbs, against the antigens on the surface of organtransplantrejection(destroys the foreigncells
cancer cells,are usefulfor the treatmentof cancer. in the host). This is preventedby use of MAb
T he ant i b o d i ebs i n d to th e c a n c e rc e l l sa nd destroy agai nstC D , anti gen.
them. This is broughtout by antibody-dependent
cell-mediatedcytotoxicity,complement-mediatedln the tleatment of AIDS
cytotoxicity and phagocytosisof cancer cells
l mmunosuppressi ion s the hal l mark of A ID S.
(coatedwith MAbs) by reticuloendothelial system.
Thi s i s caused by reducti on i n C D o (cl ust er
The patientssufferingfrom leukemia,colorectal
determinantantigen4) cellsof T-lymphocytes. The
c anc er , l y m p h o m a a n d me l a n o m a have been
human i mmunodefi ci ency vi rus (H l V ) bi nds t o
treatedwith MAbs. However,there was a wide
specificreceptorson CDo cells by using surface
v ar iat io n i n th e s u c c e s sra te . A monocl onal
membraneglycoprotein(gp120). Ceneticengineers
ant ibod vs p e c i fi cto th e c e l l so f l e u k e mi ai s usedto
have been successful to attach Fc portion of mouse
des t r o yth e re s i d u alle u k e m i ac e l l sw i th outaffecti ng
monoclonal antibody to human CDn molecule.
ot herc e l l s .
Thi scompl exhashi ghaffi ni tyto bi nd to membrane
MAbs are used ln vitro to remove the residual glycoprotein9p120o( virus infectedcells.The Fc
tumor cells prior to autologousbone marrow fragment induces cell-mediated destruction of HIV
transplantation (transplantation of the patient'sown infected cells (Fig. 17.7).
 (HY B R ID OMATE C H N OLOC Y )
Chapt er17 : M ON O C L O N ALAN T IBOD IES 223

\/

gpl20

-----* Cell-mediated
lysis
Fc
Fc

T-cellsinfected
with HIV

Fig. 17.7 : Modified monoclonal antibody in the treatment of AIDS.

ln the treatment of autoimmune Ricin is a cvtotoxic protein derived from castor

diseases o i l p l a n t . l t i s c o r n p o s e do f t w o p o l y p e p t i d e c h a i n s
( A a n d B ) h e l d t o g e t h e r b y a d i s u l f i d e l i n k a g e .T h e
A ut oim m un ed i s e a s e sl i k e rh e u ma to i da rthri ti s
B-chain of ricin binds to the cell surface. This
and multiplesclerosisare of greatconcern.Some
b i n d i n g f a c i l i t a t e st h e A - c h a i n o f r i c i n t o e n t e r t h e
successhas been reported in the clinical trials
cell and inhibit the function of ribosomes (r.e.
of rheumatoid arthritis patients by using
b i o s y n t h e s i so f a l l p r o t e i n s i s b l o c k e d ) . T h i s r e s u l t s
MAbs directed against T-lymphocytes and
in the death of cells (Fig, 17.8A). Ricin can be
B-lymphocytes.
subjected to oxidation to separate to A and B
chains. The toxic A-chain can be conjugated to
lBl MAbs AS TABGETTNG AGENTS tw MAb that is specific to cancer cells. The tumor-
T}lERAPY
specific MAb bound to A-chain of ricin binds ro
Toxins, drugs, radioisotopesetc., can be c a n c e r c e l l s a n d n o t t o n o r m a l c e l l s . O n c e t h e
attached or conjugated to the tissue-specific A-chain enters the cells, it blocks ribosomal
monoclonalantibodiesand carriedto targettissues function, leading to the death of cancer cells
f or ef f ic ient a c ti o n . T h i s a l l o w s h i gher (Fig. 17.88).
concentrationof drugs to reach the desiredsite
wit h m inim alt o x i c i ty .In th i s w a y , M Ab s a re used MAbs in drug delivery
for the appropriatedeliveryof drugsor isotopes.
ln general,the drugs are lesseffective in vivo (in
the living body) when compared to in vitro (in
MAbs in use as immunotoxins l a b o r a t o r yw h e n t e s t e d w i t h c u l t u r e d c e l l s ) . T h i s i s
The toxins can be coupledwith MAbs to form mainly due to the fact that sufficient quantity of the
im m unot ox inasn d u s e di n th e ra p ye .g .,d i p h th eri a drug does not reach the target tissue.This problem
toxin, Pseudomonasexotoxin, toxins used for c a n b e s o l v e d b y u s i n g t i s s u e - s p e c i f i cM A b s . T h e
cancertreatment. drugs can be coupled with MAb (directed againsta
cell surfaceantigen of the cells, say a tumor) and
Anti-TacMAb raisedagainstlL2-R(T-cellgrowth
specifically targeted to reach the site of action
factorreceptor)can be conjugatedwith exotoxinof (Fig.
17.eA).
Pseudomonas sp. This immunotoxincan be usedto
des t or y t he m a l i g n a n t T -c e l l s i n th e p a ti ents In the treatment of certain diseases,a prodrug
sufferingfrom T-cell leukemia (Note : lL2-R is (an inactive form of the drug) can be used. This can
ex pr es s edin a b n o rm a l T -c e l l s w i th l y mphoi d be enzymatically converted to active drug in the
m alignanc ies). target tissues. For this purpose, the enzyme (that
224 B IOTE C H N OLO CY

converts prociruBto drug) is coupled with MAb that

(A) i s d i r e c t e d a g a i n s t a s p e c i f i c c e l l s u r f a c e a n t i ge n
(Fig. 17.9A). This approach, referred to as
antibody-directed enzyme prodrug therapy
(ADEPTI, allows an effective delivery of the drugto
the cells where it is required.

T h e f o l l o w i n g a r e s o m e e x a m p l e s o f e n z y me s
that have been used in ADEPT.

. Alkaline phosphatase for the conversion of

p h o s p h a t ep r o d r u g s .
. Carboxy peptidase for converting i n a c ti ve

I
Normal cell death
carboxyl prodrugs to active drugs.
o Lactamase for hydrolysing B-lactam ring
containing antibiotics.

MAbs in the dissolution of blood clots

'" ' E l € - o x i d a t i o n , E . € A great majority of natural deaths are due to a

J
Discarded
blockage in cornary or cerebral artery by a blood

(A) {-Drug

Tumor-specific Conjugation
MAb Monoclonal
antibody

Cell surface
protein

(B)
--'-}- Enzyme
Prodrug Drug

Monoclonal
antibody

Cell surface
protein

Fig. 17.8 : Use of ricin as a cytotoxic agent

(A) Action of ricin on a normal cell; Fig. 17.9 : Monoclonal antibody based drug delivery
(B) A-chain of ricin conjugated to MAb to the target cells. (A) The drug is bound to MAb
causing cancer cell death (B) The enzyme that converls prodrug to drug is
(Note : A-chain of ricin cannot enter normal celi) bound to MAb.
 (H Y B R ID OMA
Chapt er17 : M O N O C L O N ALA N T IB O D IES TE C H N OLOC Y )
Monoclonal
antibody
Fig. 17.10 : Monoclonal antibody in the dissolution
of blood ctot (tPA-Tissueplasmlnogen aetivato).
clot (thro mbu s ) .Fibr in is t he m ajor c ons t it ue n t o f
blo od clot whic h get s dis s olv ed by plas m i n .
Plasmin in tu r n is f or m ed by t he ac t iv at io n o f
pla smin og en by plas m inogen ac t iv at or T h e
blockage of arteries occurs due to inadequate
d issolu tion o f blood c lot s . Tis s ue plas m in o g e n
activator (tPA)can be used as a therapeutic agent to
remove the blood clots.
A monoclonal antibody directed against fibrin
can be coupled to tPA and used for degradation of
blood clots. MAb-tPA complex due to a high
affinity gets attached to fibrin (Fig. 17.10). Due to
the concentration of tPA at the target spots,there is
more efficient conversion of plasnrinogen to
pla smin wh ich in t ur n dis s olv esblood c lot ( f ib r i n )
Good success of clot lysis has beer.rreported by
u sin g MAb-tP A c om plex in ex per im ent al anim a l s .
Drug delivery through liposomes
coupled to tissue,specific MAbs
Liposomes afe sacs or vesicles formed
sp on tan eo usly when c er t ain lipid m olec ules a r e
expo se d to a queous env ir onm ent . Dr ug ent r a p p e d
in liposomes that are coated with MAbs directed
ag ain st tissu e- s pec if icant igens ar e being t r ied f o r fact that a small quantity of MAb leaks into the
drug delivery. Unfortunately, the progress in this e l u t i o n . F u r t h e r ,M A b s c a n n o t d i s t i n g u i s h b e t w e e n
a pp roa ch h as been lim it ed, s inc e s uc h iiposo m e s the intact target protein and a fragn.rentof it with
do not reach the target cells. They are retained t h e a n t i s e n i c s i t e .
Biotechnology [15]
225
mostly in the liver and spleen (reticuloerrdothelial
cells), and degraded.
MAbs in radioimmunotherapy (RAlTl
f he radioisotopes can.be coupled to MAbs that
are directed against tumor cells. This allows the
concentration of radioactivity at the desired sites
and a vety efficient killing of target cells (tumor
c e l l s ) . T h e a d v a n t a g ew i t h r a d i o i r n m u n o t h e r a p yi s
that conjugated complex need not penetrate the
c e l l s , a s i s r e q u i r e d i n i m r r r u n o t o x i nt h e r a p y . T h e
l i m i t a t i o n i s t h a t t h e n e i g h b o u r i n gn o r m a l c e l l s r n a y
aiso get damagedor killed. This can be minimized
by using radioisotopes with short half-lives.
Y t t r i u r n - 9 0 w i t h a h a l f - l i f eo f 6 4 h o u r s i s a s u i t a b l e
isotope to be employed in RAIT. Due to shortagern
the srrpply of yttrium-90, indium-l 11 is more
commoniy used.
3. PROTEIN PURIFICATION
M o n o c l o n a l a n t i b o d i e sc a n b e p r o d u c e d f o r a n y
protein. And the so produced MAb can be
c o n v e n i e n t l y u s e d f o r t h e p u r i f i c a t i o no f t h e p r o t e i n
a g a i n s tw h i c h i t w a s r a i s e d .M A b s c o l u m n s c a n b e
prepared by coupling them to cyanogen bromide
activated Sepharose(chromatographicmatrix). The
immobilized MAbs in this manner are very useful
for the purification of proteins by immunoaffinity
method.
Advantages
There are certain advantagesof using MAbs for
p r o t e i n p u r i f i c a t i o n .T h e s e i n c l u d e t h e s p e c i f i c i t yo f
the MAb to bind to the desired protein, very
e f f i c i e n t e l u t i o n f r o m t h e c h r o m a t o g r a p h i cc o l u m n
and high degree of purification. lmnrunoaffinity
chromatography is routinely used for the
ourification of recombinant interfcrons. The
e f f i c i e , . r c yo f t h i s t e c h n i q u e w i l l b e o b v i o u s f r o m
the fact that by a single step, it is possible to
achieve more tl-ran 5.000 fold ourification of
interferon-cr.
Disadvantages
I t i s n o t p o s s i b l et o a c h i e v e 1 0 0 % p u r i y o f t h e
target protein by immunoaffinity.This is due to the
226 B IOTE C H N OLO CY

4. MISCELLANEOUS APPLICATIONS L e r n e r a n d h i s a s s o c i a t e sc a r r i e d o u t p i o n e e ri n g
CATALYTTC MAbs (ABZYMES, work in the development of abzymes. They could
c r e a t e a l a r g e n u m b e r o f i m m u n o g l o b u l i n - ge n e
Catalysis is the domain of enzymes. The most l i b r a r i e sf o r t h e p r o d u c t i o n o f a n t i b o d i e st h a t c o u l d
important common chdracter between enzymes be screened for their catalvtic function. Abzymes
and antibodies is that both are proteins. Further.rne represent a major biotechnological advancement
bin din g of an ant ibody t o it s ant igen is c o m p a r a b l e t h a t w i l l h a v e a w i d e r a n g e o f a p p l i c a t i o n s( c u t ti n g
to the binding of an enzyme to its substrate.In both of peptides and DNAs, dissolution of blood clots,
in sta nc es ,t he binding is s pec if ic wit h h i g h a f f i n i t y k i l l i n g o f v i r u s e se t c . )
and involves weak and non-covalent interactions
(electrostatic,hydrogen and van der Waals forces).
Advantages of abzymes
The striking difference is that the enzyme alters the
substrate(to a product) while the antigen bound to T h e n u m b e r o f n a t u r a l l yo c c u r r i n g e n z y m e s a n d
an tibo dy r em ains unalt er ed. t h e i r c a t a l y t i c f u n c t i o n s a r e l i m i t e d . A n t i b o d i e s ,o n
and may be
Certain similarities between enzyme-substrate t h e o t h e r h a n d , a r e u n l i m i t e d ,
developed to possess recognized site structures,
interaction and antibody-antigen interaction have
tempted researchersto explore the possibility of appropriate for the catalytic functions. This makes
using ant ibodies in c at aly s is . T h e a n t i b o d y a b z y m e s v e r s a t i l e w i t h w i d e r a n g i n g c a t a l yti c
technology
enzymes, appropriately regarded as abzymei, are applications. Thus, the area of al:zyme
i s v e r y promising, although the studies are at
the catalytic antibodies.
preliminary stages.
There is a difference in the antibody recognition
of an antigen and enzyme recognitionof a substrate.
Limitations of abzymes
While the antibodies recognize in ground sfate, the
enzymes recognize in a transition state (associated Despite the progress made in the production
with a conformational change of protein). In fact, it and utility of abzymes, it is doubtful whether they
is in t he t r ans it ionc ondit ion t he c at al y s i so c c u r s . l f w i l l e v e r m a t c h t h e n a t u r a l e n z y m e s i n . t he i r
a molec ule r es em bling t he t r ans it i o n s t a t e a n d catalytic function. However, the abzymes will be
conformation (betweensubstrateand product)could certainly useful for a variety of reactionswhere the
be used as a hapten the antibodies so produced natural enzymes do not have the desired
sh ou ld br ing about c at aly s is .This is w h a t p r e c i s e l y s p e ci f i c i t i e s .
is done to create abzymes.

Researchers have produced a hapten-carrier AUTOANT' BODY F' NG EBPBI NT ING

complex which resembles the transition state of
T h e o c c u r r e n c e o f a u t o a n t i b o d i e s a n d t he r r
an ester undergoing hydrolysis. This hapten
i n v o l v e m e n t i n c e r t a i n d i s e a s e si s w e l l k n o w n ( e.9 .
conjugate is used to generate anti-hapten
rheumatic arthritis).A new category of individual
monoclonal antibodies. These MAbs could bring
specific (lS) autoantibodies have been discovered
about hydrolysis of esters with great degree of
in recent years. These lS-autoantibodies are
specificity (to the transition state to which MAbs
oroduced after birth and reach maximum in
were raised).
number bv 2 years, and then remain constant for
Besidesester hydrolysis,there are several other t h e l a t e r o a r t o f l i f e . M o n o c l o n a l a n t i b o d i e s
types of reactionswherein antibodies can be used. produced against lS-autoantibodiescan be used for
Th ese inc lude hy dr oly s isof am ides an d c a r b o n a t e s , their detection, and identification of individuals.
cycliz at ion r eac t ions , elim inat ion r e a c t i o n s a n d This technique referred Io as autoantibody finger-
bio molec ular c hem ic al r eac t ions .Cer t a i n e n z y m e s printing, is particularly useful for the detection of
require cofactors for their catalytic function. MAbs criminals, rapistsetc. The autoantibodiescollected
incorporating metal ions have been developed to from samples such as blood, saliva, semen and
carrv out catalvsis. tears can be used.
J raditiorral methods of breeding animals Thus, the ruminant female manrmals cannot
',rrere
I o nly mea ns of im pr ov ing t he genet ic qua l i t i e s . produce more than one offspring in a year, in the
Despite the several advances made in normal circumstances.
bio techn olo gy ,genet ic m anipulat ionsin r elat i o n t o
The current approaches for the artificial
rep rod uction of anim al and hum ans ar e v e r y
breeding of animals with particular reference to the
limited for ,obv ious r eas ons , t he m os t im po r t a n t
following are briefly described.
be ing the un k nown r is k s inv olv ed in t he out c o m e
. Artificial insemination.
The manipulations of reproduction in animals and
humans are collectively considered under assisted . Embryo transfer.
reproductivetechnology(ARI), and dealt with in this o ln vitro fertilization.
chapter. The objective of ART is to increase the
. Embryo cloning.
number of progeny of the desired species.

Some o f the im por t ant as pec t sof m anipul a t i o n ARTIFICIAL INSEMINATION

o f re pro du cti on in anim als ar e f ir s t dealt wit h, a n t l
As the male produces millions of sperms
th en the sa lientf eat ur esof m anioulat ionsin hu m a n
daily, the semen can be used to produce several
reoroduction are described
offsprings. This is made possible by artificial
i n s e m i n a t i o nt A l ) o f i e m a l e s . F o r a n e f f e c t i v eA l t o
produce the desired results,the following aspects
must be considered.

Semen collection and storage

The semen ejacuiate is collected, appropriately

All th e m ale m am m als or oduc e m illion s o f
diluted and examined under nricroscopefor the
spe rms (male gam et es )dailv and t hus t heor eti c a l l y ,
n u m b e r o f m o t i l e s p e r m s .N o r m a l l y , 0 . 2 m l o f b u l l
can insemin at e m any f em ales t o pr oduc e a l a r g e
sernen contains about 10 million motile soerms.
number of offsprings On the other hand, the female
The diluted seinen can be used fresh rryithinfew
ma mmals pro duc e a lim it ed num ber of eggs( f e m a l e
d a y s , o r c r y o p r e s e r v e da t - 1 9 6 'C i n l i q u i d n i t r c l g e n
g ame ts), usua lly , one at a t ir ne ( ex c ept ior r - p i g ) ,
for long-term storage and transport
a pp roxinra tel yat m ont hly int er v als .The f em ale a l s o
h as th e re sp ons ibilit yof dev eloping t he f er t i l i z e d Artificial insemination is done on standing
e gg , b eside spr ov iding nour is hm entt o t he new b o r n . animals through a technique, known a rectal
224 B IOTE C H N O LO CY

palpation.Eachsemenejaculateof a male bull, in cell sorter (FACS),two populationsof sperms(X or

theory;can be usedto inseminate as many as 500 Y chromosome containing)can be separated. Using
cows. Anotheradvantageof Al is that the semen this approach and in vitro fertilizationtechnique
can be transported(in cryopreservedform) to has producedpre- sexedcalves.
different places/countries and used to develop
FACSseparationof spermsis expensiveand
s uo e ri o a
r n i ma l s .
time (about 24 hours)-consuming. Many sperms
may die before they are actually separated.
Synchronization of ovulation
Attempts are being made to develop better
The female animals are inseminatedafter separation techniquesof sperms.
ovulation which can be detected by behavioural
estrous.lt is ratherdifficultto detectestrous,since EMBRYO TRANSFEB
it mostlyoccursduring night and lastsfor only a
As the ruminant animal producesone egg at
few hours.
time, it can carry one pregnancyat a time. lt is
T h e a n i m a lb re e d e rs w o u l d l i k e t o i nsemi nate a possible to increasethe production of female
largenumberof femalessimultaneously, the numberof matureeggs
so that the ani mal sby i ncreasi ng
managementbecomeseasy.For this purpose,the from a given female, fertilize them and transfer
femalesare induced(synchronized) to ovulateat a (implant)the embryos(fertilizedeggs)into a foster
set time. This is achieved by administrationof mother (recipien}. The fostermother servesas an
pfogesterone and/orprostaglandins, which regulate incubator, and does not make any genetic
ov u l a ti o n c y c l e . A l th o u g h to ta l synchronyof contribution to the offspring.
ovulationis not possible,aboutBO%o{ the females
Embryotransferis a costly technique,and is
could be made to respondby this approach.
selectivelyused for the productionof animalsof
high geneticor economicvalve.
Sperm sexing
The livestockindustriesoreferto have animals Superovulation (multiple ovulationf
belongingto one sex. For instance,the dairy and
In the normal reproductivecycle of a non-
bee fi n d u s tri edse ma n dm o refe m a l es thanmal es.l t
pregnantfemale,one ovarianfollicle(outof the 20
is possible to produce the animals of desired sex
that develop)maturesand ruptures,releasingone
throughspermsexing.
fertileeggat a time. The time of ovulationvariesin
Sperms and ova contain half of the differentanimals-21 daysfor cow and horse;16
chromosonres of a somaticcell, Thus, an ovum daysfor sheepdnd goat.
c on ta i n sa u to s o m easn d o n e X c h ro mosome w hi l e
The circulating gonadotrophic hormone is
the sperm contains autosomes and one Y
closely associatedwith ovulation and releaseof
chromosome. Sex is determinedgeneticallyby the
egg. By increasingthe concentrationof this
sex chromosomes(X and Y). X chromosomeis
hormone, more ovarian follicles can be induced to
presentin all the ova, whereashalf of the sperm(of
ripen and produce more eggs.This process,known
a semenejaculate)possessX and the other half
as superovul ati on or mul ti pl eovul ati onmay yield
Y chromosomes.The sex of the embryo is
not less than B-.1 0 eggs at a time. Some animal
det e rm i n e bd y th e s p e rm(X o r Y c o ntai ni ng)
that i s
breederswere successful in superovulating animals
successfulin fertilization.Thus, if the embryo
to yield as many as 60 eggsat a time. This largely
containsX chromosome,it is a female;while it is
dependson the breed,nutritionand healthof the
a male if it possesses Y chromosome. In the natural
animal,besidesthe environmental factors.
breeding,the sex ratio of progenyis closeto 1:1.
B y admi ni steri ng prostagl andiFr" n (P C Fru) and
It is possibleto separatethe spermscontaining
hormone(FS H ),estrouscan be
fol l i cl esti mul ati ng
eitherX or Y chromosome, and usethemselectively
i nduced.A rti fi ci ali nsemi nati on i s carri edout i n t he
for the desiredsex of the progeny.
superovulated females.Al is preferredto natural
By employinga fluorescent dye (Hoechst33342) mating,since the purposeof superovulation is to
and an instrument namelv fluarescent activated geneticallyimprovethe progeny.
 Chapt eT
1B : AS SIS T ED
R E PR O D U C T IVE
T EC H N oLocY 229

Fertilization Embryo Two cell stage 8-cellmorulastage Blastocytestage

(0 day) (1day) (2 day) (3 day) (a day)

Fig. 18.1 : The early stages of embryo development.

As the eggs are fertilized, they undergo a n d b o v i n e s e r u m a l b u m i n ( B S A )c o n t a i n i n gc u l t u r e

'f h i s
developmentto form embryos(Fig. lS.1). medium. resultsin shrinking of embryo cells
and settling of the embryos to the bottom of the
Multiple ovulation with embryo transfel container (usually a petridish) due to increased
(MOEr) density. Further, the embryos stick to the bottom
Some times , t he pr oc es s of m ult iple ov ul a t i o n due to electrostatic interaction of the negative
(superovulation described above) and embryo c h a r g e s o n i t ( d u e t o a t t a c h m e n t o f a l b u m i n ) a n d
transferare consideredtosether which is referredto t h e p o s i t i v e c h a r g e so f t h e p e t r i d i s h .T h e i n n e r c e l l
as MOET. mass (lCM) of the blastocyte can be bisected by
using a surgical blade and a micromanipulation
The embryos developed in the superovulated
technique (with the lrelp of an inverted
animals (described above) are recovered after 6-6
microscope). Splitting of each embryo results rn
days of insemination.In case of cattle, the recovery
two ectuai halves.
pro ce ss is ea s y and c an be done by us i n g a il

ca the ter. For c er t ain anim als wit h s m a l r e r As the embryos are split, each hemispherical I
reproductive tract, surgical procedures may be cell mass reforms spheres.These split embryos can {
needed to expose the oviduct and recover the be transferred into the oviducts of synchronized
embryos. These embryos are examined recipients The pregnancy rate with split embryos is
microscopically and icjentified. a b o u t 5 - 1 0 % l e s st h a n t h e t h e o r e t i c a lc a l c u l a t i o n s .
Thus, about 9 progencies (insteadof only 5 in the
The embryos are then transferred into a
original embryo transfer)can be developed through
synchronized recipient (i.e. the foster mother) by
MOET
using a pro ce dur e,whic h is c om par ablet o ar t i f i c i a l
insemination. Alternately, the embrvos can oe By MOET, it is possible to produce at least four
frozen and storedfor use at an appropriatetime and i d e n t i c a l o f f s p r i n g si n a n i m a l s a t a t i m e .
place later. About 50-60% of pregnancy could be
achieved in cattle with transferredembryos.Thus, a Embryo biopsy
superovulatedfemale may result in 5-6 pregnancies.
Embryo biopsy involves the removal of a ferv
The enrbryos may also be subjected to cells frorn the embryo (mostly from the trophoblast
ma nip ula tion s .as des c r ibed below. cells of the trophectoderm)for analysisto determine
t h e s e x . I n r e c e r r ty e a r s ,t h i s t e c h n i q u e i s b e c o m i n g
Embryo splitting popular for the detection of genetic diseases.By
It is po ssiblet o inc r eas et he num ber of pr og e n i e s using embryo biopsy, it is possible to stop tl're
(a lmost to do uble) by s plit t ing t he em br y o s . I n transferof embryos with genetic abnormalitiesand
a dd ition , e mbr y o s plit t ing r es ult sin ident ic al t w r n s undesirable traits.
for various purposes(particularlygenetic researcn).
Embryo sexing
Embryo splitting techniques have been refined,
and are routinely used these days. The embryos are Before the transfer or implantation of the
su sp en de d in a hy per t onic ( high os m ot ic ) s u c r o s e embryo, the progeny sex can be determinecl.
230 B IOTE C H N OL O CY

E m b ry o s e x i n g i s b a s e d o n th e pri nci pl e of
detecting the presence or absence of Y
c hr o mo s o m ei n th e e mb ry o b i o psy cel l s. The
absence of Y chromosome indicates that the
embryo is a female.
In recentyears,the presenceDNA sequences
specificto Y chromosomeare being detectedfor
embryosexing.Anotherdevelopmentis the use of
polymerasechain reactionto amplify the DNA
sequence(of Y chromosome)even from a single
c ell a rrdd e te rm i n a ti oth
n e sei.
The occurrenceof a large number of antral
folliclesthroughoutreproductive life in the ovaries
of cattle and sheepare known. lt is theoretically
possibleto remove them by biopsy and induce
oocyte maturationin vitro. This may ultimately
resultin the productionof hundredsand thousands
of eggsfrom a singlefemale.However,the potential
of in vitro maturationof oocvtes is limited for
variousreasons-degeneration of follicles,unknown
metabol i c
and hormonalcondi ti ons.
Fertilization of eggs

Limitations of embryo transfer ln vitrofertilizationof the eggsis carriedout by

usi ngsemenobtai nedfrom a superi ormal eani mal.
r The supplyof embryofrom superovulated donors For lVF, the eggs are carried in small droplets
is l i m i te d . (microdroplets) of culture medium. Each
. Freezingand thawingof embryosrequiresa lot of mi crodropl et usual l ycarri esabout10 eggs.A dose
care to keepthem functionallyintact. of spermapproxi matelone y mi l l i oncel l sper ml is
. Er-nbryo adequate for lVF. The penetration of the sperminto
transferrequirestechnicalskill, besides
high cost factor. the egg i s faci l i tated
by suppl ementi the medium
ng
w i th certai n compounds (peni ci l l am ine,
TN V'TBO FERTILIZATION epi nephri ne etc).

The limitationsof embryo transfercould be Embryo culture

successfully overcomeby usingin vitrofertilization
(lVF).IVF basicallyinvolvesfertilizationof oocytes The IVF embryosmust be maintainedin the in
vltro conditionsfor a few days (about7 days for
of a female arrimal in the laboratoryconditions
sheepand 8oat,B daysfor cattle).This allowsthe
unde ra rti fi c i acl o n d i ti o n sT. h i si s i n contrast
to tne
naturalfertilization, developmentof embryosto blastocytestage.
which occursin the uterus.IVF
is reasonably successful as it resultsin 7O-BO"h of Approximately,6O"/"of the IVFembryosin vitro
the feftilizedeggs. culturecan form blastocytes.
Through IVF technology,a large number of lmplantation of embryos
offspringscan be producedfrom a singleanimal.
T hus ,a fe rn a l ea n i m a l ,w h i c h n o rm al l yproduces The 7 or B day old embryosfrom the in vitro
4-5 offsprings in her lifetime,can produceas many culttlreare implantedin the reproductivetract of
as 50-20 offsprirrgs by employinglVF. the recipientfemalewhich actsas a fostermother
or surrogatemother.
In vitrofertilizationinvolvesthe followingstages.
Limitations of M
Oocyte recovery
The pregnancyiossof IVF embryos,particularly
Duringthe courseof estrouscycle,the ovarian duringthe first two months,is very high, although
f ollicl e sg ro q B e t fi l l e d w i th fl u i d and become the reasonsare not clearly known. The following
Craafian(antral)follicles.The oocytesfrom these parameters, which may be consideredas limitations
follicls can be recoveredby using laparoscopic of lVF,may contributeto fetal lossesof IVFembryos.
surgery.it is possibleto recovermore numberof
oocytesfrom superovulateddonors. o Cenetic defectsin oocytes.
o Cenetic defect in {ertilizingsperms.
In vitro maturation (lVMl of oocytes
. Environmentalmutagenesis.
The immatureoocytesrecoveredin the above
. Inadequatesupplyof nutrientsand hormones.
step,when incubatedin vitro will resultin mature
oocytes(eggs). . Exposureto toxic agentsand free radicals.
Chapt er1B : A SS IST ER
DEP R OD U C T IVE
T EC H N OLOC Y 231

In recent years/ some improvements have been Fallopian

ma de in IVF t o m inim iz e t he f et al los s es . tube

EMBRVO C LO NI NG
A clone represents a population of cells or ry
org an isms d er iv ed f r om a s ingle anc es t o r c e l l Ovarian
Clon ing ba si c allym eanst he pr oduc t ion of ide n t i c a l ligament
co pie s of an indiv idual.
It wo uld be ac iv ant ageoust o inc r eas e . t h e
number of embryos from a pai'ticularembryo which
possessesthe desired characters.Two approaches
Cervix
are in use fo r em br y o c lonr ng.
1 . Nuclear transfer. Vagina

2 . Use o f em br y onic c ells .

Fig. 18.2 : An outline of the female repraduction
For more inf or m at ion on nuc lear t r ans fe r a n d
organs.
embryonic cells, the reader must refer the chapter
o n tran sg en icanim als ( Chapt er 41) .
Non-functional ovaries : Some women may
possessovaries that are non-functional, or in some
cases the ovaries may be totally absent. These
women may serve as surrogate or foster mothers.
The oocyte has to be obtained from a donar ancl
Assisted reproductive technology (ART) In ferlilized (in vitro) with the husbands' sperm.
humans is one of the greatest advances in Non-functional uterus : In some w,omen, the
reproductivemedicine.ART has now becomean u t e r u s m a y b e a b s e n t o r n o n - f u n c t i o n a l .I n s u c h a
acceoted method for the treatmentof infertile case, the oocvtes of these women can be fertilizeo
couplefor whom there is no alternativetherapy. by the husband'ssperms, and then transferredinto
the uterus of surrogate mothers.
CA US E S OF IN F ER T IL IT Y A N D
APPLICATION OF ART ldiopathic infertility : Some women may be
infertile for unknow,n reasonswhich is regarded as
Infertilityin men and women may be due to a idiopathic infertility.ART is useful for such women
varietvof reasons. arso.

Male infertility The important techniques employed in assisted

reproductive technology are listed 6elow.
Oligospermia : Reduced concentration of
s per m sin s e me n(n o rma l1 5 -2 0 mi l l i o n /ml ). o I n t r a u t e r i n ei n s e m i n a t i o n ( l U l ) .

Azoospermia : Total lack or very low o ln vitro fertilization and embrvo transfer(lVF and
concentration
of motile sperm. ET).

It is now possibleto carryout ARTer.,en

with a . C a m e t e i n t r a f a l l o p i a nt r a n s f e r ( C I F T ) .
low concentration of motile sperms. . ZyBoIe intrafallopian transfer (ZIPT).
. I n t r a v a g i n a lc u l t u r e ( l V C ) .
Female infertility
r Cytoplasmic transfer (CT).
An outline of the female reproductivesystem
with the essentialorgansis depictedin Fig 18.2. o M i c r o m a n i p u l a t i o n (lntracytoplasmic sperm
i n j e c t i o n ( l C S l ) ,s u b z o n a l i n s e r t i o n ( S U Z I ) .
Tubal infertility : This occurs due to non-
. Cryopreservation.
f unc t ionalor d a ma g e dfa l l o p i a ntu b e s T
. h i scan be
correctedsurgicallyor by tubal transplantation. . Assisted hatching (AH).
232 B IOTE C H NO LO G Y

Among these techniques, the most commonly r Endometriosis

used procedure is ln vitrc fertilization and embryo
o ldiopathic infertility (in men and women).
transfer. lmportant features of different types of ART
are briefly described.
ldeal subjects for IVF

TNTRAUTERTNE (rUrl
TNSEMTNATTON Although it is not always possible to have a
choice in the selection of subjects, the follovring
The infertile women (due to endometriosis, criteria are preferred.
idi opat hic inf er t ilit y ) wit hout bloc k a g e o r d a m a g e
to fallopian tubes can be effectively treated by o Woman below 35 years.
intr aut er ine ins em inat ion. The w o m e n w i t h . Presenceof at least one functional ovary.
adequate ovulation and below the age of 40 years
are c ons ider ed f or I Ul. . H u s b a n d w i t h n o r m a l m o t i l e s p e r m c o u n t ( i .e .
normal seminogram).
The wom en ar e us ually s u p e r o v u l a t e d b y
ad m inis t er ing gonadot r ophins . T h i s r e s u l t s i n . The couple must be negative for HIV and
mult iple egg dev elopm eni. The IU I i s t i m e d t o hepatitis.
co inc ide wit h ov ulat ion.
METHODOLOGY OF
The s em en is was hed and t he h i g h l y m o t i l e 'VF
sperms are separated. By using a thin and soft The in vitro fertilization boardlv involves the
catheter,the sperms are placed either in the cervix following steps.
or in uterine cavity. The women subjects are 1. lnduction of suoerovulation.
advised to remain lying down for about 15-30
minut es f ollowing lUl. 2. Monitoring of ovarian response.

3. Oocyte retrieval.
I ns em inat ions hould be c ar ef ull y t i m e d f o r g o o d
success. lf it is done, a little before the expected 4. Fertilization in vitro.
time of ovulation, the chances for fertilization are
5. Embryo transfer.
muc h higher lUl is us ually s uc c e s s f u li n t h e f i r s t
3-4 attempts. In any case, this approach is not
Induction of superovulation
recommended for more than a maximum of 6
o vu lat ion c y c les . It is well known that the success rate IVF is
much higher when more embryos (3-5) are
The successrates of lUl vary considerably and
transferred. This is possible only with controlled
are in t he r ange of 15- 30% . (COH). The other
ovarian hyperstimulation
advantages of COH include improvement in the
IN V'TRO FERTILIZATION AND quality of oocyte, control of ovulation timing,
EMBRYO TRANSFER (lVF and ETf besidesovercoming the ovulatory dysfunction. The
following drug regimes are in use to induce
ln vitro fertilization broadly deals with
superovulation.
the removal of eggs from a women, fertilizing
them in the lahoratory, and then transferring the . Clomiphene citrate (CC).
fertilized eggs (zygotes) into the uterus a few days
. CC + human menopausal gonadotrophin (hMC).
Iater.
. CC + follicle stimulatinghormone (FSH).
Indications for IVF
. H u m a n m e n o p a u s a lg o n a d o t r o p h i n .
Int-ertilitydue to the following causes may be
. Follicle stimulatinghormone.
considered for lVF.
. Conadotrophin releasing hormone agonists
. Failed ov ulat ion induc t ion
(CnRHa) + hMC (or FSH).
. Tubal diseases
It is now common to use CnRH agonists to
. C er v ic al hos t ilit y induce ovulation.
Chapt er1B : A SS IST ER
DE PR O D U C T IVE
T E C H N OLOC Y
These compounds act through a process called
do wnre gu lation of t he phy s iologic hy pot ha l a m i c -
p ituitary-ovarian feedback mechanism to effectively
suppressspontaneousovulation.
Monitoring of ovarian response
Th e follicular gr owt h or ov ar ian r es pons eca n b e
monitored by increase in serum estradiol level,
in cre ase in f ollic ular diam et er and t hic k en i n e o f
endometrial bed.
Oocyte retrieval
The most common method for oocyte retrieval is
ca rried o ut th r ough v aginal r out e under ult r a s o u n d
gu ida nce. Th is m et hod is s im ple and les s inv a s i v e ,
an d can b e per f or m ed wit h analges ic sonly. l t i s
ea sy to recogniz e t he ooc y t e as a s ingle c e l l
su rrou nd ed by a m as s of c um ulus c ells . T h e
recovered oocytes are maintained in vitro culture
for 4-6 hours.
Fertifization in vitro
The semen specimensare collected (just prior to
oocyte retrival) via masturbation, processed, and
in cu ba ted in pr ot ein- s upplem ent ed m edi a f o r
3-4 ho urs pr ior t o f er t iliz at ion. The inc ub a t r o n
results in sp er m c apac it at ion.
The retrieved oocytes are also cultured in
protein-supplementedmedia for about 6-8 hours.
For th e pur pos e of lVn 50, 000- 1 , 0 0 , 0 0 0
ca pa cita ted s per m s ar e plac ed in c ult ur e w i t h a
single oocyte. The signs of fertilization may be
.l
demonstrated 6-20 hours later bv the presenceof
two p ron uclei wit hin t he dev eloping em br y o .
There is no need to change the regime for a
single failure of lVF. Many a times, successoccurs
in th e sub se quentc y c les
The two most importanl criteria for the success
of IVF are sperm density and motility.
Embryo transfer
Embryo at a stage between pronuclei and
blastocyststage are transferred.Conventionally, 4-
B cell stageembryos are transferredbetween 48-60
ho urs fo llowing ins em inat ion. The t r a n s f e r
procedure is carried out by use of a catheter Not
more than three embryos are transferred(per cycle)
to min imize m ult iple pr egnanc ies .Howev er , i n t h e
women above the age of 40 years, higher number
233
of embryo may be transferred. (Note : Excess
oocytes and embryos are cryopreservedfor further
u s e . T h i s w i l l r e d u c e t h e c o s t , b e s i d e st h e r i s k o f
ovarian hyperstimulation).
Luteal phase support is given by administration
of progesteronefor about two weeks. By this time,
the diagnosis of pregnancy can be assessedby
e s t ! m a t i n gh u m a n c h o r i o n i c g o n a d o t r o p h i n ( h C C ) .
sUCCESS NATES OF IVF
Success of IVF varies from programme to
programme and within the same programme, the
successrate is dependent on the correct diagnosrs
of the patient, and age.
T h e o v e r a l l p r e g n a n c yr a t e i n I V F i s i n t h e r a n g e
of 25-35"h per oocyte retrival.The take home babv
rate is ahout t5-20% per procedure. The success
rate of IVF is rather low, due to the followine
reasons.
. Increasedrisk of abortion
. Multiple pregnancy
o Ectopic pregnancy
. Low birth weigh baby
r Prematuredelivery.
THE WORLD'S PTCTUBE OF
TEST TUBE BAB'ES
By employing in vitro fertilization and embryo
transfer, the world's first test tube baby (Lourse
Brown) was born in UK on 2}th luly 1978. fhe
w o r l d 's s e c o n d t e s t t u b e b a b y ( K a n u p r i y a a l r a s
Durga) was born in Kolkata on 3td Cctober 1978.
A t e a m l e d b y S u b h a s hM u k h e r j e e c a r r i e d I V F a n d
E T i n I n d i a . S c i e n t i s t sr e s p o n s i b l ef o r t h e b i r t h o f
test tube babies were severely criticized then
ln fact, IVF turned out to be one of the major
achievements of medical sciences in the last
century. lt has become a novel way of treating
i n f e r t i l i t y .T o d a y ,t h e r e a r e m o r e t h a n a m i l l i o n t e s t
tube babies born all over the world.
I n 2 0 0 3 , t h e w o r l d c e l e b r a t e dt h e s i l v e r l u b i l e e
of IVF with much fanfare.
GAMETE INTRAFALLOPIAN TRANSFER
(GrFTl
Camete intrafallooian transfer involves the
transfer of both sperm and unfertilized oocyte into
234
th e fallopian t ube. This allows t he fe r t i l i z a t i o n r o
naturally occur in vivo. The prerequisitefor CIFT
procedure is that the woman should have at least
o ne nor m al f allooian t uoe.
The induc t ion of ov ulat ion and t h e m o n i t o r i n g
p!'ocedLrresfor CIFT are almost the same as
describedfor lVF. A couple of hours prior to oocyie
re triev al, s em en s pec im ens ar e c o l l e c t e d . T w o
oocytes along vrith 2-5 lakhs motile sperms for
e ach f allopian t ube ar e plac ed in a p l a s t i c t u b e
container. lt is then inserted (by laparoscopy)4 cnr
into t he dis t al end of t he f allor : ian t u b e , a n d t n e
oocyte sperm combinaiion is injected.
The ov er all pr egnanc y r at e is as h i g h a s 3 0 -
4O% fhe take home baby rate is about 25"h. This
is mtrch higher when compared to lVF. But the
major limitation is the requirernent of laparoscopy
(a major surgical procedure)to transferoocytes and
spe rm s int o t he f allopian t ubes .
ZYGOTE INTRAFALLOPIAN TRANSFER
(zrFTl
The procedureof cytoplasmictransferis tedious
ZlFf is s uit ablewhen t he inf er t ili t y I i e s i n m e n ,
and technicallydifficult,besidesthe cost factor.At
or in case of failure of ClFl-. least two viable pregnancieshave been so far
The wife's oocytes are exposed to her husband's reported in Iiteratureby this approach.
sperms in the laboratory. The fertilized eggs
(zygotes) within 24 hours are transferred to the
fa llop ian t ube by us ing lapar os c opy .
ZlPf has an advantage over CIFT with male
factor infertility. Further, it can be known whether
the wife's oocytes have been fertilized by her
h usbands ' s oer m s .
CULTURE(rVCl
TNTRAVAGTNAL
The body'sown environmentis appropriately
utilized in intravaginal culture. The retrieved
o ocy t es and s per m sar e plac ed in a c u l t u r e m e d i u m
in sid e a s ealed c ont ainer .This is ins e r t e d i n t o t n e
vag ina. The c ont ainer is held b y a v a g i n a l
d iap hr agm . Thus , t he ooc y t es and s p e r m s a r e
maintained at the normal body temperature (in
contrast to any incubator in the laboratory).Two to
3 three days later,the container is opened, and the
fertilized and dividing zygotes are transferred into
the uterus.
This procedure appears simple) but the success
rate is very low. Only a few centers practice
this.
B IOTE C H N OLO CY
CYTOPLASMIC TRANSFEF (CTI
C ytopl asmi ncl udes many thi ngs, the most
i mportant bei ng n.i tochondri aw hi ch pro vide
energyto the cel l . l t i s possi bl ethat defi ci encyr n
the mitochondriamay leavethe oocytewithoutthe
necessary powerfor cell division,afterfertilization.
-i hi smay resul ti n abnormalcel l di vi si onand poor
developmentof ernbrvo.
It is thereforelogicalto think of the transferof
cvtoplasmfrom a donor (with activemitochondria)
into the oocyte of a woman. The advantagewith
cytoplasmictransfer is that Ihe mother's own
genetic material is passedon to the offspring.
Two methodsof cytoplasmictransferhavebeen
developed.
of a smallanrountof cytoplasmby a
l. Transfer
tiny needlefrom a donor to a recipientoocyte.
of a largeamountof cytoplasmwhich
2. Transfer
is fusedwith the recipient'scytoplasmby applying
electricity.
MIC R OMA N IP U LA TION
Micrc.manioulation involves in vitro
microsurgicallyassistedfertilization procedures.
Thi s i s requi redw hen the spermsare unablet o
penetrate the zona pellucidaof oocvteand fertilize.
Mi cromani pul ati onsare usual l y done i n se ver e
casesof male factor infertility.
A diagrammatic representationof micro-
mani pul ati on
i s depi ctedi n Fi g 18.3.
Intracytoplasmic sperm injection (lGSl)
!ntracytoplasmic spernrinjectionis a new and
novel i nferti l i ty treatment uti l i zi ng t he
mi cromani pul ati ontechnol ogy. Many of t he
previoustreatmentprocessesfor male infertility
have been abandonedin favourof lCSl.The mare
factorinfertilitycould be due to low spermcounts,
poor spermmotility,arrd poor quality of spermto
peneirateoocyte.
By partial zona dissection (PZD), the zona
pel l uci da i s opened usi ng ei ther chem ical
di ssol uti onor a sharp i nstrument.A single
 Chaot er1B : A S S IST ER T IC HN OLOC Y
DE PR O D U C T IVE
{- Subzonal
sperm iniection
I CRYOPRESERVATION
Intracytoplasmic
sperm injection
Zona oellucida
Fig. 18.3 : Micromanipulation for fertilization of an
egg (micrcsurgically assisted fertitization).
spermatozoon can be directly injected into the
cytoplasm of the oocyte through the micropuncture
oI zona pellucida. A micropipette is used to hold
the oocyte while the spermatozoon is deposited
inside the ooplasm of the oocyte.
Beside s using nor m al s per m s , r ound- hea d e d
sperrns, sperms collected directly form the
ep idid ymis an d pr ev ious ly c r y opr es er v ed s pe r m s
ca n be u se d in lCSl.
Arrong the micromanipulation techniques ICSI
is the most successful one with a fertilization rate
of about 65%. Attempts are on to improve this
further. In fact, lCSl has revolutionized assistant
reproductive technology by utilizing the sperms of
husbands who were once considered to oe
un su itab lefo r fer t iliz at ion pr oc es s .
Sub zo na l in s er t ion ( SUZI l
In sub zo na l ins er t ion, t he z ona pelluc ida i s
cun ctu red a nd s per m s ( 1- 30 in num ber ) a r e
^iected into an area between the zona and the
=rl. lt is expected that one of the sperms will
'-': .:ze th e e 88
-lre ma ior lim it at ion of SUZI is poly s per n r y
^ ce it is no t o os s ible t o c ont r ol t he num ber o f
-
;:€11s that enter the egg.
Ro un d spe rmid nuc leus iniec t ion
(ROSNT)
Trere are a few men who cannot manufacture
::,.-'-r's and therefore they have a zero sperm
----ri. For the se m en, it is pos s iblet o t ak e out t h e
' ( im m at ur e c ells ) dir ec t ly f r om t h e
--a spe rmati ds
235
testicle, isolate the nucleus (containing the genetic
material) and inject it into the partner's eggs.
ROSNI is a recent exciting breakthroughto solve
the problem of male infertility through
micromanioulation.
Preservation in a frozen state is regarded as
cryopreservation.Cryopreservationis verlzu5sfLrl1n
assistedreproductive technology.
. Semen can be cryopreserved.This may be frorn
the donors, cancer patients (before the
commencement of treatment).
. Fertilizedeggs after IVP or lCSl can be preserved.
. Embryos can also be preservedfor transfer at a
later stage.
Human enrbryos have been successfully
preserved in the presence of cryoprotectants ('l,
2-propanediol/dimethyl sulfoxide/glycerol) and
s t o r e d a t - 1 9 6 'C u n d e r l i q u i d n i t r o g e n . A t
appropriate time, the embryos are thawed,
crvoprotectants removed and then transferreo.
Many test tube babies in fact have been born as a
result of application of freezing technology.
ASSISTED HATCHING (AHI
lmproper implantation of the embryo in the
uterus is one of the limiting factors in the success
of ART in humans. Assisted hatching is a novel
approach for the proper implantation of the embryo
in the endrometrium.
The embryos in the uterus possess an outer
coating namely zona pellucida (the shell). These
embryos must be hatched to remove the shell,
a step necessary for implantation. In certain
women, particularly above 40 years age, natural
hatching does not occur, and requires outside
assistance.
A s s i s t e dh a t c h i n g i s c a r r i e d o u t b y u s i n g a L a s e r
to make a small hole in the shell of the embryo.
These embryos when transferred into the uterus,
hatch and get implanteo.
During the course of AH for 3-4 days, the
women are kept on steroids (to suppressmother's
immunity) and antibiotics (to counter infections).
Better results are reported with this approach.
236 B IOTE C H NO LO C\

P R E IM PL A N T A T IO N G E N E T I C THE NEGATIVE ASPECTS OF ART

DTAGNOSTS(PGDI
There are certain limitations/disadvantages
The geneticdefectsin ovum beforefertilizatron associated with assisted reproductive technologyrn
or in the embryo before implantationcan be humans. S ome hi ghl i ghtsare gi ven. l t m ust
identified by a new medical tool namely however, be,noted that the advantagesof ART
pr e i m p l a n ta ti ogne n e ti cd i a g n o s i s.l t i s esti mated outweigh the disadvantages.
that about 60'/' of the ART driven pregnancies
ar e Io s t d u e to c h ro m o s o malabnormal i ti es.Ovarian hyperstimulation syndrome
T h i s c a n b e mi n i mi z e d o r p re ventedby usi ng (oHssl
PCD. Due to administration of hormonesand drugs,
A d i re c t d e te rm i n a ti o n o f chromosomal ovarian hyperstimulation is frequentlyassociated
ab n o rma l i ti e sp ri o r to i m p l a nti on ensures a w i th compl i cati ons, someti mes even lif e-
s u c c e s s fupl re g n a n c ya n d u l ti mate del i very of threatening. OHSS is more severe in women who
a healthybaby.One groupof workershasreported conceived in the same cvcle, and receivedhCC as
luteal support (followingembryotransfer)
an increasein the pregnancy rate from 15 to
30 % b y e m p l o y i n g p re i m p l antati ongeneti c
Risks associated with pregnancy
dia g n o s i s .
ART is associatedwith multiple pregnancy,
DNA amplification and analysis increasedrisk for anemia,gestational diabetesand
prematurelabour.Low birthweightand prematurity
T h e l a te s ti n PC D i s th e d i re c t D N A anal ysi s.
are cl osedl i nkedw i th mortal i tvand morbi dit v.
T h i s c a rrb e c a rri e do u t b y re mo vi nga si ngl ecel l
from 6-B-cell embryo. The DNA is removed Premature menopause
and a m p l i fi e d b y e m p l o y i n gp ol ymerasechai n
reaction. C ontrol l ed ovari an hypersti mul ati on(CO H)
causesmul ti pl efol l i cul aruti l i ty.Therei s a risk of
D i re c tD N A a n a l y s i si s u s e fu lfor the di agnosi s prematuremenopauseas COH may reduce the
of several genetic diseasese.g. cystic fibrosis, ovarianfollicles,besidesfasteraging.Sometimes, a
s ic k l e -c e l l a n e m i a , h e m o p h il i a, D uchene' s si ngl eC OH may useovari anfol l i cl es,w hi ch in t he
musculardystrophy,Tay-Sachs disease. normal course are equivalentto two years of
ovul ati onduri ngthe naturalmenstrual cycl e .
Ethical advantages of PGD

PCD is highly advantageous from the ethical Ovarian cancer

point of view since the embryos with genetic The use of ferti l i ty drugs and i nj uries t o
disorderscan be discarded in the very stages eoi thel i umi ncreasethe ri sk of ovari ancancerat
withoutthe formationof offspringswith undesirable least by three times when comparedto normal
characteristics w omen.
 (t)
1 9 Bi oprocess/Fermentati on
Technology 239
20 D ow nstream P rocessi ng 27l J
21 E nzyme Technol ogy 281
22 B i otransformati on 306
23 Mi crobi al P roducti onof Organi c
S ol ventsg$1 t
24 Mi t robi al P roducl i on oI Organi c
A r-i ds 318
25 Mi crobi al P roducti onof
A nti bi oti cs 329
26 Mi crobi al P roducti on of A mi no
A ci ds 344
27 Mi crobi al P roducti on of
vttamrns .J55
M!CROBIAL/ 28 Mi crobi al P roducti onof Foods
and Beverages 362
INDUSTRIAL 29 S i ngl e-C el lP rotei n, and
BIOTECHNOLOGY Mushrooms 373
30 P ol ysacchari des,
P ol yhydroxyal kanoates, and
Li pi ds 382
31 B i omassand B i oenergy 393
32 Mi crobi al Mi ni ng and Metal
B i otechnol ogy 400

237
 i-, o r ma ny c ent ur ies , m an has been ex ploi t i n g
: microo rga nis m s f or t he pr oduc t ion of f o o d s
(bread, cheese, yoghr,rrts,pickles) and beverages
(beer, wine). Howevet; the organized use of I he heart of fermentatiorr (or bioprocessing)
micro org an ismf or indus t r ial pur pos esis about o n e technology is the fermenter (or bioreactor) A
an d a ha lf ce nt ur y old. bioreactor is basicallv a device in which the
organisms (cells) are cultivated and motivated to
The word fermentation originates from a Latin form the desired product(s). lt is a containment
verb fervere which literally means to boil. During system designed to give right environment for
the p rod uction of alc ohol ( t he f ir s t t r u e l y optimal growth and metabolic activity of the
ind ustrialisedp r oc es s ) ,t he gas bubbles ( of C O r ) orSanrsm.
ap pe ar at th e s ur f ac e of t he boiling liq u i d .
A fermenter usually refers to the containment
Fermentation in a strict sense rs a biological
system for the cultivation of prokaryotic cells
process that occurs in the absence of oxygen
(bacteria, fungi), while a bioreactor grows the
(anaerobic). This definition however, is no more
eukaryotic cells (mammal ian, insect).
valid, since the term industrial fermentation is now
used for large-scalecultivation of microorganisms, Traditionalfermentersare open vats made up of
even though most of them are aerobic (use wood or slate. In recent years, stainless steel
oxyg en ). b i o r e a c t o r sa r e i n u s e . A h i g h q u a l i t y s t a i n l e s ss t e e l
that does not corrode or leak toxic metals into the
Bioprocess technology is a more recent usage
growth medium is used.The size of a bioreactoris
to replace fermentation technology. Bioprocessing
highly variable, ranging from 20 litres to 250
broadly invo'lvesa multitude of enzyme-catalysed
million litres or even more.
re action s ca rried out by liv ing c ells ( or c ell- f r e e
systems) for industrial purposes. Some workers T Y P E S O F B I O R E A G T O R S
prefer to use bioprocess technology for industrial
use ol higher plant and animal cells while Based on the designs of the bioreactors,
fermentation technology is confined to microbial they can be grouped into the following types
( F i g s . 19.1-19.4)
use. This demarcation is however, not very rigid.
1. Continuous stirredtank bioreactors
Bioprocessfermentation technology is very
ivid ely e xp loited f or indus t r ial applic at ions . T h e 2. Bubble column bioreactors
broad range of fermentation products are listed rn 3. Airlift bioreactors
Table 19.1. 4. Fluidized bed bioreactors
 24(, B IOTE C H NO LO CY

Group Products
Foods Dairyproducts(cheese,
yogurt)
Vitamins(Br,Brz)
Amino (glutamic
acids acid,lysine)
Glucoseandhighfructosesyrup
Mushroom products
Baker'syeast
Foodadditives(antioxidants, flavours)
colours,
Beverages(beer,wine,whisky)
Chemicals
(bulk)
Organic Ethanol,butanol,acetone, acids(citricacid,gluconic
organic
acid,lacticacid)
(fine)
Organic Enzymes, polymers (xanthan,
dextran)
Inorganic Bioaccumulationandleaching(Cu,U)
Pharmaceuticals( healthcare) Antibiotics
Vaccines
Steroids
Diagnostic
enzymes
Monoclonalantibodies
Enzyme inhibitors
Agriculture protein
Single-celi
pesticides
Microbial
Composting processes
Plantcellandtissueculture

5. Packed bed bioreacrors Different types of impellers (Rustom disc, concave

6. Photobioreactors. bladded, marine propeller etc.) are in use.

ln all types of bioreactors,the ultimate aim is to
ensure that all parts of the system are sublected to
the s am e c ondit ions .
Gontinuous stirred tank bioreactors
A continuous stirred tank bioreactor consistsof
a cyiindrical vessel with motor driven central shaft
that supports one or more agitators (impeller) fhe
shaft is fitted at the bottom of the bioreactor
(Fig. 19.1A). The number of impellers is variable
and depends on the size of the bioreactor i.e.,
height to diameter ratio, referred to as aspect ratio.
The aspect ratio of a stirred tank bioreactor is
usually. between 3-5. However, for animal cell
culture applications,the aspect ratio is less than 2.
Th e diam et er of t he im peller is usu a l l y {r d o f t h e
vessel diametei'. The distance between two
impeller s is appr ox im at ely 1. 2 im p e l l e r d i a m e t e r .
In stirred tank bioreactors or in short stirred tank
reactors (STRs), the air is added to the culture
m e d i u m u n d e r p r e s s u r e t h r o u g h a d e v i c e ca l l e d
sparger.The spargermay be a ring with many hotes
o r a i u b e w i t h a s i n g l e o r i f i c e . T h e s p a r g e ra l o n g
with impellers (agitators) enables better gas
distribr,rtion system throughout the vessel. The
bubbles generated by sparger are broken down to
s m a l l e r o n e s b y i m p e l l e r sa n d d i s p e r s e dt h r o u g h o u t
t h e r n e d i u m . T h i s e n a b l e st h e c r e a t i o n o f a u n i fo r m
a n d h o m o g e n e o u s e n v i r o n m e n t t h r o u g h o ut th e
bioreactor.
Advantages of STRs : There are many
advantagesof STRs,overother types. These incluoe
the efficient gas transfer to growing cells, good
mixing of the contents and flexible operating
c o n d i t i o n s , b e s i d e s t h e c o m m e r c i a l a v a i l a b i l i ty o f
the bioreactors.
 ChA P I CT
19 : B I O P R OC ES S/F E R ME N T A TTIO N N OLOC Y
E CH 241

means of a baffle or draft tube. In one of the rwcr

zones referredIo a riser, the air/gasis pumped. The
other zone that receives no gas is Ihe downcomer.
The dispersionflows up the riser zone while the
lmpeller down flow occurs in the downcomer. There are
two tvpes of airlift bioreacrors.

Culture f nternal-loop airlift bioreactor (Fig. 11.1C) has

medium a single container with a central draft tube that
creates interior liquid circulation channels. These
b i o r e a c t o r sa r e s i m p l e i n d e s i g n , w i t h v o l u m e a n d
circulation at a fixed rate for fermentation.
External loop airlift bioreactor (Fig. 19.1D)
possesses an external loop so that the liquid
(B) circulates through separate independent channels.
These reactorscan be suitably modified to suit the
Down- requirementsof dilferent fermentations.In generar,
the airlift bioreactorsare more efficient than bubble
c o l u m n s , p a r t i c u l a r l yf o r m o r e d e n s e r s u s p e n s i o n s
J} o f m i c r o o r g a n i s m sT . h i s i s m a i n l y b e c a u s ei n t h e s e

lll bioreactors, the mixing of the contents is better

comoared to bubble corumns

Jll
t
Airlift bioreacfols are commonly employed for
aerobic bioprocessing technology. They ensure a
controlled liquid flow in a recycle system by
pumping. Due to high efficiency, airlift bioreactors
are sometimespreferrede.g., methanol production,
waste water treatment, single-cell protein
(D) production. In general, the performance of the
, airlift bioreactors is dependent on the pumping
Fig. 19.1 : Types of bioreactors (A) Continuous (injection.o) f air and the liquid circulation.
stirred tank biareactar (B) Bubble column bioreactor
(C) lntemalloop airlift bioreactor (D) Externalloop Two.stage airlift bioreactors
r ,, r ,.' :,airt!ftbioreactgT , i ,, ,
,,; Two-stage airlift bioreactors are used for the
temperature dependent formation of products.
G r o w i n g c e l l s f r o m o n e b i o r e a c t o r ( m a i n t a i n e da t
Bubble column bioreactors
temperature 30'C) are pumped into another
th e a i r o r g a s bioreactor (at temperature 42oC). There is a
In t he bubblec o l u m nb i o re a c to r,
is introducedat the base of the column through necessityfor the two-stageairlift bioreactor,since it
perforatedpipes or plates,or metal microporous is very difficult to raise the temperature quickly
spargers(Fig. 19.18).The flow rate of the airlgas from 30oC to 42"C in the same vessel. Each one
influencesthe performancefactors-O, transfer, of the bioreactors is fitted with valves and they
mixing. The bubble column bioreactorsmay be are connected by a transfer tube and pump
fitted with perforated plates to improve (Fig. 19.2A). The cells are grown in the first
performance.The vesselused for bubble column bioreactor and the bioprocessproper takes place in
bioreactorsis usually cylindricalwith an aspect the second reactor.
ratio of 4-6 (i.e.,heightto diameterratio).
Tower bioreactors .'
Airlift bioreactors
A pressure-cycle fermenter with large
In the airlift bioreactors,the medium of the dimensions constitutes a tower bioreacror
vesselis divided into two interconnected
zonesbv (Fig. 19.28). A high hydrostatic pressuregenerated

Biotechnology
[16]
 242
Transfer
tube
Airinlet
Fig. 19.2 : Types of bioreactors (A) Two-stage airlift bioreactor (B) Tower bioreactot.
at the bottomof the reactorincreases the solubility
of O, in the medium.At the top of the riser,(with
expanded top) reduces pressureand facilitates
ex p u l s i o no f C O r. T h e m e d i u mfl ow s back i n the
downcomer and completes the cycle. The
advantage with tower bioreactoris that it has high
aerationcapacitieswithout havingmoving parts.
Fluidized bed bioreactors
Fluidized bed bioreactor is comoarable to
bubble column bioreactor except the top
positicnis expandedto reducethe velocityof the
f luid . T h e d e s i g n o f th e fl u i d i zed bi oredttors
(expandedtop and narrow reaction column) is
such that the solids are retainedin the reactor
while the liquid flows out (Fig. 19.3A). These
bioreactorsare suitable for use to carry out
reactions involving fluid suspended biocatalysts
such as immobilized enzymes,irnmobilizedcells,
m icro b i a fl
l ocs.
Foran efficientoperationof fludizedbeds,gasis
s pa rg e dto c re a tea s u i ta b l eg a s -l i qui d-sol fi
i dui d
bed. lt is also necessaryto ensure that tne
suspendedsolid particlesare not too light or too
dense(too light ones may float whereasto dense
ones may settleat the bottorn),and they are in a
good suspendedstate.Recyclingof the liquid is
B IOTE C H NO LO C\
Air inlel
(B)
importantto maintaincontinuouscontactbetween
the reactioncontentsand biocatalysts.
This enable
good efficiencyof bioprocessing.
Packed bed bioreactors
A bed of solid particles, with biocatalysfson or
w i thi n the matri x of sol i ds.packedi n a co lum n
constitutesa packed bed bioreactor(Fig. 19.38).
The solidsusedmay be porousor non-porousgels,
and they may be compressible or rigid in nature.A
nutrient broth flows continuously over the
immobilisedbiocatalyst. The productsobtainedin
the oacked bed bioreactorare releasedinto the
fl ui d and removed.W hi l e the fl ow of the fl ui d can
be upwardor downward,downflow undergravity
is oreferred.
'fhe
concentration of the nuirients(andtherefore
the products formed) can be increased by
increasingthe flow rate of the nutrient broth.
Becauseof poor mixing, it is rather difficult to
control the pFl of packedbed bioreactorsb1,the
addition of acid or alkali. However, these
bioreactors are preferred for bioprocessing
technologyinvolving product-inhibitedreactions.
The packed becl bioreactors do not allow
accumulationof the productsto any significant
extent.
 19 : BIOP R OC ES S/F E R ME N T A TION
TE C H N OLOC Y

out
Settlingzone

Fluidizedbiocatalyst

Nutrientbroth

Packedbed with
biocatalyst

Outlet
(B)

Fig. 19.3 : Types of bioreactors (A) Fluidized bed

bioreactar (B) Packed bed bioreactor.

Photobioreactors
These are the bioreactors specialisedfor
fermentation that can be carried out either by
exposing to sunlight or artificial illumination. Since
ar t if ic ialillumi n a ti o ni s e x p e n s i v eo, n l y th e out
door photobioreactorsare preferred. Certain
importantcompouncis are producedby employing
photobioreactors e.g.,B-carotene, asthaxanthin.
The different types of photobioi'eactors are
depictedin Fig. 19.4.Theyare madeup of glassor
more commonlytransparentplastic.The array of
t ubes or f la t p a n e l s c o n s ti tu tel i g h t re c e i vi ng
systerns(solar receivers).The culture can be
circulatedthroughtlre solar receiversby methods
s uc has us ingc e n tri fu g aplu m p so r a i rl i ftp u m ps.l t
is es s ent ialth a t th e c e l l s a re i n c o n ti n uous
c ir c ulat ionw i th o u t fo rm i n g s e d i m e n ts .F u rther
adequat e pe n e tra ti o no f s u n l i g h t s h o u l d be (D)
maintained.The tubes should also be cooled ro
preventrise in temperature. Fig. 19.4 : Types of phatobiarcactors (A) Continuous
run tubular loop (B) Multiple parallel tube (C) Helical
Photobioreactors are usually operated in a
wound tubular loop (D) Flat panel conliguration.
continuousmode at a temperature in the rangeof
244 B IOTE C HNO LO CY

Air exhaust

Removabletoo
Sightglass

Foam beaker
Connectionsfor acid, alkaliand
antifoamchemicals
lnoculumconnection

Sparger
Jacket -
conneclton

Motor
Reducinggear box
Harvestnozzle

Fig. 19.5 : Diagrammatic representation of a typical bioreactor.

25-4O'C. Microalgae and cyanobacteria are
nor r nally us ed. The or ganis m s g r o w d u r i n g d a y
light while t he pr oduc t sar e pr od u c e d d u r i n g n i g h t .
A COrtNxiESlTICIESALBIOREACTOR - and i nocul umare l ocated.
G O M M O f { FEATI I RES
The different types and designs of bioreactors
are described. The most common features of a
typical bioreactor are diagrammaticallyrepresented
in Fig. 19.5, and briefly described hereunder.
Conv ent ional bior eac t or sar e c y l i n d r i c a l v e s s e l s
r,vith domed top and bottom. The reaction vessel,
surrounded by a jacket, is provided with a sparger
at the bottom through which air (or other gases
s uc h as CO , and NH, f or pH m a i n t e n a n c e )c a n b e
introduced. The agitator shaft is connected to a
motor at the bottom.The reactionvesselhas side
portsfor pH, temperature and dissolvedO, sensors.
Above the liquid level of the reaction vessel,
connecti onsfor aci d, al kal i , anti foamchem icals
The bioreactoris usuallydesignedto work at
higher temperature(150-1B0"C), higher pressure
377-412 kP a).The reacti onvesseli s al sod esigned
to w i thstandvaccum,or el sei t may col l apsewhile
cooling.The matei'ialsusedfor the construction of
bioreactormust be non-toxicand must withstand
with high pressure steam.
the repeatedsterilization
The bioreactorvessel is usually made up of
steel.lt shouldbe free from crevicesand
stainless
accumulate.
stagnantareasso that no solids/liquids
Easyto cleanchannelsand weldedjoints(instead of
 Chapt er19 : BIOP R OC ES S/F E R ME N TA TION
TE C H N OLOC Y 245

arepreferred.
couplings) Transparent materialshould I N O C U L A T I O N A N D S A M P L I N G
be usedwhereverpossible,
sinceit is advantageous
The bioreactor with the growth nredium under
to inspectmediumanclculturefrequently.
aseptic conditions is ready for inoculation with the
production organism.The size of the inoculum is
generally'l-10% of the total volume of the medium.
A high yielding production strain of the organism
taken from a stock culture (lyophilized and stored
in a deep freezer or irr liquid nitrogen)is used.

D u r i n g t h e c o u r s e o f f e r m e n t a t i o n ,s a r n p l e sa r e
The o pe r at ion of a bior eac t or bas ic ally in v o l v e s
regularly drawn from the bioreactor.-fhis is
the following steps.
required to check the contamination (if any) and
1 . Ste riliz at ic ln measurementof the oroduct formed.
2. Ino cu lat ion and s am pling
AERATION
3. Aera t ion
Aeration of the fermentationmedium is required
4. Control systerns to supply O, to the production organisms and
renrove CO, frotn the bioreactor. -lhe aeration
5. Clea ning.
system is designed for good exchange of gases.
Oxygen (stored in tanks in a compi'essedform) is
STERIL IZAT I O N
introducecjat the bottom of the bioreactor through
Aseptic conditions are the basic requirernents a sparger.The small bubbles of the air passthrough
for successfulfermentation That is the bioreactor t h e m e d i u m a n d r i s e t o t h e s u r f a c e T h e b i o r e a c t o r
and its accessories,the grol,r,thmediurn and the arr usually has about 20"k of its volume as vacant
su pp lied d ur ing f er m ent at ion m us t be s t er il e . space on the upper part which is referred to as
head space.The bioreactor has about BO'h working
In situ sterilization v<tlume. The gases released during fermentatron
accumulate in the headspace which pass out
Th e b iore ac t or f illed wit h t he r equir ed m e d i u m
through an air outlet.
is injected with pressurizedsteam into the jacket or
coil surrounding the reaction vessel fhe whole
Air-lift system of aeration
system is heated to about 120"C and held at this
temDerature for about 20 minutes. ln situ ln this type of aeration, sparging of air is done at
ste riliza tionhas c er t ain I im it at ions .lt is not e n e r g y - the bottom of the fermenfer. This allows an uoward
e fficie nt (i.e . , ener gy is was t ed)s inc e t he bio r e a c t o r flow of air bubbles. The more is the aeration
has to be heated fr,rr a long period to rise the capacity of the fermenter,the more is the dissoived
te mpe ratu re of t he whole s y s t em t o 1 2 0 'C . O, in the nredium. Further,the aeration capacity of
Prolonged heating may destroy vitamirrs, besides the air-lift system is directly proportional to the arr-
pre cip itatin gt he m edium c om ponent s . flow rate and the internal pressure Oxygen demand
refers to the rate at n,hich the growing culture
Gontinuous heat sterilization requires Or. For all the aerobic organisms, the
aeration capacity should be more tlran ihe oxyg,en
In th is tec hnique, em pt y bior eac t or i s f i r s t
demand or else the growth of the organismswili be
ste rilize d by injec iing pr es s ur is ed s t eam . T h e
inhibited due to oxygen depletion (starvation).
medium is rapidly heated to 140"C for a short
period, by injecting the pressurised steam
Stirred system of aeration
Alternately, the medium can be sterilized by
p assin g th rough a heat ex c hanger heat e d b y The aeration capacity of the mediurn can be
pressurisedsteam. Subjecting ihe rnediunr to high enhanced by stirring. This can be dc-,neby using
temperature for a short period does not precipitate impellers driven by a motor. The aeration capacity
-'ec/iurrt components. Further there is no enerqy of the stirred fermenter is proportional to the stirring
.: -i-. ;n cont inuous heat s t er iliz at ionm et h o d . speed, rate o{ air {low and the i'nternal pressure
246 B IOTE C H N OLO CY

Stirred fermenters are better suited than air-iift nutrients and O2, besides preventing the
fermentersto produce better aeration capacities, accumulationof toxic metabolic byproducts(if
any). C ood mi xi ng (by agi tati on)al so creat es
CONTRO L SYSTEM S favourable environment for optimal and
It is essential to maintain optimal growth
homogeneousgrowth environment, and good
product formation. However,excessiveagitation
environment in the reaction vessel for maximum
product formation. Maximal efficiency of the may damage microbial cells and increasethe
fermentation can be achieved by continuously
ternperatureof the medium,besidesincreasedfoam
formation.
monitoring the variahles such as the pH,
ternperature, dissolved oxygen, adequate mixing,
nutrient concentration and foam formation.
Nutrient concentration
lmp r ov ed s ens or sar e now av ailable f o r c o n t i n o u s The nutrient concentrationin a bioreactoris
a nd aut om at ed m onit or ing of t hes e v a r i a b l e s ( i . e . , limitedso that its wastageis prevented. In addition,
on line m eas ur em entof oH) . l i mi ti ng concentrati onsof nutri ents may be
M os t of t he m ic r oor ganis m s e r n p l o y e d i n advantageous for optimal productformation,since
fermentation grow optimaliy beiween pH 5.5 and high nutrient concentrations are often associated
8.5. In the bioreactor,as the microorganismsgrow, with inhibitory effect on microbial growth.lt is now
they reiease metabolites into the medium w,hich
possi bl e
to do on-l i ne moni tori of the nut r ient
ng
cl.range pH. Therefore, the pH of the mediurn concentrati on,and sui tabl y modi fy as per t he
sh ou ld be c ont ir r uous lym onit or ed a n d m a i n t a i r . r e d reouirements.
at th e opt inr al lev el. This c ar r be d o n e b y t h e
addition of acici or alkali base (as r.reeded)ano a Foam formation
tlrorough nrixirrg of the fernrentation contents. The media used in industrialfermentationis
Som et ir r es ,an ac id or alk aline m ediu m c o m p o n e n t
general i yri ch i n protei ns.W hen agi tateddu r ing
ca n be us ed t o c or r ec t pH, besi d e s p r o v i d i n g
aeration. it invariably results in froth or foam
n utr ient st o t he gr owing nr ic r oor gan i s m s .
formati on that bui l ds i n head space of t he
bioreactor.Antifoamchenlicalsare usedto lower
Temperature surfacetensi onof the medi um, besi descau sing
Temperaturecontrol is absolutely essentialfor a foam bubblesto collapse.Mineral oils basedon
good fermentation process. Lower temperature si l i coneor vegetabl e oi l s are commonl yused as
causes reduced product formation while higher antifoamagents.
ternperature adversely affects the growth of Mechanicalfoam controldevices,referredto as
micr oor Ranis m s . ' i- he bior eac t or s a r e n o r m a l l y mechanicalfoam breakers, can also be used.Such
e qu ipped wit h heat ing and c ooling s y s t e m s t h a t devices.fittedat the too of the bioreactorbreakthe
can be usecias per the requirement,io maintain the foam bubbles and the throw back into the
reaction vessel at optima! temperature. fermentati onmedi um.

Dissolved oxygen C LE A N IN G
O x y gen is s par ingly s oluble in w a t e r ( 0 . 0 0 8 4
As the fermentationis compiete,the bioreactor
g/1 at 25'C\. Continuous suppiy of oxygen in the
is harvested i.e. the contents are removed for
form of s t er iliz edair is done t o t he c u l t u r e m e d i u m .
processing. The bioreactoris then prepar€dfor the
Th is is c ar r ied out by int r oduc ing a i r i n t o t h e
next round of fermentation after cleaning
bioreactor in the form of bubbles. Continuous
(technicallycalled turn round).The time taken for
rnonitoring of dissolved oxygen concentration is
turn round, referredto as down time, should be as
done in the bioreactor for optimal product
short as possi bl e(si nce i t i s non-produ ct ive) .
folmation.
Due to large size of the bioreactors,it is not
possi bl eto cl ean manual l y.The cl eani ngof t he
Adequate m:xing
bioreactorsis carried out by using high-pressure
Cont inuous and adequat e m i x i n g o f t h e water jets from the nozzlesfitted into the reaction
micr obial c ult ur e ens ur es opt im a l s u p p l y o f vessel.
ChA O t Cr TE C H N OLOC Y
19 : BIOP R OC ES S/F E R ME N T A TION 247

Tlru 19.2 A setected list of solid state fernentatlons

Product Substrate Mic roorganism(s) i nvolved

Edible
mushrooms Strawmanure Agaricus
bisporus
Lentinula
edodes
Cheeses Milk,curd roquefortii
Penicillrum
Soysauce Soybeans,wheat Aspergillus
oryzae
Sauekraut Cabbage Lactic
acidbacteria
Enzymes Wheatbran Aspergillus
niger
Organic
acids Canesugar,molasses Aspergillus
niger
Leaching
of metals Lowgradeores Thiobacillus
sp.
Composting Mixedorganic
material Fungi,
bacteria,
actinomyceles
Sewage
treatment Sewagecomponents fungi,
Bacteria, protozoa

For sol i d substratefermentati on,si ngl e pure

Thereare certainfermentation processes that do
not inv olv e l i q u i d me d i u m . F o r th e s e bi o-
technological processes,the growth of the
microorganisms is carried out on solid substrates
in the complete absence or almost complete
absenceof free water. The presenceof some
moisture (about 15%) is necessaryfor solid
substrate(or solid state)fermentation(55D. The
most commonly used solid substrates for SSFare
cerealgrains,wheat bran,sawdust,wood shavings
and severalotherplantand animalmaterials. These
solid substrates are polymericin nature,insoluble
or sparingly soluble in water, and contain
concentrated sourceof nutrientsfor the growth of
m ic r oor gani s m s .
S S Fis a v e ry o l d tra d i ti o n ate
l c h n i q u ec a rri ed are depicted.
out in many countries.lt is usedfor the productron
of edible mushrooms,cheese,soy sauceand many Advantages of SSF
other fermentedproducts(includingenzymesand
organic acids). A selected list of solid state
fermentationsis given Table 19.2. Compostingis a
good example of SSF.
Solid substratefermentationhas been very .
popular for the production of fermented foods
(idli, dosa, dhokla, bread, beverages,fermented
fish, meat, yogurt, cheese,pickles).Fermentation . Y i el dof the productsi s reasonablhi
often makes the food more nutritious,
digestableand betterin flavour.
cultures,mixed culturesor mixed organisnrs rnay
be used.Pretreatment raw materialsrs
of substrate
sometimesdone to facilitatethe availabilityof
nutri ents.
S ol i dsubstrate
fermentati on i s normal l vcarri ed
out as a non-aseptic process. This saves
sterilizationcosts.lt is imoortantthat the substrares
used in SSFhave adequatespacesin betr.reen to
al l ow goodai r ci rcul ati on.
Thi sfaci l i tates
adequate
exchange of gases, besides oromoting heat
el i mi nati on.Forcedai r ci rcul ati onmay be done to
mai ntai nopti malcondi ti onsi n S S F.
Bioreactors for SSF
Bioreactors designedfor solid statefermentation
are much si mpl er compared to l i qui d-state
fermentation.In the Fig 19.6, tower reactor,drum
reactor and forced aeration reactor, used in SSF
. Solid substratefermentationemploys simple
naturalsol i dsas the medi a.
r Low technology,lour energy expenditureand
requireslesscapital investment.
N o need for steri l i zati on, l ess mi crobi al
contaminationand eas,r, downstreamprocessing.
y gh.
easily o Bioreactordesign, aerationprocess, and effluent
treatmentare ouite simole.
244 B IOTE C H N OLO CY

r P r e c i s e m o n i t o r i n g o f S S F ( e . g . , O z a n d C O,
levels, moisture content) is not possible

. T h e o r g a n i s m s g r o w s l o w l y a n d c o n s e q u e n tl y
there is a limitation in product formation.

o Heat prodr,rction creates problems, and it

is very difficult to regulate the grovrth
envrronment.

The media used for the Erowth of

m i c r o o r g a n i s m s i t . t i n d u s t r i a l f e r m e n t a t i o n m u sf
contain all the elements in a suitable form for the
synthesis of cellular substances as well as the
Tower reactor netabolic products. While designing a medium,
several factors must be taken into consideration.
T h e m o s t i m p o r t a n t a n t o n g t h e m l s th e
u l t i m a t e p r o d u c t d e s i r e d i n t h e f e r m e n t a t i o n . Fo r
growth-linked products (primary metaboliies
e . g . e t h a n o l , c i t r i c a c i d ) , t h e p r o d u c t f o r m a t i o n s ts
d i r e c t l y d e p e n d e n to n t h e g r o w t h o f t h e o r g a n i sm s,
hence the medium should be such that it
supports good growth. On the other hand, for
p r o d u c t s w h i c h a r e n o t d i r e c t l y l i n k e d t o tn e
g r o w t h ( s e c o r r d a r l r m e t a b o l i t e s e . g . a n t i b i o ti cs,
a l k a l o i d s , g i b b e r e l l i n s ) ,t h e s u b s t r a t er e q u i r e me n ts
for product formation must also be considered.

Fig. 19,6 : Bioreactors for solid-substrate
fermentatian.
. Manv dom es t ic , indus t r ialand agr ic u l t u r a lw a s t e s
can be f r uit f ully us ed in SSF
Limitations of SSF
. The m ic r oor ganis m s t hat t oler a t e o n l y
mois t ur e c ont ent c an be us ed.
I n t h e l a b o i 'a t o r y ,p u r e d e f i n e d c h e m i c a l s m a y
b e u s e df o r c u l t u r i n g m i c r o o r g a n i s m sH . o w e v e r , fo r
i n d u s t r i a l f e r m e n t a t i o n s , u n d e f i n e d a n d c o m pl e x
substrates are frequently used for economic
reasons.Cheaper substratesare advantageoussince
t h e y m i n i m i z e t h e p r o d u c t i o n c o s t o f t h e f e r m e nte d
products. Wastesfrom agriculture,and byproducts
of other industriesare generally preferred,although
they are highly variable in composition. Raw
materialsused in fermentation largely depend on
their cost at a particular time, since there are
s e a s o n a lv a r i a t i o n s .
T h e c h o i c e o f t h e m e d i u m i s v e r y c r i t i c a l fo r
s u c c e s s f u l p r o t l u c t f o r m a t i o n . F o r i n d u str i a l
[ e r n r e n t a t i o n , t h e m i c r o o r g a n i s m s , i n g e n er a l ,
utilize a luxury metabolism. Therefore, good
production yields are expected with an aburrdant
s u p p l y o f c a r b o n a n d n i t r o g e n s o u r c e s , b e si d e s
low requisite growth factors. The media used in
fermentation processes rnay be synthetic or crude.
 TE C H N OLOGY
Chapt er1 9 : B IO P R OC ES S/F E R ME NTA TION 249

Synthetic media They are frequently used for the industrial

producti onof al cohol .D ue to i ts w i de avai l abi l i ty
Media with all the requisiteconstituentsin a
and low cost, the use oi cellulose for alcohol
pure form in the desired proportion represents
productionis extensivelystudied.
syntheticmedia. Use of this type of media rn
fermentationsis not practicable. Whey

Grude media Whey is a byproduct of dairy industry and is

producedworldrn'ide.Most of it is consumedby
T henon -s y n th e timce d i aw i th n a tu ra l l avai
y l abl e humansand ani mal s.W hey i s a reasonablgood y
sourcesare better suited for fermentation. source of carbon for the productionof alcohol,
ln practice,crude media with an addition of si ngl e-celprotei l n,vi tami n B 1r, l acti c aci d and
requisitesyntheticconstituentsis ideal for good gibberellicacid.Storageof whey is a limitingfactor
productyield in fermentation. for its widespreaduse in fermentationindustry.

The mostfrequentlyusedsubstrates for industrial Methanol and ethanol

fermentationwith specialreferenceto the supplyof
carbonand nitrogensourcesand growthfactorsare Some of the microorganisms are capable of
brieflydescribedbelow. utilizingmethanol and./or ethanolas carbor.rsource.
Methanol is the cheapest substrate for
S UB S T RAT E S U S ED fermentation- Ho'rvever,it can be utilized by only
A S CA RB O N SOU R C ES a few bacteriaand yeasts.Methanolis commonly
used tbr the production of single-cellprotein.
Carbohydrates constitutethe most predominant Ethanolis ratherexpensive.Florvever, at presentit
sourceof energyin fermentationindustry.Refined is usedfor the oroductionof aceticacid.
and pure carbohydrates suchas glucoseor sucrose
are rarelyusedfor economicreasons. S U B S TR A TE S U S E D
A S N ITR OGE N S OU R C E S
Molasses
The nitrogen supply to the fermentation
Molassesis a byproductof sugarindustryand is mi croorgani sms may come from i norgani c or
one of the cheapest sources of carbohydrates. orSanrcsources.
Sugarcane molasses(sucrosearound 4B%) and
sugar beet molasses(sucrosearound 33o/olare Inorganic nitrogen sources
c om m onl y u s e d . Be s i d e sb e i n g ri c h i n sugar,
Ammonium salts and tree ammonia are cheap
molassesalso contain nitrogenoussubstances,
inorganic nitrogen sources, particularly in
vitaminsand tracgelements. Thereoccursvariation
industrialisedcountries. However, not all the
in the compositionof the molasseswhich mostly
production mi croorgani sms are capabl e of uti l i si ng them,
dependson the climaticconditionsand
hence thei r use i s l i mi ted.
process.Hydrol molasses,a byproduct in glucose
productionfrom corn, is alsousedas a fermentation
Organic nitrogen sources
substrate.
Urea is fairly a good source of nitrogen.
Malt extract However,other cheaperorganicforms of nitrogen
sourcesare preferred.
Malt extract, an aqueous extract of malted
barely,containsaboutB0%carbohydrates (glucose, Corn steepliquor : This is fornredduringstarch
fructose,sucrose,maltose).Nitrogen compounds productionfrom corn. Corn steepliquor is rich rn
constitutearound4.5% (proteins,peptides,amino nitrogen(about4%) and.is uery efficientlyutilized
ac ids ,puri n e s p, y ri m i d i n e s ). by nri croorgani sms. It i s ri ch i n severalami noaci ds
(al ani ne,val i ne, methi oni ne,argi ni ne,threoni ne,
Starch, dextrin and cellulose glutamate).

The polysaccharides-starch, dextrin and Yeastextracts: ThevcontainaboutB% nitrogen

cellulosecan be metabolisedby microorganisms.and are ri ch i n ami noaci ds,pepti des
and vi tami ns.
25tJ
Glucose formed from glycogen and trehalose
during yeast extraction is a good carbon source.
Yeast extracts are produced from baker's yeast
th rough aut oly s is ( at 50- 55' C) o r t h r o u g h
p ias m oly s is ( high c onc ent r at ion o f N a C l ) . Y e a s t
extractsare very good sourcesfor many industrially
important microorganisms.
Soy meal : After extractingthe soy bean oil from
the so1,bean seeds,the left out residue is soy rneal.
It i s r ic h in pr ot eins ( about 50% ) a s w e l l a s
carbohydrates (abor-rt307o) contents. Soy meal is
otien used in antibiotic production.
Peptones : I he protein hydrolysates are
collectively referred to as peptones, and they are
good sources for many microorganisms. The
sourcesof peptonesinclude meat, soy meal, peanut
seeds, cotton seeds and sunflower seeds. The
proteins namely casein, gelatin and keratin can
also be hydrolysed to yield peptones. In generat,
peptones derived from animal sources have more
nitrogen content while those frorn plant sources
have more carbohydrate content. Peptones are
relatively more expensive, hence not widely used
in i ndus t r ies .
SOURCES OF GROWTH FACTORS
Sorne of the microorganismsare not capable of
synthesisingone or more growth factors such as
vitamins. These growth factors are very expensive
in pure form, hence crude sources are preferred.
Yeast extract is a rich source of almost all growth
factors.
Cenerally, the substratesderived from plant or
animal sources in a crude form are reasonablyrich
in mineral conteht. Sometimes, however mineral
(phosphate, sulfate) supplementation may be
reouired.
For successful fermentation, it is absolutely
esserrtialto ensure.
. Sterility of the media containing the nutrients.
o St er ilit y of inc om ing and out goin g a i r .
. Sterility of the bioreactor.
.. Preventionof contamination during fermentation.
B IOTE C H N O LO CY
A brief account on the sterilization of tne
bioreactor has already been described (See p...). A
bioreactor can be sterilized by destroying the
organisms by heat/chemicals/radiation or sometimes
by physical procedures such as filtration.
Sterilizationof media and air are discussedbelow.
STERILIZATION OF CULTURE MEDIA
The constituents of culture media, water and
containers contribute to the contamination by
vegetativecells and spores.The mecjiamust be free
from contamination before use in fermentation.
Sterilization of the media is most comnronly
achieved by applying heat, and to a lesser extent
by other means (physical methods, chenrical
treatment, radiation).
Heat sterilization
Heat is the mosf widely used sterilization
technique. The quality and quantity of
contamination (i.e., the type and load of
microorganisms),compositiorr of the media and its
pH and size of the suspended particles are the
imoortant factors that influence the suciess of heat
sterilization. In general, vegetative cells are
destroyed at lower temperature in a short time
(around 60'C in 5-1 0 minutes). However,
destruction o{ spores requires higher temperature
and relatively longer time (around BO"C for 15-20
minutes). Spores of Bacillus stearothermophilus are
the most heat resistant. In fact, this organism is
exploited for testing the sterility of fermentation
equipment.
Physical methods
The physical methods such as filtration,
centrifugation, and adsorption (to ion-exchangers
or activated carbon) are in use. Among these,
filtration is most widely used. Certain constituents
(vitamins,blood components, antibiotics)of culture
media are heat labile and therefore, are destroyed
by heat sterilization. Such components of the
medium are completely dissolved (absolutely
essentialor eise they will be removed along with
microorganisrns) and then subjected to filter
sterilization. There are a couole of limitations of
filtration technique.
1 . A p p l i c a t i o n o f h i g h p r e s s u r e i n f i l t r a t io n r s
u n s u i t a b l et b r i n d u s t r i e s .
 TE C H N OLOC Y
Chapt er19 : BIOP R OC ES S/F E R ME N T A TION 251

15 0 bioreactorto attain the requisitetemperature(i.e.

120' C ). A nother 20-60 mi nutesfor the actuar
125 processcf sterilization,followed by cooling for
1-? hours. All this processinvolves wastageof
I roo energy,and thereforebatch sterilizationis quite
E costly.
l
c
(d
q) f
Q)
o Continuous sterllization
E c o
o
P5 0 c
o Continuoussterilizationis carriedout at 140oC
o
for a very short period of time ranging from
o
E - 30 to 120 seconds.(Thisis in contrastto the batch
(! o
o) o) fermentationdone at 121"C for 20-60 minutes).
I (E
This is basedon the principlethat the time required
J
for ki l l i ngmi croorgani sms
i s muchshbrterat hi gher
Stages
temperature. Continuoussterilizationis carriedout
by directlyinjectingthe steamor by meansof heat
Fig. 19.7 : Different stages in continuous sterllization exchangers. In eithercase,the temperatureis very
process in relation to tempenturc.
qui ckl y rai sedto 140" C ,and mai ntai nedfor 30-
I20 seconds.The stagesof continuoussterilization
processand the correspondingternperatures are
2. Some of the media components may be lost
depicted in Fig: 19.7. fhe different stagesare-
fo rm th e med ia dur ing f ilt r at ion.
exchanger,heater, heatmaintenanceunit, recovery
Sometimes,a combination of filtration and heat of residual heat, cooling and fermenter.
sterilization are applied. For instance, the water
ln the continuoussterilizationprocess,3 types
used for media preparation is filtered while
of heat exchangersare used. The first heat
concentrated nutrient solution is subiected to heat
exchangerraisestemperatureto 90-120"C within
sterilization. The filtered water is now added for
20-30 seconds.The second exchangerfurther
ap pro pria te dilu t ion of t he m edia.
rai sestemperatureto 140" C and mai ntai nsfor
The chemical .methods (by using disinfectants) 30-120 seconds.The third heat exchangerbrings
and radiation procedures (by using UV rays, y rays, down the ternperatureby cooling in the next
X-rays) are not commonly used for media 20-30 seconds.The actual ti me reoui red for
ste riliza tion . steri l i zati on
dependson the srzeof the suspended
particles.The bigger is the size, the more is the
Batch
ti me requi red.
sterilization
The main advantage with continuous
The culture media are subjected to sterilization
sterilizationis that about 80-900/6of the energyis
at 121oC in batch volumes, in the bioreactor.
conserved.The limitation however,is that certain
Batch sterilization can be done by injecting the
compounds i n the medi um preci pi tate(e.9.,
steam into the medium (direct method) or injecting
calcium phosphate,calcium oxalate)due to very
the steam into interior coils (indirect method). For
high temperaturedifferencesthat occur in a very
the d irect b atc h s t er iliz at ion, t he s t eam s ho u l d
short time betweensterilizationand cooling.The
be pure, and free from all chemical additives
(that usually come from stearn manufacturing
starch-containing culture media becomesviscous
in continuoussterilization and thereforeis not used.
process). fhere are two disadvantages of batch
sterilizatio n.
S TE R ILIZA TION OF A IB
1. Damage to culture media : Alteration rn
In general, the industrial fernrentationsare
nu trien ts,ch an ge in pH and dis c olour at ion of t h e
carriedout undervigorousand continuousaeration.
cu lture med ia a r e c om m on.
For an effective fermentation, the air should be
2. High energy consumption : lt takes a few completely sterile, and free from all micro-
hours Q-4 hrs) for the entire contents of the organisms and suspended particles.Thereis a wide
252 B IOTE C H N OLO CY

---)A i rout

Diffusion

Fig. 19-8 : Use of depth filter in air sterilization.

varia tion in t he quant it y of s us pendedp a r t i c l e sa n d
microb es in t he at m os pher ic out do o r a i r . T h e
microo r ganis m s m ay r ange f r om 1 O- 2 , O O O / n 3
wh ile t he s us pendedpar t ic les m ay be 2 O - 1 0 0 , 0 0 1
m3 . Am ong t he m ic r oor ganis m spr es e n t i n t h e a r r ,
the fungal spores(50%) and Cram-negativebacteria
(4 0% ) dom inat e.
Air or ot her gas esc an be s t er iliz ed b y f i l t r a t i o n ,
h ea t, UV r adiat ion and gas s c r ubb i n g . A m o n g
th ese, heat and f ilt r at ion ar e m os t c om m o n l y u s e d .
Air sterilization by heat
In the early years, air was passed over
e lectric ally heat ed elem ent sand s t er il i z e d .B u t t h i s
is qu it e ex pens e, henc e not in us e t he s e d a y s .
recent years, glassfiber filter cartridges(that do not
h a v e t h e l i m i t a t i o n s o f g l a s s w o o l f i l t e r ) a r e b e in g
useo.
Membrane cartridge filters : These are
removable pleated membrane filters made up of
cellulose ester, nylon or. polysulfone. Membrane
c a r t r i d g e f i l t e r s a r e s m a l l e r i n s i z e , s i m p l e r fo r
operation and replacement.
T h e m o s t i m o o r t a n t l i m i t a t i o n o f a i r s t e r i l i z a ti o n
'is
that there is no filter that can remove
bacteriophages. Bacteriophages are capable of
crippling the i n d u s t r i a l f e r m e n t a t i o n . e .8 .,
bacteriophages interfere in the production of
glutamic acid by Corynebacteriumglutamicum.

Air sterilization by filtration

Filtr at ion of air is t he m os t c om m o n l y u s e d

sterilizat ion in f er m ent at ior rindus t r ies. There are over a mi l l i on speci esof mi cr o-
organi sms w i del y di stri butedi n nature.Lessth an
Depth filters : When the air is passedthrough a
1" k of the w orl d' s mi croorgani sms have be en
g lass w ool c ont aining dept h f ilt er s th e p a r t i c l e s
studied. In fact, only a few hundred speciesare
are trapped and removed (Fig. l9.B). This filtration
important for industrial use. A selected list of
te ch ni que pr im ar ily inv olv es phy s i c a l e f f e c t s
organi smsal ong w i th thei r productsi s gi ven r n
such as inertia, blocking, gravity, electrostatrc
Table 19.3.
attraction and diffusion. Class wool filters can be
subjected to steam sterilization and reused. But The good sources for the isolation of
th ere i s a lim it at ion in t heir r eus e s inc e g l a s sw o o l aresoi l s,l akesand ri vermuds.l t is
mi croorgani sms
shrin k s and s olidif ies on s t eam s t e r i l i z a t i o n . r n estimatedthat a eram of soil contains 106-108
 .19 TE C H N OLOGY 253
Chapt er : B IO P R OC ES S/F E R ME N TA TION

4 A erosoldi l uti on
Tasrr 19.3 A selected list of important
Flotation
fiiicyoorganismsand their products
o. C entri fugati on.
Microorganism Product
F o r f u l l d e t a i l s o n t h e i s o l a t i o n p r o c e d u r e s ,t h e
Algae reader must refer books on microbiology. The
Chlorellasorokiniana protein
Single-cell actual technique for the isolationof nricroorganisms
maxima
Spirulina protein
Single-cell depends on the source and the physiological
properties of microorganisms.The general scheme
Bacteria
adopted for isolating microorganismsfrom soil or
Acetobacteraceti Aceticacid
water source is given below.
Acetobacterwoodii Aceticacid
Bacillussubtilis Bacitracin . The sample (soil or water) is diluted with sterile
w a t e r t o w h i c h a n e m u l s i f y i n g a g e n t ( T w e e n )i s
B. brevis Gramicidin
added.
B. thuringiensis Endotoxin
Clostridiumaceticun Aceticacid . Sample is throughly mixed and allowed to stand
at room temperature
Methylophil
us nethylotroph
us Glutamicacid
Pseudomonas denitrificans VitaminB.,, . S u p e r n a t a n ti s d i l u t e d , 1 6 - 1 1 o 1 6 - 1 0 .

Actinomycetes o V a r i o u sc u l t u r e m e d i a a r e i n o c u l a t e dw i t h d i l u t e d
aureof
Streptomyces aciens Tetracycline s a m p l e sa n d i n c u b a t e d .
S. griseus Streptomycin . Colonies fronr the plates are isolated and
S. tradiae Neomycin i d e n t i fi e d .
Nocardianediterranei Rifamycin o The required pure strains are maintained and
M!,!g'?l??l?i?
P!w7? Gentamycin preserved.
Fungi
Enrichment methods
Aspergillus
niger Citricacid
for isolation of microorganisms
A oryzae Amylase, cellulase,
'l-he culture conditions can be appropriately
protein
single-cell
Candidalipolytica Lrpase modifiedto isolatecertaintypesof microorganisms.
protein The typesof organisms that can be isolatedby use
C. utilis Single-cell
of enrichmentmethodsis given in Table19.4.For
Penicillium
chrysogenum Penicillin
instance,thermophilescan be isolatedby using
Saccharomyces cerevisiae Ethancl,wine, hi ghtemperature w hi l e aci dophi l es
can be i sol ated
protein
single-cell i n aci di c pH . E nri chmentmethodsare certai nl y
S. lipolytica Citricacid, useful for quick isolation of specific types of
protein
single-cell orS anrsms.
Rhizopusnigricans Steroids
Gibberellafujikuroi Gibberellin Strains of microorganisms
Trichoderna vuide Cellulase from unusual environments
Biotechnologistsoften prefer to isolate
bacteria, 104-106 actinomycete spores and
microorganisms from very extreme and unusual
102--104fungal spores. The common techniques environments. This is done with a hope that such
e mplo ye d f or t he is olat ion of m ic r oor gan i s m sa r e strainsmay be capableof
producingnew products
given below. of i ndustri al
i mportance. The unusualenvi ronments
such as cold habitats,high altitudes, deserts,deep
1 . Dire c t s ponge of t he s oil
seaand petroleum fields areconstantlybeing tried
2 . Soil dilut ion for this purpose.The enrichmentmethodsdescribed
3 . Crad ient plat e m et hod ( pour plat e an d s t r e a k above(Iable 19.4)will be very usefulfor isolating
p late techn ique) Lrnusual strai ns.
254 B IOTE C H N OLOCY

P R E S E R V A TION OF MIC R OOR GA N IS MS

Tlau 19.4 Types of nlcoorganisns that
can be isolated by enrichnent methods There are distinct methodsfor preservation of
microorganisms. The most importantbeing storage
Type of organisms Enrichmentmethod by refrigeration, freezing and lyophilization.

Thermophiles (42-100"C)
Hightemperature
Psychrotroohs Lowtemperature
(5-15'C)
Acidophiles LowpH (2-4)
Halophiles HighNaClconcentration
The microorganisms possess tremendous
Anaerobes N, atmosphere
capacityto producea wide rangeof productsthat
Actinoplanes grains
Pollen havecommercialvalue.The primaryand secondary
metabolisms and bioconversions of microorganisrns
Myxobacteria Woodbark
with specialreferenceto their importancefor the
formationof biotechnologicallyimportantproducts
Screening of metabolites for isolation are di scussedhereunder(Fi g. 19.9). Mi crobial
of microorganisms
growth in relation to primary and secondary
metabolismsis depictedin Fig. 19.10.
'fhe
microorganisms can be tested directly for
the product formation, and isolated. In fact, the PRIMARY METABOLITES
water or soil samples can be directly used or
Primary metabolism, also referred to as
su itab ly dilut ed f or m et abolit e s c r e e n i n g . A g a r
trophophase,is characterizedby balancedgrowth
plates can t-'eused for screeningmetabolitesforrned
l t occursw hen al l the nutri e nt s
of mi croorgani sms.
from the m ic r oor ganis m s . For ins t a n c e , i f t h e
needed by the organismsare provided in the
required product is an antibiotic, then the test
medium. Primarymetabolismis essentialfor the
system consists of the strains of organisms which
verlr existenceand reproductionof cells. In the
inh ibit t he z ones , on t he agar plat es .Th e i n h i b i t o r y
trophophase, the cells possess optimal
activitv indicates the possible presence of some
concentrations of almost all the macromolecules
an tibio t ic being pr oduc ed by t he m icr o o r g a n i s m s .
(proteins,DNA, RNA etc.).
Ano the r ex am ple is t he is olat ion of m ic r o o r g a n i s m s
p rod ucing am y las es .W hen gr own on a g a r p l a t e s It i s duri ng the peri od of trophophasea, n
co nta ining s t ar c h, and t hen s t ained w i t h i o d i n e , exponential growth of microorganismsoccurs.
amylase- or oduc ingor ganis m sc an be id e n t i f i e da n d Severalmetabol.ic products,collectivelyreferredto
isolated. as primary metabolites, are produced in
trophophase (i.e.,duringthe periodof growth).The
Screening for new metabolites, primarymetabolitesare divided into two groups.
and isolation of microorganisms 1. Primaryessentialmetabolites: Thesearethe
Ind us t r ial m ic r obiologis t sc ont inue t h e i r s e a r c h
compounds produced in adequate quanties to
for newer metabolitesproduced by microorganisms.
sustain cell grawth e.g. vitamins, amino acids,
Research work is particularly directed for
nucleosidesThe nativemicroorganisms usuallydo
ide ntify ing
not overproduceessentialprimary metabolites,
c hem ot her apeut ic ally importarrt
products for the treatment of tumors, bacterial since it is a wasteful exercise. However, for
d ise ases( newer ant ibiot ic s agains t r es i s t a n ts t r a i n s )
i ndustri al overproducti on, the regul at or y
mechani sms are sui tabl vmani oul ated.
and viral diseases,besidesseveral other substances
(e .g. h or m ones , enz y m e inhibit or s ) . I n a d d i t i o n , 2. Primary metabolic end products : Theseare
iso lati on of m ic r oor ganis m s f or im pr o v e m e n t o f the normal and traciitional end products of
fooC indusiry, and for efficient degradation of the fermentation processof primary metabolism. fhe
e nvironm ent al pollut ant s and haz ar do u sc h e m i c a l s end productsmay or may not haveany significant
a lso ass um ess ienif ic anc e function to perform in the nricroorganisms,
Chapt er19 : B IO P R OC ESF
S/ER M EN T ATION
TE C H N OLOC Y 255

S U B S TR A TE

Prima r y m et abolit es Se c o n d a r v m e t a b o l i t e s B i o c o n v er s i o n s

Es s ent ial m et abolit es Antibiotics Steroids

Am ino ac ids Alkaloids Amino acids
Nuc leos ides Gibberellins Ascorbic acid
Vit am ins Pigments

M et abolic end pr oduc t s

Et hanol, Ac et one
Lac t ic ac id
But anol

Fig. 19.9 : Dilferent types of low-molecularweight compoundsprodueedby microorganisms.

although they have many other industrial In the above example, an unbranched
applicationse.g. ethanol,acetone,lactic acid. pathway is shown. This type of manipulation for
overproduction of metabolites can be done for
Carbondioxide is a metabolicend oroduct
branched metabolic pathways also.
of Saccharomyces cerevisiae.This CO, is essential
for leaveningof dough in baking industry. 2. Mutant microorganisms with antimetabolite
limitations in growth : Due to insufficienV resistance which exhibit a defective metabolic
limited supply of any nutrient(substrate or even regulation can also overproduce primary
Or), the growth rate of microorganismsslows metabolites.
t,
down. However,the metabolismdoes not stop. lt l
a
continuesas long asthe cell lives,but the formation SECONDARY METABOLITES I
of oroductsdiffers. As the exponential growth of the
microorganisms ceases (i.e. as the trophophase
Overproduction of primary metabolites ends), they enter idiophase. ldiophase is
Excessiveproductionof primary metabolitesis characierized. lsy secondary metabolism wherein
very importantfor their largescaleusefor a variety
of purposes.
Overproduction of severalmetabolites
hasbeensuccessfully accomplished by elimirrating
the feedbackinhibitionas brieflydescribedbelow.
1. B y u s i n ga u x o tro p h i cmu ta n tsw i th a bl ock
in one of the steps in the biosyntheticpathway
concernedwith the formationof prinrarymetabolite
(this should be an intermediateand not the final
end product).In this manner,the end product(E)
formationis blocked,henceno feedbackinhibition. Hi:
But overproductionof the requiredmetabolite(C) E o
occursas illustratedbelorv. o
o
Feedbackregulation :

A-r--+ B---------* c ----=--+ D---+ E llme ---J

- ^T- ^
. Required ... Blocked Finaleno Fig. 19.10 : Microbiai growth in relation to primary
primarymetabolite reactlon Droduct (trophophase) and secondary (diophase) metabolism.
(A is startingsubstrate;B and D are intermediates)
256
the formation of certair.rmetabolites, referred to as
secondary metabolites (idiolites\ occurs. These
metabolites, although not required by the
microor ganis m s ,ar e pr oduc ed in ab u n d a n c e T h e
secondary metabolites however, are industrially
very important, and are the most exploited rn
biotechnology e.g., antibiotics, steroids, alkaloids,
g ibb er ellins ,t ox ins .
Cha r ac t er is t ic s of s ec ondar y
meta bolit es
1. Sec ondar y m et abolit es ar e s p e c i f i c a l l y
produced by selected few microorganisms.
2. They are not essential for the growth anci
reproduction of organisms from r.vhich they are
p rodu c ed.
3 . Env ir onm ent al f ac t or s inf l u e n c e the
production of secondary metaboliles.
4 Som e m ic r oor ganis m s pr odu c e s e c o n d a r y
me tabolit es as a gr oup of c om po u n d s ( u s u a l l y
struc t ur ally r elat ed) ins t ead of a s i n g l e o n e e . g .
a bo ut 35 ant hr ac y c linesar e pr oduc e d b y a s i n g l e
strain of Streptomyces.
5 . The bios y nt het ic pat hways f o r most
secondar y m et abolit esar e not c lear l y e s t a b l i s h e d .
6 The regulation of the formation of secondary
metabolites is more complex and differs from thar
of primary metabolites.
Functions of secondary metabolites
As already stated,secondary metabolitesare not
e ssent ial f or gr owt h and m ult iplic a t i o n o f c e l l s
The ir oc c ur r enc e and s t r uc t ur es v a r y w i d e l y .
Several hypotheseshave been put forth tb explarn
the role of secondary metabolites,two of them are
g ive n below.
1. The secondary metabolrtes ntay perform
certain (unknown) functions that are beneficial for
th e c ells t o s ur v iv e.
2. fhe secondary metabolites have absolutely
no function. fheir production alone is imporlanl
for the cell, whatever may be the product (which is
con s ider edt o be us eles s ) .
Overproduction of secondary
metabolites
As already stated, the production of secondary
me tabolit es is m or e c om plex th a n p r i m a r y
B IOTE C H N OLO CY
m e t a b o l i t e s .H o w e v e r ,t h e r e g u l a t o r ym a n i p u l a ti o n s
e m p l o y e d f o r e x c e s s p r o d u c t i o n o f p r i ma r y
metabolites can also be used for the secondarv
m e t a b o l i t e sa s w e l l .
S e v e r a lg e n e s a r e i n v o l v e d i n t h e p r o d u c t i o n o f
secondary metabolites. Thus, around 300 genes
p a r t i c i p a t e i n t h e b i o s y n t h e s i so f c h l o r t e t r a c ycl i n e
w h i l e 2 0 0 0 g e n e sa r e d i r e c t l y o r i n d i r e c t l y i n v o lve d
i n t h e p r o d u c t i o n o f n e o m y c i n . Wi t h s u c h c o m p l e x
systems, the metabolic regulation is equally
complex to at:hieve overproduction of secondary
metabolites. Some regulatory mechanisms are
b r i e f l y d i s c u s s e dh e r e u n d e r .
Induction: A d d i t i o n o f m e t h i o n i n e i n d u ce s
certain enzymes and enhancesthe production of
cephalosporin. Tryptophan regulatesergot alkalorcl
biosynthesis.
End producl regulation : Sonre of the secondary
n r e t a b o l i t e s i n h i b i t t h e i r o w n b i o s y n t h e s is, a
phenornenon referredto as end prociuct regulation
penicillin, p t i r o m yci n ,
e.g. streptomycin,
c h l o r a m p h e n i c o l l t i s p o s s i b l e t o i s o l a t e m u t a n ts
that are less sensifive to end product inhibition,
and in this manner the secondary metabolite
production can be increased.
Catabolite regulation : In this regulationprocess,
a key enzyme involved in a catabolic pathway is
inactivated, inhibited or repressedby adding a
commonly used substrate.Catabolic repressioncan
b e a c h i e v e d b y u s i n g c a r b o n o r n i t r o g e n s o u rce s.
T h e m e c h a n i s mo f a c t i o n o f c a t a b o l i t e r e g u l a t i oni s
not verv clearlv understood.
The most commonly used carbon source is
glucose. tt is found to inhibit the productian of
several antibiotics e.g perricillin, streptomycin,
bacitracin,chloramphenicol, purornycin.
fhe nitrogen sources such as ammonia also act
a s c a t a b o l i t e r e g u l a t o r s ( i . e . i n h i b i t o r s ) f o r th e
overproduction of certain antibiotics.
Phosphate regulation : lnorganic phosphate (Pi)
is required for the growth and multiplication of
prokaryotes and eukaryotes Increasing Pi
concentration (up to 1mM) is associatedwith an
increased production of secondary metabolites e.g.
a n t i b i o t i c s ( s t r e p t o m y c i n , t e t r a c y c l i n e ) , a l k a l oi d s,
g i b b e r e l l i n s .H o w e v e r , v e r y h i g h P i c o n c e n t r a t i o ni s
i n h i b i t o r y , t h e m e c h a n i s m o f a c t i o r r i s n o t ve r y
c tear.
 Chapt er19 : B IO P R OC ES S/F E R ME N TA TION
TE C H N OLOC Y 257

Autoregulation : ln some microorganisms

(particularlyactinomycetes), there occurs a self
regulation for the production of secondary
metabolites. A compounddesignatedas factor A
which is analogousto a hormoneis believedto be In general,the wild strainsof microorganisms
closely involved in autoregulation for the produce low quantities of commercially important
production streptomycinby Streptomycesgriseus. metabolites,althoughthe yield can be increased
More such factorsfrom other organismshave also by opti mi zi ngthe fermentati oncondi ti ons.The
been identi fi e d . potentiality of the metabolite formation ts
genetically determined. Therefore. genetic
BrocoNvERsroNs improvementshave to be made and new strains
developedfor arry substantialincreasein product
Microorganismsare also used for chemical
formationin a cost-effective manner.
transformationof unusual substratesto desired
products. This process, also referred to as There are strain deveiopment programmes
biotransformation,is very important in producing (mutati on and recombi nati on)to i ncreasetne
severalcompoundse.g. conversionof ethanolto productyi el d by 100 ti mes or even more. The
acetic acid (in vinegar), sorbitol to sorbose, nature of the desired product determinestne
synthesisof steroid hormonesand certain amino successassociatedwith strain improvement.For
ac ids . i n one or tw o genes(i .e.one
exampl e,i f al terati ons
or 2 key enzymes)can improvethe productyield.
I n bioc o n v e rs i o nmi
, c ro o rg a n i s ms
c onvert a
it is simpler to achievethe target.This type of
compoundto a structurallyrelatedproductin one
approach i s someti mespossi bl e w i th pri mary
or a few enzymaticreactions.The bioconversions
metabolites. As regardsthe secondarymetabolites,
can be carriedout with restingcells,sporesor even
the productformationand its regulationare quite
killed cells. Non-growingcells are preferredfor
complex. Hence, several genetic modifications
biobonversions, since high substrate concentration
have to be done to fi nal l y producehi gh-yi el di ng a
can be used, besideswashingthe cells easily (to if
strains. ldeally speaking, the improved strains
make them free from contamination). It
should possessthe following characteristics(as l"
Sometimes, mixed cultures are used for many as possi bl e) to fi nal l yresul ti n hi gh product
bioconversions to carry out differentreactions.In formation.
recentyears,the yield of bioconversion is increased 1. Shortertime of fermentation
by using immobilizedcells at a lower cost. For
more detailson biotransformation, the readermust 2. Capableof metabolisinglow-costsubstrates
referChapter22. 3. R educedO, demand
4. Decreased
foam formation
5. Non-productionof undesirable
compounds
6. Toleranceto high concentrations
of carbon
or nitrogensources
7. Resistant
to infectionsof bacteriophages.
Microorganisms are alsousedfor the production lt is always preferableto have improved strains
of macromo'lecules i.e. high molecular weight of microorganismswhich can produce one
compounds e.g. polysaccharides, proteins metabolite as the main product. In this way, the
( inc luding e n z y me s ). R e c o mb i n a n t D N A productioncan be maximised,and its recovery
technologyhas drasticallyimprovedthe industrial becomessimpler.
productionof variousmacromolecules in a cost-
Through genetic manipulations,it has been
effectivemanner.
possibleto develop strainsfor the productionof
The detailson the microbialproductionof high modified or new metabolites which are of
m olec ular w e i g h t c o m p o u n d s (p h a rmaceuti calcommerci al val ue e.g. modi fi ed or new er
pr oduc t sa) re g i v e n i n C h a p te r1 5 . anti bi oti cs.

Biotechnology
[17]
25ft B IOTE C H N OL O C\

Ihe major limitation of strain improvement rs

that for rnost of the industrially important Tlru 19.5 Antimetabolites used for screening
micro or ganis m s , t her e is lac k o f d e t a i l e d of some common natural metabolites
information on the genetics,and molecular biology.
Natural metabolite Antimetabolitek)
Th is hinder s t he new s t r ain dev elop m e n t .
Amino acids
METHO DS O F STRAI N DEVELOP M E N T Arginine Canavanine
There are three distinct approaches for Histidine 2-Thiazolalanine
impro v em ent of s t r ains - m ut at ion, r e c o m b i n a t i o n Valine acid,
a-Aminobutyric
an d r ec om binant DNA t ec hnology . isoleucine
Leucine 4-Azaleucine
MUTATTON Methionine a-Methylmethionine,
Any c hange t hat oc c ur s in t he DN A o f a g e n e r s ethionine
referredto as mutation. Thus, mutations result in a Phenylalanine p-Fluorophenylalanine,
struciural change in the genome. Mutations may be Tyrosine p-Fluorophenyialanine
spontaneous (that occur naturally) or induced by
Tryptophan 5 (or6)-Methyltryptophan
mutagenic agents.
Threonine B-Hydroxynorleucine
The spontaneousmutations occur at a very low
Proline 3, 4-Dehydroproline
freq uenc y ,and us ually ar e not s uit ab l ef o r i n d u s t r i a l
p urp os es .M ut at ions m ay be induc ed b y m u t a g e n i c Vitamins
a ge nt s s uc h as ult r av iolet light , v ar i o u s c h e m i c a l s Ihiamine Pyrithiamine
(n itrous ox ide, nit r os oguanidine, hy d r o x y l a m i n e ) .
Pyridoxine lsoniazid
Site-directedmutagenesis(For cietailsReferChapter
.l Niacin 3-Acetylpyridine
0) is also important for strain improvement.
p-Aminobenzoic
acid Sullonamide
Selection of mutants
Nitrogenousbases
Selectionand isolationof the appropriatemutant Adenine 2, 6-Diaminopurine
strains developed is very important for therr
Guanine 8-Azaxanthine
ind us t r ial us e. Two t ec hnique s c o m m o n l y
ernployed for this purpose are briefly described. Uracil 5-Fluorouracil

Bandom screening
2. lsolation of antimetabolite resistant strains :
The mutated strains are rarrciomlyselected and A n t i m e t a b o l i t e s w h i c h h a v e s t r u c t u r a l s i m i l a ri ti e s
checked for their ability to produce the desired with metabolites can block the norrrral metabolic
industrial oroduct. This can be done with model o a t h w a v s a n d k i l l t h e c e l l s . T h e m u t a n t s t ra i n s
fermentation units. The strains with maximum yield resistant to antirnetabolites can be selected for
can be selected. Random screening is costly and industrial purposes. In the lable 19.5, a selected
te dio us pr oc edur e. But m any a t im e s , t h i s i s t h e l i s t a n t i m e t a b o l i t e s u s e d f o r s c r e e n i n g th e
only u,ay to find the right strain of mutants metabolites is given.
develooed.
3. Isolation of auxotrophic mutants : An
Selective isolation of mutants auxotrophic mutant is characterizedby a defect in
one of the biosynthetic pathways. As a result, it
There are many rnethods for selective isolatron
requiresa specific compound for its normal growth.
of imoroved strains.
For instance, tyr rnutants of Corynebacterium
1. lsolation of antibiotic resistant strains : The glutamicus require tyrosine for their growth'while
mutated strains are grown on a selective rnedium t h e y c a n a c c u m u l a t e p h e n y l a l a n i n e .T h e i s o l a tr o n
con t aining an ant ibiot ic . The wild s tr a i n sa r e k i l l e d of such mutants can be done by grbwing them on
while t he m ut ant s t r ainswit h ant ibi o t i c r e s i s t a n c e a c o m p l e t e a g a r m e d i u m t h a t c a n s p e c i f ica l l y
can gr ow. Suc h s t r ainsm ay be us ef u l i n i n d u s t r i e s support the biochemically defective mutant.
c hapt er19 : BIo P R OC ES S/F E R ME N T A TION
TE C H N OLOC Y
GENETIC RECOMB'NATTON
The strain irnprovement can be made by
combining genetic information from two genotypes,
by a process called genetic recombination. The
recombination can be brought out by
transformation, transduction, conjugation (Chapter
6) and protoplast fusion (Chapter 44). There are
many advantagesof genetic recombination.
1. By cros s ing high pr oduc t y ielding m u t a n t fermentation orocess
strains with wild-type strains, tl.re fermentation
process can be further increased.
2. Diffe rent m ut ant s t r ains w' it h high- y ie l d i n g a d d e d c o n t i n u o u s l y . S i m u l t a n e o u s l y , a n e q u a l
pro pe rtiesca n be c om bined by r ec om binat io n .
3. The re is gr adual dec line in t he pr oduc t y i e l d
after each stage of mutation, due to undesirable
muta tion s This c an be pr ev ent ed by u s i r r g
recomb ina tion.
The gro wt h of m ic r oor ganis m s is a h i g h l y
compler and coordinated process, ultimately
e xp resse db y inc r eas ein c ell num ber or c ell m a s s .
The process of growth depends on the availability
of requisite nutrients and their transport into the
cells, an d th e env ir onm ent al f ac t or s s uc h a s
aeration, O, supply, temperature and pH.
Douhling fime refers to the time period requirecl
for douhling the rveight of the biomass while
generation fime representsthe period for doubling
the cell numbers. Doubling times normally increase
witl.r increa si ngc ell s iz e and c onr plex ic it yas g i v e n
below.
Ba cter ia 0. 30- t hour
Yea s t s 1- 2hour s
Anim al c ells 25 - 48 hour s
Plan t c ells 20 - 70 hour s
In g en era l, when all ot her c ondit ions ar e k e p t
ide al, gro rvth of t he m ic r oor ganis m sis depen d e n t
on th e subs t r at e ( nut r ient ) s upply . T h e
microorganismscan be grown in batch, fed-batch,
semicontinuous or continuous culture systems in a
bioreactor.
A diagrammatic re,presentation
of nricrobial cell
growth in relation to substrate is depicted in
259
Fig. l9.l1. In batch fermentation, the grorvth
medium containing the substratesis inoculated
rvith microorganisms, and the fermentation
proceeds without the addition of fresh growtlr
medium. In fed-batch fermentation, subsiratesare
added at short time intervals during fermentation.
In batch and fed-batch fermentation,the growth of
the cells is quite comparable.And in both cases,
growth medium is not removed until the end of
ln case of continuous fermentation, as the
fermentation proceeds, fresh growth rnediunr rs
volume of spent mediurrr containing suspended
m i c r o o g a n i s m si s r e m o v e d . T h i s e n a b l e s t h e c e l l s
to grow optimally and continuously (Fig. 19,11Q
BATCH CULTURE
OR BATCH FERMENTATION
A batch fermentaiion is regarded as a closed
s y s t e m .T h e s t e r i l e n u t r i e n t c u l t u r e m e d i u m i n t h e
b i o r e a c t o r i s i n o c u l a t e d w i t h m i c r o o r g a n i s m s .T h e
incubation is carried out under optimal
physiological conditions (pH, temperature, O2
supply, agitation etc.). lt may be necessaryto add
a c i d o r a l k a l i t o m a i n t a i n p H , a n d a n t i - f o a ma g e n t s
to minimise foam. Under optimal conditions for
growth, the following six typical phases of growth
are observed in batch fermentation (Fig. 19.12).
1. Lag phase
2. Acceleration phase
3 . L o g a r i t h m i c ( l o g ) p h a s e ( e x p o n e n t i a lp h a s e )
4. Deceleration phase
5 . S t a t i o n a r yp h a s e
6. Death ohase.
1 . Lag phase : fhe initial brief period of
culturing after irroculation is referred to as lag
phase. During the lag phase, the microorgarrisms
adapt to the nerv environment-available nutrients,
pH etc. There is no increasein the cell number,
although the cellular weight may slightly inuease.
-l
h e l e n g t ho f t h e l a g p h a s ei s v a r i a b l ea n d i s m o s t l y
determined by the new set of physioiogical
conditions, and the phase at which the micro-
organisms were existing when inoci;lated. For
instance, lag phase may not occur if the culture
i n o c u l a t e d i s a t e x p o n e n t i a lp h a s e ( i . e , l o g p h a s e ) ,
and growth may start immediately
260 B IOTE C H NO LO CY

I
o
E

E c)
c
C)
c
()o

Time--+
Time--------J
Fig. 19.12 : Pattern of microbial cell growth in batch
(A) culture or batch feimentation.

2. Accelerationphase: This is a brieftransient

II period during which cells start growing slowly. ln

phaseconnectsthe lag phaseand
fact,acceleration
l og phase.
c
3. tog phase : The most active growth of
E microorganisms and multiplication occur during
c)
log phase.The cellsundergoseveraldoublingsand
c
the cell nrassincreases.When the numberof ceils
or biomass is plotted against time on a
semilogarithmic graph, a straight /ine is obtained,
hencethe term log phase.Growth rateof microbes
Time --------J in log phaseis independentof substrate(nutrient
supply)concentration as long as excesssubstrate is
present,and there are no growth inhibitorsin the
medium. In general,the specificgrowth rate of
microorganisms for simpler substratesis greater
thanfor l ongchai nmol ecul es.Thi si s expl a inedon
the basisof extraenergyneededto split long chain
substrates.
c
Two log phases are observed when a
E comolex nutrientmedium with two substrates is
c
0)
used in fermentation,and this phenomenonis
referredto as diauxy. This happenssince one of
the substratesis preferentiallymetabolisedfirst
which represses the breakdolvnof secondsubstrate.
After the first substrateis completelydegraded
Time----------* second l ag phaseoccurs,duri ng w hi ch per iod,
(c) the enzymes for the breakdown of second
substratethe synthesized.Now a secondlog phase
Fig. 19.11 : Diagrammatic representation of microbial occurs.
cell growth in relation to substrate (A) Batch
fermentation (B) Fed-batch fermentation
More details on log phase with special
(C) Continuous fermentation. referenceto growth kineticsof microorganisms
are
discussedlater.
Chapt er19 : BIOP R OC ES S/F E R ME N T A TION
TE C H N OLOC Y 261

4. Deceleration phase : As the growth rate of Fed-batch fermentation for the

microorganisms during lo8 phase decreases, production of recombinant proteins
they enter the deceleration phase. This phase
In recent years, fed-batch fermentation has
is usuallyvery short-livedand may not be observable.
become popular, due to very high yield, for the
5. Stationary phase : As the substrate in the p r o d u c t i o n o f r e c o m b i n a n t p r o t e i n s . D e p e n d i n g
growth medium gets depleted, and the metabolic on the microorganismand the natui'eof recombinant
end products that are formed irrhibit the growth, protein, the fed-batch fermentationcan increaserne
the cells enter the stationary phase. The micrabial product yield from 25"/o Io 1000% conrpared to
growth may either slow down or completely batch fermentation. Careful monitoring of the
stop. fhe biomass may remain almost constant fermerrtationreaction and appropriate addition of
du ring sta tionar y phas e. This phas e, howe v e r , i s substrates(carbon and nitrogen sources, and trace
frequently associated with dramatic changes in m e t a l s )s u b s t a n t i a l l yi n c r e a s e st h e p r o d u c t y i e l d .
the me tab olis m of t he c ells whic h may
Limitations : The major limitation of fed batch
produce compounds (secondary metabolites) of
fermentation is that the nricroorganismsin the
bio techn olo gic al im pc r t anc e e. g. pr oduc t i o n o f
stationary phase produce proteolytic enzymes or
an tibio tics.
proteases.These enzyme attack the recombinant
6. Death phase : This phase is associatedwith proteins that are being produced. By carefully
ceasation of metabolic activity and depletion of monitoring the fermentation, the lo6 phase can be
energy reserves. The cells die at an exponential prolonged and the onset of stationary phase is
rate (a straight line may be obtained when the delayed. By this way, the formation of proteases
rrumb er o f s ur v iv ing c ells ar e plot t ed agains t t i m e c a n b e m i n i m i s e d .
o n a se milog ar it hm icplot ) . I n t he c c m r ner c i a l a n d
industrial fermentations, the growth of the Fed.batch cultures
microorganisms is halted at the end of the log for higher organisms
phase or just before the death phase begins, arrd
Fed-batch cultures are successfully employed
the cells are harvested.
for manrmalian and insect cells. This is very
advantageous for the production of hr,rman
FED.BATCH CULTURE therapeuticproteins with good yield.
OR FED.BATCH FERMENTATION

Fed-batch {ermentation (See Fig. 19.11) is an SEMI.CONTINUOUS CULTURE OR

improvement of batch fermentation wherein rne SEMI.CONTINUOUS FERMENTATION
substrate is added in increments at difierent times Some of the products of fermentation are
throughout the course of fermentation (Note : In growth-linked, and such products are formed at the
batch culture method, substrateis added oniy at the end of the log phase e.g. ethanol production. In
beginning of the fermentation).Periodical substrate semi-continuous fermentation, a portion of the
addition prolongs log and stationary phases which culture medium is removed from the bioreactor
results in an increased biomass. Consequently, and replaced by fresh medium (identical In
p rod uction o f m et abolit es ( e. g. ant ibiot ic s ) d u r i n g
nutrients, pH, teinperature etc.). This process of
stationary phase is very much increased. culture medium change can be repeateci ai
As it is difficult to directly measure substrate a p p r o p r i a t e i n t e r v a l s . I n t h e s e n l i - c o n t i n u o u s
concentration in fed-batch fermentation, other fermentation, the iag phase and other non-
indicatorsthat correlatewith substrateconsumption productive phases are very much shortened. The
product output is much higher compared to batch
are used. The formation of organic acids,
production of CO, and changes in pH may be c ulture systems. Semi-continuous fermentation
me asure d, a nd ac c or c iingly s ubs t r at e ad d i t i o n t e c h n i q u e h a s b e e n s u c c e s s f u l l 'y u s e d i n t h e
carried out. In general, fed-batch fermentation i n dustrialproduction of alcohol.
requires more careful monitoring than batch Tlrere are however, certain disadvantages of
fermentation, and is therefore not a preferred s e m i - c o n t i n u o u s f e r m e n t a t i o n . T h e s e i n c l u d e t n e
method by industrial biotechnologists. t e c h n i c a l d i t T i c u l t i e so f h a n d l i n g b i o r e a c t o r s ,l o n g
262 B IOTE C HNO LO CY

c u l tu re p e ri o d sth a t m a y l e a d to contami nati on,

mutationand mechanicalbreakdorvn.

C ON T IN U O U S C U L T U R E OR
C ON T IN U O U S F ER M EN T ATION
Continuousfermentationis an open system.lI
involves the removal of culture medium
continuously and replacement of this with a fresh
sterile medium in a bioreactor.Both addition ano
removal are done at the same rate so that the
w o rk i n g v o l u m e re m a i n s c o n stant.Further,to
m a i n ta i na s te a d ys ta tec o n d iti on i n conti nuous Nutrient
process,it is advisablethat the cell lossas a result medium
of outflow is balancedby growthof the organisms.
The two commontypesof continuousfermentation
and bioreactorsare describedbelow (Fig. 19.13).
Turbidity
Homogeneously mixed bioreactors recorder
ln thistype,theculturesolutionis homogeneously
nrixed,and the bioreactorsare of two types
'fhe
Chemostatbioreactors: concentrationof
any one of the substrates (carbohydrate,nitrogen
source,salts,C)2)is adjustedto corrtrolthe cell
growth and maintairra steadystate.
Turbidostatbioreactors: In this case,turbidity
me a s u re m e nits u s e d to mo ni tor the bi omass Nutrient
concentration.The rate of addition of nutrient medium
solutioncan be appropriately adjustedto maintarn Turbidity
a constantcell growth. recoroer

Plug flow bioreactors

In plug florv bioreactors, the culture solution
flowsthrougha tubularreactionvesselwithoutback
mi x i n g . T h e c o mp o s i ti o no f tl re medi um, the
q u a n ti tyo f c e l l s ,O, s u p p l ya n d productformatron
vary at different locations in the bioreactor.
M i c ro o rg a n i s masl o n g w i th n utri entmedi um are
continuously addedat the entrance of the bioreactor.

Industrial applications of
continuous fermentation
Continuousfermentationprocesseshave been
used for the production of antibiotics, organic
solvents, single-cell protein, beer and ethanol,
besideswaste-water treatment.

Advantages of continuous fermentation

.l
. The size of the bioreactor and other
Fig. 19,13 : Continuous fermentation bioreactors
e o u i p me n tu s e d i n c o n ti n u o u sfermentati onare (A) Chemostat bioreactor (B) Turbidostatbioreactor
relativelysnrallercomlraredto batch fermentation (C) Flug flow bioreactor.
for the productionof the samequantityof product.
c hapt er1 9 : B Io PR O C ES S/F E R ME NTA TTON
TE C H N OLOC Y 263

2. The yield of the product is more consistent After 2nd generation, the cell number will be
sincethe physiological stateof the cells is uniform. N o x 2 2 .
3. The 'down time' between two successrve After 3rd generation, Nn x 23, and so on. Thus,
fermentationsfor cleaning and preparing the the number of cells after a given time (Nt) will be
bioreactor for reuse is avoided in continuous as follows :
fermentation. N t =N o x 2 n
4. Continuousfermentationcan be run in a where n is the number of generations.
cost-effective manner.
The term douhling firne (td) ot mean generation
fime (MCT) refers to the time taken for doubling
Disadvantages of the cell number or biomass. The specific growth
continuous fermentation rate constant expressedby [, is the direct measure
Despite many advantages of continuous of rate of growth of the organism. If N is the
fermentation above),it is not veryvvidely number of ceils at a given time, then the increase
(described
used in industries.Some of the drawbacksare in the number of cells (growth rate) with time rs
Iisted. given by the formula.
.l dN =
(1)
. Continuous fermentation may run lrN
dt
continuouslyfor a period of 500 to 1,000 hours.
Maintenance of sterile conditions for such a long lf X is the biomassconcentrationat a given time,
period is difficult. then the increase in the biomass (growth rate) with
time is given by
2. T he re c o mb i n a n t c e l l s w i th pl asmi d
constructs cannot function continuously and dx =
pX (2)
thereforethe productyield decreases. dr
lrr

3. It is not easyto maintainthe samequalityof In general, the specific growth rate (p) is a lrtl

t he c ult u rem e d i u mfo r a l l th e a d d i ti o n s.N utri ent function of the concentration of limiting substrate l ll
1, ll
variationswill alter the growth and physiologyof (S),the maximum specific grorvth rate (p-u*) and a t i r.
the cells,and consequently the productyield. substratespecific constant (Kr). Their relationship
was expressed by Monond by the following
In addition to the disadvantageslisted above, equation
industrialbiotechnologistsare rather reluctantto
S
switch over to continuousfermentationfrom the !t = F.u* (3)
K r+S
batch fermentation.However; it is expectedthat
continuousfermeltationwill alsobecome,popular Both S and K, are expressedas concentrationse.g.,
in due course. in moles or grams per liter.

The growth rate (p) of an organism is not fixed

GROWTH KINETICS but it is variable depending on the environmental
OF MICROORGANISMS conditions such as concentration of substrateano
The different types of fermentation processes- temperature. At a low concentration, the substrate
batch,fed-batch,semi-continuous and continuous is the limiting factor for growth (Fig. 19.1 A).
are describedabove. The kinetics of microbial The Fig. 19.148 represents the growth rate for
growthwith specialreference to log phaseof batch a given substrate concentration (by plotting p
fermentationare brieflydiscussedhere. a g a i n s tS ) .

Aftercompletionof lag phase,the cell enterslog In batch culture, the substrateis initially present
phase which is characterizedby exponential at a higher'concentration i.e. (5) > Kr, hence the
growth (See Fig. 19.12). lf the initial number of equation (3) is approximately 1.
cells is No, then s 1 -=
K +S
Atter 1st generation,the cell number will be
Nn x 21. Thus, p = F_u*.
26,4 B IOTE C H N O LO CY

CLASSIFICATION OF
FEBMENTATION PROCESSES
There are different ways of classifyingthe
s5 fermentation processes.
The major classification as
S, batch,fed-batch,semi -conti nuousand conti r r uous
S3 fermentationprocessesare already describedin
6 some detai l (S eep. 259). A nothercl assi fi c at ion,
-cl basedon the productformationin relationto energy
5
c metabolismis brieflydiscussed below (Fig. 19.15).
I
Type I fermentation
When the product is {ormed directly from the
primary metabolismusedfor energyproduction,it
Time _____+ is referredto as type I and may be representedas.

(A) SubstrateA ----+ Product

SubstrateA -----+ B ------+C .--+ D -) Product
Growth, energy metabolism and product
formati onal most run i n a paral l elmanner( Fig,
19.15A). ln this type, trophophaseand iodophase
are not separated from each other e.g. production
, uconi caci d and si ngl e-celprotein.
of ethanol gl l

Type ll fermentation
!n type ll category,the product is also formed
from the substrate used for primary energy
] u."* l- metabolism.However.the product is produced in
0)
g
the secondarypathway, as illustratedbelow.
-c
= A + B -+ C -+ D'..Primarymetabolism
Substrate
(5 I
+ E + F+ C -+ P roduct
Ks
Substrateconcentration(S) At the beginning, the gi'owth of the
microorganisrns is accompaniedby high substrate
(B)
utilizationwith littleor no productformation.Now
the growth is slowed down but the substrate
Fig. 19.14 : Growth curves for unicellular organism in consumpti oni s hi gh, and thi s i s coupl ed wit h
batch culture (A) tllith increasing ccncentrations of a productformation.As is evidentfrom Fig. 19.158,
substrate (S7<S?<E<S4<S) (B) The effect of a given in type ll fermentation,the trophophaseand
substrate concentration on growth rate (Ks = substrate idiophaseare separate.Productionof some amino
concentratjon to prodyce half-maximal growth rate) aci ds, ci tri c aci d and i taconi c aci d are g ood
examplesof type ll fermentation.

Wherr the substrateconcentration is lorv, Type lll fermentation

as usually occurs at the end of growth p n a s e /
t hen , Thereis a cleardistinctionbetweenthe primary
metabolismand product formation in type lll
S
< 1 fermentation (Fig.19.150 as they occurat separate
Kr + S
times. Substrateconsumptionand rapid growth
H e n ce p L ( !t."*. occur in the first phaseand the product formatian
 TE C H N OLOC Y
Chapt er1 9 : BIOP R OC ES S/F E R ME NTA TION 265

occursin the second phase.The product is formed

from amphibolicmetabolicpathwaysand not from

I primary metabolisme.g. productionof vitanrins

and antibiotlcs.

(U Overlap of different types

o of fermentations
a
o Types l , l l and l l l fermentati ons,
ori gi nal l y
o
categorized by Carden(in 1959)are not very rigid.
There are intermediateforms based on the
compositionof the nutrientculturemedium,strain
of the microorganism usedand productformation.
For instance,industrialproductionof lactic acid
falls betweentype I and ll, while productionof the
antibioticamyloglycoside is intermediatebetween
typesl l and l l l .
I It is sometimesdifficult to categorize the

o
I t
jit
industrialfermeniationsunder anv one of these
types(1,ll, lil) due to complexnatureof the process
e.g. mycel i um produci ng mi croorgani smsi n
i:
rel ati onto anti bi oti coroducti on.
-c
o
()
o TH E FE R ME N TA TION P R OC E S S
The fermentationprocessbasically consistsof
inoculum preservation, inoculum build-up,
T]me-----*
prefermenter culture and finally production
(B)
fermentation A brief accountof the four stagesof
fermentationis given below.

Inoculum preservation
(culture maintenancef
The preservationof high-yieldingstrains of
microorganisms for fermentationis very important
for productformationin substantial amounts.The
ultimatepurposeof preservation is to maintainthe
o strains,as long as possible,without cell division.
g
Thereare differentmethodsof preservation.
E
o
o
Storage at low (2-6'C) temperature : In this
o
o method,the microorganisms can be stored in a
refrigeratorin liquid culture or as stab culture.
Althoughthis is the easiestmethodof preservation,
llm€ -_--) therei s a hi gh ri sk of contami nati on.
(c)
Storageby freezing: The nricrobialculturescan
be frozen and preservedfor severalyears.In the
Fig. 19.15: Typesof fermentations in rclationto freezers,the preservationcan be done at - 1BoCor,
product formation(A) Type I fermentation(B) TVpell
at -B0oC. For preservationat -1 96'C, liquid
fermentation(C) Typelll fermentation(- represents
nitrogenmust be used.It is very importantthat the
grawthrate; -------correspondto substrate
freezing(and laterthawingwhen required)is done
consumption;- indicatesproduct fomation).
slorvly (usually with a change of 1'Clmin) to
266 B IOTE C H N OLO G Y

pre v ent danr ageanc i k illing of t he m i c r o o r g a n i s m s . Prefermentel culture

lf p r oper c ar e is not t al< en,as m any a s 9 5 % o f t h e
Fermenter preculture or prefermenter culture is
ce lls m ay be k illed by f r eez ing and t h a r , t , i n g .
o f t e n r e q u i r e d f o r i n o c u l a t i n g l a r g e si ze d
Storage by lyophilization : Preservation of b i o r e a c t o r s .I n a d e q u a t eq u a n t i t v o f i n o c u l u m w i l l
nric r c or ganis nr s by ly ophiliz at ion ( i . e , f r e e z e n o t o n l y d e l a y t h e p r o d u c t f o r m a t i o n , b u t a l so
drying) is t he bes t r r . r et hod,alt hou g h , i t r e q u i r e s r e d u c e t h e y i e l d d r a s t i c a i l y . B y c u l t u r i n g th e
sirecial equipment. In fact, lyophilization is the m i c r o o r g a n i s m s( t h e i r r o c u l u m b u i l d - u p ) i n sn r a l l
methocl of chaice by many' fermentation f e r m e n t e r s , t l r e s i z e o f t h e i n o c r - r l u m c a n b e
bio t ec hnologis t s increased for large-scale industrial u se .
The s t or ageof r r i< : r oor ganis m cs a n b e d o n e b y B i o t e c h n o l o g i s t s h a v e w o r k e d o u t t h e r e qu i si te
an )/ one oi t he t hr ee t ec hniques de s c r i b e c la b o v e i n o c u l u r r r c o n c e n t r a t i o n sf o r o p t i m a l f e r m e n ta ti o n
Flowever, fclr each method, optimal conclitionsfor e . g , f o r b a c t e r i a l f e r m e n t a t i o n , t h e i n o cu l u m
pre s er v at ionm us t be wor k ed out f o r e a c h s t r a i n ccrncentration should be betr,veen0.2 ro 3.0'k;
separately.ln general, the preservedmaster strains ior fungal fermentatron,it rs in the range of 5-10"/,'
are c ult iv at ed onc e in t wo y ear s f o r c h e c k i n g o f
their activity. When needecl ior use, the working Production fermentation
.sfrainscan be obtained from the master strains.
The general features and the different types of
b i o r e a c t o r sa r e a l r e a d y d e s c r i b e d( S e ep . 2 3 9 -2 4 4 )
Ino c ulum build up
T h e s i z e o f t h e f e r r n e r r t e ru s e d m a i n l y d e p e n d s o n
The preserved cultures have to be revived for t h e p r o d u c t F o r e x a m p l e , a s m a l l b i o r e a c t o r (1 - 2 0
Lr s e This c an be do n e b y g r o w i n g l i t r e s i z e ) c a n b e u s e d f o r p r o d u c i n g d i a g n o sti c
th ci r indr - r s t r ial
tlre c ult ur es in liquic l or on s olid m e d i a T h e a c t u a l e n z y n r e sa n d s u b s t a n c e sf o r m o l e c u l a r b i o l o g y b y
pro c es s and t he c ondit ions us ed f o r i n o c u l u m r e c o m b i n a n t m i c r o o r g a n i s n r s , w h i l e l a r g e b r o -
bu ild- up lar gelT- depend on t h e p r c s e r v a t i o n r e a c t o r s ( > 4 5 0 l i t r e s ) a r e e m p l o y e d f o r p r o d u ci n g
te ch nique us ed. Ther e ar e wide v a r i a t i o l r s i n t h e s i r . r g l e - c e lpl r o t e i n a n d a m i n o a c i d s .
growth tinres rarhich depend on the type of
pre s er v at ion and t he or ganis m s u s e d a s S i v e n A d i a g , r a m m a t i cr e p r e s e n t a t i o no f a g e n e r a l i ze d
be low. f e r m e n t a t i o n p r o c e s s i s d e p i c t e d i n F i g . 1 9 . 16 .

Refrigerated cultures (2-6'C) : F o r a p p r o p r i a t e p r o d u c t i o n b y f e r m e n ta ti o n ,

several parametersneed to be carefully considerecl
Bac t er ia 6- 24 hour s
a n d o p t i n 'r i z e d . T h e s e i n c i u d e c o m p o s i t i on o f
r \ c t ir r om y c et es1- 3 day s n u t r i e n t m c d i u r n , c a r b o n a n d n i t r o g e n s o u r ce s/
.l batch to batch variations, effect of sterilization on
Fungi -.5 days
n u t r i e n t sa n d o n p H , a n d a l t e r a t i o n si n t e r n p e r a tu r e
Frozen cultures (1BoC,-B0oC, *195"C) :
and aeration.
Bac ier ia 6 48 hour s
The parameters-temperature,pressure,aeration
Ac t ir r or r y c et es1- 5 day s and stirring are briefly described.
.l
Fungi -7 oays
T e m p e r a t u r e : T h e t e m p e r a t L t r em u s t be so
Lyophilized cultures : m a i n t a i n e d t h a t t h e r e o c c u r s m a x i m a l g r o w th t- tf
For all or ganis r ns4 10 c lay s n r i c r o o r g a n i s m sw i t h o p t i m a l p r o d u c t f o r m ati o n ,
a l t h o u g l . rt h i s i s n o t a l w a y s p o s s i b l e . I n g e n e r a l ,
F or pr oper gr or v t h, anc l t o ob t a i n s u f f i c i e n t
there are two temperatLrre ranges to run the
qu ant iiy of ir - r oc ulur n,a s er ies o f c u l t u r e s a r e
fermentations a mesophile range (2O-45'C) and a
preoa'ed For g,oodfern'rentationyielcl, the number
thermophile range (> 45'C). Sometimes, two
o f c ells and s por es , nLr t r ientm ediu m , t e m p e r a t u r e
different temperatures are used for the same
an c Jagc < lf ihe inc c ulut r l c 1r €im pod a n t
fermentation process-a higher temperature ts
The inoc ulr : m build- up is s u s p e n d e d i n a e.nrployedfor good growth (in trophophase), and
su rf ac e- ac t iv e agent s uc h as Tw e e n B 0 a n d t h e n t h e t e m p e r a t u r e i s d e c r e a s e df o r o p t i mi zi n g
transferredto the bioreactor for fermentation. product formation (in idiophase)
 TE C H N OLOC Y
Chapt er19 : B IO P R OC ES S/F E R ME N T A TION 267

Inoculum Seed Bioreactor Culturefluid

fermenter

Fig. l9-16 : Diagrammatic representation of a generalized fermentatian process.

Pre ssure : Appr opr iat e m aint enanc e o f (reference electrode, glass electrode) are being
hydro sta tic p r es s ur e, par t ic ular ly in lar ge s i z e d used. In fact, electrodes are also available for
bio rea cto rs is v er y im por t ant . This is beca u s e m c a s u r i n g s e v e r a lo t h e r i n o r g a n i c i o n s .
pre ssureinflu enc est he s olubilit y of O r and COr i n
O, and CO, measurement : Oxygen electrodes
th e cultu re medium . An ov er pr es s ur ein t he r a n g e
and CO, electrodes can be used to measure O,
C).2-0 .5b ar is gener ally us ed
and COz concentrations respectively. The
Aeration : A bioreactor gets aerated by the electrodes are amperometric in nature They are
su pp lv o f O, and t her ef or e, adjus t m ent m us t b e h o w e v e r , s u s c e p t i b l ef o r d a m a g e o n s t e r i l i z a t i o n
ma de to furn is h r equir ed am ount of O " t o t h e
In a commonly used technique, O, and CO,
micro org an is m s .Us ually ,t he aer at ion r at e is i n t h e
respectively can he measured by the magnetic
ra ng e of 0.2 5- 1 . 25 v v m ( v olum e of air / v olum e o f
liq uid /minu te) .
Stirring : The type and the speed of impellers Tns$ 19.6 Inportant parametersthat can be
de termin es the s t ir r ing r at e in a f er m ent e r . I n neasured durlng bloprocessing
ge ne ral,th e im peller s peed dec r eas esas t he s i z e o f
the fermenter increases.Thus, for a small bioreactor Physical parameters
(size 1-2 0 litr es ) ,t he im peller s peed is in t he r a n g e Temperature
of 25 0-3 50 rpm , while f or a lar ge bior eac t or ( s i z e Pressure
around 450 litres, the i.'npeller speed is 60=120 Flowrates
r0 m. Viscosity
OF
l urD r0[y
MEASUREME NT AND CO NTRO L
BIOPROCESS PARAMETERS Powerconsumption

Th ere are a lar ge num ber of phy s ic al, c hem i c a l Chemical parameters
and biological parameters that can be measured pH
during fermentation/bioprocessing(Table | 9.6) for Substrateconientration
data analysisand appropriatecontrol. Some specral
Productconcentration
sensors have been developed to carry out
(dissolved)
O, concenlration
mea su reme nts in t he bior eac t or s . The b a s i c
Waste gasesconcentration
(e.9.CO2)
req uire men to f all t he s ens or sis t hat t hey m u s t b e
sterilizab le. The m eas ur em ent sof t he par am e t e r s lonicstrength
(liste d in Tab le) c an be done eit her dir ec t ly in t h e
Riological parameters
bioreactor or in the laboratory.
Activities
of specific
enzymes
pH measurement : There are pH electrodesthat
Proteinconcentration
can withstan d high t em per at ur e ( s t er iliz a t i o n )
Energetics(ATPconcentration)
p ressu rean d m ec hanic al s t r es s esand
, y et m ea s u r e
DNAiRNA content
tb e o H a c c ur at elv . Com binat ion elec t r o d e s
268 B IOTE C HNO LO CY

property of O, and the infrared absorption of COr.

This c an be done by us ing s enso r s .

Use of rflass speetrometer In genera techn iques

l, fermentation/bioprocesses
are developedin stages,startingfrom a laboratory
The mass spectrcmeter is a versatile technique.
Thephen om enon
andfi nal l yl eadi ngto an i ndustry.
It can be used to measurethe concentrationsof Nr,
of developing industrial fermentation process in
N Hr , et hanol and m et hanol s i m u l t a n e o u s l y . I n
sfages is referred to as scale-up. Scale-up is
addition, mass spectrometeris also useful to obtain
necessaryfor implementinga new fermentation
i nf or m at ion on qualit at iv e a n d q u a n t i t a t i v e
techni quedevel oped, and usi ngmutantorganism s.
exchange of O, and COr.
The very purpose of scale-up is to develop
Use of gas'permeahle mernbranes optimal environmental and operating conditions at
different levels for a successful fermentation
The measurement of dissolved gases, up to B
industrv.The conditionsthat need to be studied
s inr ult aneous lyc, an be done alm o s t a c c u r a t e l y b y
i ncl ude substrateconcentrati on,agi tat ion and
us ing gas - per m eablem em br anes.T h e a d v a n t a g ei s
mi xi ng,aerati on,pow er consumpti on
and r at e of
that s uc h m eas ur em entis pos s i b l e t o c a r r v o u t I n
O, transfer.
the nut r ient m edium .
In a conventi onalscal e-up,a ferment at r on
Use of computers techni quei s devel opedi n 3-4 stages(i .e. ,scales) .
proc ess
The i ni ti alstagei nvol vesa screeni ng using
Com put er s ar e us ed in indus t r i a l b i o t e c h n o l o g v
petri dishesor Erlenmeyer flasks.The next stageis
for dat a ac quis it ion, dat a analy s i s a n d d e v e l o p i n g
a pi l ot proj ectto determi nethe opti maloper at ing
fermentation models.
conditiorrsfor a fermentationprocess with a
Data acquisition : By employing on-line sensors capacityof 5-200 litres.The final stageinvolves
and computers in fermentation systern,data can be the transfer of technology developed in the
obtained with regard to the concentration of O, laboratory to industry.
and COr, pH, temperature, pressure, viscosity,
It has to be continuously noted that a
turbidity, aeration rate etc. Certain other parameters
fermentation process that works well at the
( e. g. nut r ient c onc ent r at ion, p r o d u c t f o r m a t i o n ,
Iaboratoryscale may work poorly or may not work
biom as s c onc ent r at ion) c an be m e a s u r e d i n t h e
at all on industrialscale.Therefore,it is not always
laboratory i.e. off-line measurements. The
possi bl eto bl i ndl yappl ythe l aboratory
co ndit ions
inf or m at ion c ollec t ed f r om on - l i n e a n d o f f - l i n e
of a fermentation techniquedevelopedto industry.
rneasurementscan be entered into a computer. ln
At the laboratoryscale,one is interestedin the
this f as hion, t he ent ir e da t a r e g a r d i n g a
maxi mumyi el dof the productfor uni t ti me.At t he
fermentation can be processed, stored and
i ndustryl evel ,besi desthe productyi el d, m inim al
retneved.
operating cost is another important factor for
Dat a analy s is : The dat a c o l l e c t e d o n a A n i ndustri albi otechnol og ist
consi derati on. hast o
c om put er c an be us ed f or v ar iou s c a l c u l a t i o n se . g . appl y hi s/her i ntel l ect and techni cal skill f or
rate of substrate utilization, rate of product establishing a successful fermentationset up in a
formation, rates of O, uptake and CO, formation, cost-effectivemanner.
heat balanc e, r es pir at or y qu o t i e n t . T h r o u g h
c om put er dat a analy s is ,it is pos s i b l et o a r r i v e a t t h e
o pt im al pr oduc t iv it y f or a gi v e n f e r m e n t a t i o n
system.

Development of fermentation models : The F o o d t e c h n o l o g y i s a v a s t f i e l d . T h e sa l i e n t

computer can be used to develop mathetnatical features of food processing which has some
models of fermentation processes.These models in relevance to bioprocess engineering and
turn will be useful to have a better control over technology are given here. In general, the food
fermentation systems with high productivity in a p r o c e s s i n gi s c a r r i e d o u t t o a c h e i v e t h e f o l l o w i n g
cost-effective man ner. obiectives.
Chapt er19 : B IO P R OC ES S/F E R ME N T A TTON
TE C H N OLOC Y
o For the purpose of storage and transport.
. To proiect from contamination
r To increase the shelf-life
. To make it attractive for the consumers.
For appropriateprocessingof food, many criteria
ne ed to b e taken int o ac c ount . Thes e inc lude th e
a bility o f th e m ic r oor ganis m sand pes t s t o inv a d e
a nd g row on fnods . and t he c hem ic al ins t abi l i t y
and biological activity of foods.
FOOD PRESERVATION
Amon g th e m any f ood pr oc es s ingt ec hniqu e s ,
food preservation is the most important one.
Selected food preservation processes are briefly
de scribe d.
Drying
Exp osureof f oods t o s unlight and dr y ing is a
natural, and an earliest method of fooo
preservation.Drying involves the removal of water.
Consequently, the moisture content of the food
falls, an d the inv ading m ic r oor ganis m s c ann o t
grow.
Hot air is most frequently used to remove
moisture. In actual practice, foods are dried by
usin g d iffere nt ty pes of equipm ent .
. Shallow-bed dryers (drying is carried out In
perforated conveyor beos,).
. Deep-bed dryers (drying occurs in bins).
o Spray dryers (to dry liquid foods and food
slu rrie s).
26,9
o Freeze dryers (to dry soup ingradients, beverage
extracts).
Ghilling
The technique of chilling is commorrly used to
preserve foods without freezing. The process of
chilling is carried out around 0"C with humidity
between 85-95%. The shelf-life of fresh foods like
meat, fish and dairy products can be extended oy
chilling (usually less than a month). lt is reported
that the if chilling is done under lorv oxygen
conditions, the shelf-life of foods increases.
Chilling is frequently used for long distance
shipping of meat, apples, vegetablesetc
Freezing
Foods can be preserved in a frozen state (-30.
to -10'C). At a very low, temperatures,the water
activity and reactant mobility are low, hence the
c h e m i c a l r e a c t i o n sa r e r e d u c e d .
Foods can be frozen by using a stream of
refrigerated air, or by passing inert refrigerant or
cryogenic gas. RapiC freezing is advocated to
minimize the adverseeffectson the texture of fooos.
Thermal processing
By applying heat (comparable to sterilization),
majority of the microorganismsand spores present
i n t h e f o o d s c a n b e k i l l e d . T h e r m a l p r o c e s s i n gi s
carried out in sealed containers devoid of oxygen,
and thus aerobic organisms cannot grow. Further,
higher temperature also leads to inactivation of
enzymes, and consequentlymicrobial growth will
be retarded.
 I r c om ple t e ( d e s c r i b e d r n
s t he f er m ent at ior . is S o m e t i m e s ,t h e m i c r o o r g a n i s mm a y i t s e l f be th e
Achapt er 19) , it is nec es s ar y r o r e c o v e r t h e d e s i r e d e n d p r o d u c t f o r i s o l a t i o n e . g . s i n gl e - ce l l
de s ir ed end pr oduc t The end p r o d u c t s i n c l u d e protein. The product recovery as a biomass rs
an t ibiot ic s , am ino ac ids , v it am ins , o r g a n i c a c i d s , simpler When subjectedto heat, the cells aggregate
industrial enzynres, and vaccines fhe extraction a n d f o r m c l u m p s w h i c h c a n b e s e p a r a te d b y
and purification of a biotechnological praduct s e di m e n t a t i o n .
from fermentation is referred to as downstream
processing (DSP) or product recovery. DSP is as
co m plex and im por t ant as f er m ent a t i o n p r o c e s s .l t
often r equir es t he ex per t is eand t e c h n i c a l s k i l l s o f
ch em is t s , pr oc es sengineer s /bes id e s b i o s c i e n t i s t s .
In the present day biotechnology, the
fermentation and dpwnstream processing are Downstream processing of metabolites is a
considered as an integratedsystem.The methociology multistage operation, and may be broadly divided
adopted for downstreamprocessingdepends on the into the following stages.
nature of the end product, its concentration,stability 1. Solid-liquid separatron
and the degree of purification required, besidesthe 2 . R e l e a s eo f i n t r a c e l l u l a r o r o d u c t s
presence of other products. The product recovery
3. Concentration
yield in general is expected to be higher, if the
number of steos in DSP is lower. 4. Purification

The des ir ed pr oduc t s f or is ola t i o n b y D S P a r e 5. Formulation.

mo s t f r equent ly m et abolit eswhic h m a y b e p r e s e n t ln Fig. 20.1, an outline of the major steps In
as f ollows downstream processing is given, and they are
1 . Intracellular metabolites : These oroducrs d e s c r i b e d i n s o m e d e t a i l i n t h e f o l l o w i n g p ag e s.
are loc at ed wit hin t he c ells e. g. v it a m i r . r se, n z y n r e s
2. Extracellular metabolites : Thev are Dresenr S O L I D . L I Q U I D S E P A R A T I O N
o uts ide t he c ells ( c ult ur ef luids ) e g. m o s t a n t i b i o t i c s The first step in product recovery is the
(pe nic illin, s t r ept om y c in) , am ino a c i d s , a l c o h o l , s e p a r a t i o n o f w h o l e c e l l s ( c e l l b i o m a s s)
and
citr ic ac id, s om e enz y m es ( am y las e s ,p r o t e a s e s ) o t h e r i n s o l u b l e i n g r a d i e n t sf r o m t h e c u l t u r e b r o th
3 Both intracellular and extracellular e.i.. ( N o t e : l f t h e d e s i r e d p r o d u c t i s a n i n t r a ce l l u l a r
vilam in B, , , f lav om y c in. metabolite, it must be releasedfrom the cells before

270
 P R OC ES SIN C
Chapt er20 : D OW N ST R EA M 271

s u b j e c t i n gt o s o l i d - l i q u i d s e p a r a t i o n .D e t a i l s o f c e l l
disruption are described later). Some authors use
the term harvesting of microbial cells for the
seoaration of cells from the culture medium.

Several methods are in use for solid-liouid

EXTRACELLULAR
PRO DUCT separation. These include flotation, flocculation,
filtration and centrifugation.

FLOTAT'ON
CEL LDISRUP TI O N
(physical,chemical Wh e n a g a s i s i n t r o d u c e d i n t o t h e l i q u i d b r o t h ,
enzymaticmethods) i t f o r m s b u b b l e s .T h e c e l l s a n d o t h e r s o l i d p a r t i c l e s
get adsorbed on gas bubbles. These bubbles rise to
the foam layer which can be collected and
BROTHWITHSOLIDS
AND LI Q UI D removed. The presence of certain substances,
referred to as collector substances,facilitates stable
foam formation e.g., long chain fatty acicis,amines

SO LI D_LI Q UI D FLOCCULATION
SEPARATION
(flotation,f locculation, ln flocculation, the cells (or cell debi'is) form
filtration,centrifugation) large aggregatesto settle down for easy removal.
The process of flocculation depends on the nature
o f c e l l s a n d t h e i o n i c c o n s t i t u e n t so f t h e m e d i u m .
Addition of flocculating agents (inorganic salt,
CONCENTRATION organic polyelectrolyte, mineral hydrocolloid) is
(evaporation,liquid- often necessaryto achieve appropriateflocculation.
liquidextraction,
membranefiltration, FILTRAT'ON
precipitation,
adsorption)
F i l t r a t i o ni s t h e m o s t c o m m o n l y u s e d t e c h n i q u e
for separatingthe biomass and culture filtrate. The
efficiency of filtration depends on many factors-
the size of the organism, presence of other
PURI FI CATI OBY N organisms, viscosity of the medium, and
CHROMATOGRAPHY
temperature. Several filters such as depth filters,
(gel{iltration,
absolute filters, rotary drum vacuum filters and
ion-exchange,affinity,
hydrophobicinteraction) membrane filters are in use.
Depth filters : They are composed of a
filamentous matrix such as glass wool, asbestosor
filter paper. The particles are trapped within the
FORMULATION matrix and the fluid passesout. Filamentousfungi
(drying,
freeze-drying, can be removed by using depth filters.
crystallization)
Absolute filters : These filters are with soecific
pore sizes that are smaller than the particles to be
removed. Bacteria from culture nredium can be
removed by absolute filters.
FI NALPRO DUCT
Rotary drum vacuum filters : These filters are
frequently used for separation of broth containing
10-40% solids (by volume) and particles in the size
Fig. 20.1 : A summary of the major steps in
of 0.5-1 Opm. Rotary drum vacuum filters have
downstream processing.
been successfullyused for filtration of yeast cells
272 B IOTE C H N O LO C\

Types of filtration processes

Thereare 3 major typesof filtrationsbasedon
the particlesizesand othercharacters(Table20.1).
These are microfiltration, ultrafiltration and
reverce osmosis.

Fig. 20.2 : Diagrammatic representation of a rotary
drum vacuum filter.
and fi l a m e n to u sfu n g i . T h e e q u i p menti s srmpl e
with low power consumptionand is easy to
operate.The filtration unit consistsof a rotating
drum partiallyimmersedin a tank of broth (Fig.
20.2).As the drum rotates,it picks up the biomass
which gets depositedas a cake on the drum
surface.This filter cake can be easilyremoved.
Membrane filters : ln this type of filtration,
membraneswith specificpore sizescan be used.
However,cloggingof filters is a major limitation.
Therearetwo typesof membranefiltrations-sfatic
filtration and cross-flow filtration (Fig, 20.3). ln
cross-flowfiltration,the culturebroth is pumpedin
a crosswisefashion acrossthe membrane.This
reducesthe cloggingprocessand hencebetterthan
the staticfiltration.
CENTBIFUCAT'ON
The techniqueof centrifugaticn is basedon the
principle of density differences between the
particlesto be separatedand the medium.Thus,
centrifugationis mostly used for separatingsolid
particles from liquid phase (fluid/particle
separati on).U nl i ke the centri fugati onthat is
convenientlycarried out in the laboratoryscare,
therearecertainlimitationsfor largescaleindustrial
centrifugation. However, in recent years/
continuousflow industrialcentrifugeshave been
deveiopeci.There is a continuousfeedingof the
sl urryand col l ecti onof cl ari fi edfl ui d, w ' hi let he
solidsdepositedcan be removedintermittently. The
different types of centrifugesare depicted in
Fig. 20.4, and brieflydescribedhereunder.
Tubular bowl centrifuge(Fig.20.44) : This is a
si mpl eand a smal lcentri fuge,
commonl yused r n
pilot plants. Tubular bowl centrifuge can be
operatedat a high centrifugalspeed,and can be
run in both batch or continuousmode.The solids
are removedmanually.
Disc centrifuge (Fig. 20.48) : lt consists of
severaldiscs that separatethe bowl into settling
zones.The feed/slurry
is fed througha centraltube.

Culturesolution

Biomass
concentrate
Membrane
Membrane

Filtrate

(A)

Fig. 20.3 : Filter systems for separation of biomass and culture fiitrate (A) Static-tlow filtration
(B) Crass-flovt filtration.
 Chapter20 : DOWNSTREAMPROCESSINC 273

Tmr.r 20.1 Major types of flltration processeswith characteristic features

Type Sizes of particles separated Compound or particle separated

Microfiltration pm
0.1-10 Cellsor cellfractions,
viruses.
Ultrafiltration pm
0.001-0.1 Compoundswithmolecular greater
weights
than1000(e.9.enzymes).
3. Reverse
osmosis pm
0.0001-0.001 Compounds weights
withmolecular less
(hyperfiltration) than1000(e.9.lactose).

T he c lar if ie dfl u i d mo v e su p w a rd sw h i l e th e sol i ds R E LE A S E OF IN TR A C E LLU LA R

settleat the lower surface.
Multichamber centrifuge (Fig. 20.aA : This is
basicallya modificationof tubular bowl type of
centrifuge. lt consists of several chambers
connectedin such a wav that the feed flows in a
zigzag fashion. There is a variation in the
centrifugalforce in differentchambers.The force is
m uc h highe ri n th e p e ri p h e ryc h a m b e rsa, s a resul t
smallestoarticlessettle down in the outermost
chamber.
Scroll centrifugeor decantet (Fig. 20.4D) t lt is
composedof a rotatinghorizontalbowl taperedat
one end. The decanter is generally used to
concentratefluids with high solid concentration
(biomasscontent5-80%).The solidsare deposited
on the wall of the bowl which can be scrappedand
removedfrom the narrowend.
P R OD U C TS
As already stated, there are several
biotechnological products (vitamins, enzymes)
which are located within the cells. Such
compounds have to be first released (maximally
and in an active form) for their further processing
and final isolation. The microorganisms or other
cells can be disintegratedor disrupted by physical,
chemical or enzymatic methods. The outline of
different techniques used for breakage of celis is
given in Fig. 20.5. The selection of a particular
method depends on the nature of the cells, since
there is a wide variation in the property of cell
disruption or breakage.For instance,Cram-negative
bacteria and filamentous fungi can be lnore easily
broken compared to Cram-positive bacteria and
yeasrs.

Clarified
fluidout Fluid
tn

Cells

Cells
ano
Fluidin solids Clarified Gellsand
(A) (c) fluid out solids
(B) (D)

Fig. 20.4 : Centrifuges commonly used in downstream processing (A) Tubular bowl centrifuge (B) Disc
centrifuge (C) Multichamber centrifuge (D) Scroll centrifuge (decante).

Biotechnology [18]
274 B IOTE C H NO LO CY

*
Ultrasonication Alkalies
I
Osmoticshock Organicsolvents L Glucanase,mannanase
and protease
Heat shock Detergents
(thermolysis)

High-pressure +
*
homogenisation

lmpingement*
Grindingwith glass beacls*

Fig. 20.5 : Major melhods for cell disruption to release the intracellular prcducts
(* indicate fieahanicat methods while all the remaining are non-mechanical).

CELL DTSBUPT'ON i o s t r i k e o r h i t ) . T h e c e l l s a r e d i s r u p t e d b y th e
forces created at the point of contact.
Physical methods of cell disruption
Microfluidizer is a device developed based on the
The m ic r oor ganis m sor c ells c a n b e d i s r u p t e d b y p r i n c i p l e o f i m p i n g e m e n t l t h a s b e e n s u c ce ssfu l l y
c er t ain ohv s ic al m et hodst o r elea s et h e i n t r a c e l l u r a r used for breaking F. coli cells. The advantagewith
oroducts. i m p i n g e m e n t t e c h n i q u e i s t h a t i t c a n b e e f fe cti ve l y
used for disrupting cells even at a low
Ult r as onic at ion : Ult r as onic d i s i n t e g r a t i o n r s
concentratron.
widely employed in the laboratory. However, clue
to high cost, it is not suitable for large-scaleuse rn Grinding with glass beads : The cells mixed
indus t r ies . with glass beads are subjectedto a very high speed
i n a r e a c t i o n v e s s e l . T h e c e l l s b r e a k a s t he y a r e
O s m ot ic s hoc k : This m et h o d i n v o l v e s t h e
forced against the wall of the vessel b1' the beads.
s us pens ionof c ells ( f r ee lr om gr o w t h m e d i u m ) r n
'fhe Severalfactors influence the cell breakage-sizeand
20% buffered sucrose. cells are then transferreo
quantity of the glass beads, concentration and age
to water at about 4'C. Osmotic shock is used for
of cells, temperature and agitator speed. Under
the r eleas e of hy dr oly t ic enz y m e s a n d b i n d i n g
o p t i m a l c o n d i t i o n s , o n e c a n e x p e c t a ma xi m a l
proteins from Cram-negative bacteria.
breakage of about BO% of the cells
Heat shock (thermolysis) : Breakageof cells by
A diagrammatic representation of a cell
subjecting them to heat is relatively easy and
disrupter employing glass beeds is shown In
c heap. But t his t ec hnique c an be u s e d o n l y f o r a
Fig. 20.6. It contains a cylindrical body rn,ith an
v er y f ew heat - s t ableint r ac ellula rp r o d u c t s .
inlet, outlet and a central motor-drivenshaft.To this
High pressure homogenization : This technique shaft are fitted radial agitators.The cylinder is fitted
inv olv es f or c ing of c ell s us pens io na t h i g h p r e s s u r e w i t h g l a s s b e a c j s . T h e c e l l s u s p e n s i o n i s a d d e d
through a very narrow orifice to come out to t h r o u g h t h e i n l e t a n d t h e d i s r u p t e d c e l l s c om e o u t
a t m os pher ic pr es s ur e.This s udde n r e l e a s eo f h i g h t h r o u g h t h e o u t l e t . T h e b o d y o f t h e c e l l d i s ru p te r r s
pressure creates a liquid shear that can break the kept cool while the operation is on.
c el Is .
Mechanical and non-mechanical
lmpingement : In this procedure, a stream of
methods
s us pendedc ells at high v eloc it y a n d p r e s s u r ea r e
forced to hit either a stationarysurfaceor a second Among the physical methods of cell disruption
s t r eam of s us pendedc ells ( im pin g e l i t e r a l l y m e a n s d e s c r i b e d a b o v e , u l t r a s o n i c a t i o n , h i g h - p r e ssu r e
 Cha pte r 20 : D O W NSI REAM PRO CTSSI NC 275

Cellsuspension
Disruptedcells
I
I I
+
{- Coolant
O0
oo
o
o oo Glass beads
Oo
0
{- Shaft
0
0
o
o
ni Bodyof disrupter

Radialdisc
Baffleto
preventoutflow

Fig. 20.6 : Diagrammatic representation of a cell disruptet.

ho mog en isa tion, im pigem ent and gr inding w i t h membrane proteins and lyse the cells. Non-ionic
glass beads ate mechanical while osmotic shock cietergents(although less reactive than.ionic ones)
and heat shock are non-mechanicnT.rh" chemical are also used to some extent e.g., Tritdn X-l00 or
and enzymatic methods (described belc.rw)are non- lween. The problem with the use of detergentsis
mechanical in nature that they affect purification steps, particularly the
s a l t p r e c i p i t a t i o n .T h i s l i m i t a t i o n c a n b e o v e r c o m e
Ghemical methods of cell disruption by using ultrafiltration or ion-exchange chromato-
graphy for purification.
Tre atme nt wit h a lk alies , or ganic s olv e n t s
and detergents can lyse the cells to release the
contents. Enzymatic methods of cell disruption

Alkalies : Alkali treatment has been used for the Cell disruption by enzymatic methods has
extraction of some bacterial oroteins. However. the c e r t a i n a d v a n t a g e si . e . , l y s i s o f c e l l s o c c u r s u n d e r
alkali sta bilityo f t he des ir ed pr oduc t is v er y c r u c i a l m i l d c o n d i t i o n s i n a s e l e c t i v em a n n e r .T h i s i s q u i t e
fo r th e su cces sof t his m et hod e. 9. , r ec om bi n a n t advantageous for product recovery
growth hormone can be efficiently released from
Lysozyme is the most frequently used enzyme
E. coli by treatment with sodium hydroxide at
and is commercially available (produced from hen
p H 1 1.
egg white). lt hydrolysesB-1, 4-glycosidic bonds of
Orga nic solv ent s : Sev er al wat er m is c i b l e the rnucopeptide in bacterial cell walls. The Cram-
orq .rnic so lve nt s c an be us ed t o dis r upt t he c e l l s positive bacteria (with high content of cell wall
: q , meth an ol, et hanol, is opr opanol, br , r t a n o l . mucopeptides)are more susceptibiefor the action
Th ese comp ou nds ar e inf lam m able, henc e r eq u i r e of lysozyme. For Cram-negativebacteria, lysozyme
.:re cia lise de qu ipm ent f or f ir e s af et y .The or ga n i c i n a s s o c i a t i o nw i t h E D T A c a n b r e a k t h e c e l l s A s
: .e'rt tolu en e is f r equent ly us ed. lt is belie v e d the cell wall gets digestedby lysozyme,the osmotic
--.- :oiu en e d is s olv esm em br ane phos pholipidsa n d effects break the periplasmic membrane to release
- -:-:i€Snreffibrane pores for releaseof intracellular t h e i n t r a c e l l u l a rc o n t e n t s .
Certain other enzymes are also used, although
Detergents : Detergentsthat are ionic in nature, less frequently, for cell disruption. For the lysis of
:atio nic-ce tvl tr im et hy l am nr onium br om ide o r yeast cell walls, glucanase and mannanase in
rrro nic-.od ium laur y l s ulf at e c an denatu r e combination with oroteasesare useo
276 B IOTE C HNO LO CY

Combination of methods L'QU'D.LTQUID EXTRACTION

I n or der t o inc r eas e t he e f f i c i e n c y o f c e l l
The concentration of biologicalproductscan be
disintegration in a cost-effective manner, a achieved by transferringthe desiredproduct (solute)
combination of physical, chemical and enzymatic from one liquid phaseto another liquid phase,a
methods is employed. phenomenon referredto as liquid-liquidextraction.
B esi des concentrati on, thi s techni quei s al sousef ul
COhSCENTRATION for partialpurification of a product.Theefficiencyof
The filtrate that is free from suspendedparticles
extractionis dependenton the partition coefficient
(cells, cell debris etc.) usually contains BO-98"/"of i.e. the relativedistributionof a substance between
product the tw o l i qui d phases.The processof l i quid- liQ uid
water. The desired is a very minor
constituent. The water has to be removed to
extractionmay be broadlycategorizedas extraction
achieve the product concentration.The commonly
of low molecular weight products and extraction of
us ed t ec hniques f or high molecular weight products.
c onc en t r a t i n g b i o l o g i c a l
products are evaporation, Iiquid-liquid extraction,
Extraction of low molecular
membrane filtration, precipitation and adsorption.
weight products
The actual procedure adopted depends on the
n at ur e of t he des ir ed pr oduc t ( qu a l i t y a n d q u a n t i t y
B y usi ng organi c sol vents,the l i p ophilic
to be retainedas far as possible)and the cost factor.
compounds can be conveniently extracted.
However,it is quite difficultto extracthydrophilic
EIfAP&HATION
compounds.E xtracti on of l i pophi l i cprod uct scan
Water in the broth filtrate can be rernoved by a be done by the fol l ow i ngtechni ques.
simple evaporation process. The evaporators, In
general, have a heating device for supply of steam,
Physicalextraction: The compoundgets itself
and unit for the separationof concentratedproduct
distributedbetweentwo liquid phasesbasedon the
physi cal properti es.Thi s techni quei s u sed f or
and vapour, a condenser for condensing vapour,
compounds.
extracti onof non-i oni si ng
accessoriesand control equipment. The capacity of
t hb equipm ent is v ar iable t hat m a y r a n g e f r o m Dissociationextraction : This technique is
sm all labor at or y s c ale t o indus t r i a l s c a l e . S o m e o f suitablefor the extractionof ionisablecompounds.
the important types of evaporatorsin common use Certain antibiotics can be extracted by this
are briefly described. oroceoure.
Plate evaporators : The liquid to be
Reactiveextraction: In this case,the desired
concentrated flows over plates. As the steant is
supplied,t he liquid get s c onc ent r a t e da n d b e c o m e s
oroduct is made to reactwith a carriermolecule
(e.g.,phosphorus compound,al i phati camine)and
vr s c ouS.
extractedinto organicsolvent.Reactiveextraction
Falling film evaporators l In this case, the liquid procedure is quite useful for the extractionof
flows down long tubes which gets distributed as a certai ncompounds that are hi ghl ysol ubl ein wat er
t hin f ilm ov er t he heat ing s ur f a c e F a l l i n g f i l m (aqueousphase)e.9.,organi caci ds.
evaporators are suitable for removing water from
viscous products of fermentation. Supercritical fluid (SCF) extraction : This
techniquediffersfrom the aboveprocedures, since
Forced film evaporators : The liquid films are
the materialsused for extractionare supercritical
m ec hanic allydr iv en and t hes e d e v i c e s a r e s u i t a b l e
fluids (SCFs).SCFsare intermediatesbetween gases
for producing dry product concentrates.
and liquids and exist as fluids abovetheir critical
Centrifugal forced film evaporators : These temperature and pressure. COr, with a
Supercritical
equipm ent ev apor at e t he liquid v e r y q u i c k l y ( i n low criticaltemperature and pressureis commonly
seconds), hence suitable for concentrating even usedin the extraction. fluid extraction
Supercritical
h eaf labile s ubs t anc es . I n t hes e e v a p o r a t o r s , a is ratherexpensive, hencenot widely used(SCFhas
centrifugal force is used to pass on the liquid over been usedfor the extractionof caffeinefrom coffee
heated plates or conical surfacesfor instantaneous beans,and pigmentsand flavor ingradientsfrom
evaporaton. bi ol ogi calmateri al s).
 Chapt er20 : D O W N ST R EA M
P R OC ES SIN G 277

Extraction of high molecular The other types of membrane filtration techniques

weight compounds are discribed briefly.

Prote ins ar e t he m os t pr edom inant h i g h Membrane adsorbers : They are micro- or

mo lecula r w eight pr oduc t s pr oduc ed in macroporousmembraneswith ion exchangegroups
fermentation industries.Organic solventscannot be and/or affinity ligands.Membrane adsorberscan bind
used for protein extraction, as they lose their to proteins and retain them. Such proteins can be
biological activities.They are extractedby using an eluted by employing solutionsin chromatography.
aqueous two-phase systems or reverse micelles
Pervaporation : This is a technique in which
formation.
volatile products can be separatedby a processof
Aqueous two-phase systems (ATPS) : They can permeation through a membrane coupled with
b e pre pa red by m ix ing a poly m er ( e . g . , evaporation. Pervaporation is quite useful for the
p olyeth yle neg ly c ol) and a s alt s olut ion ( am m on i u m extraction, recovery and concentration of volatile
sulfate) or two different polymers. Water is the products. However, this procedure has a limitation
main component in ATPS, but the two phases are since it cannot be used for large scale separationof
no t miscible . Cells and ot her s olids r em ain in o n e volatile products due to cost factor.
phase while the proteins are transferred to other
Perstraction : This is an advanced technique
p ha se . Th e dis t r ibut ion of t he des ir ed pr oduct i s
working on the principle of membrane filtration
based on its surface and ionic character and the
c o u p l e d w i t h s o l v e n t e x t r a c t i o n .T h e h y d r o p h o b i c
n atu re o f ph ases .Th s epar at iont ak es m uc h lon g e r
compounds can be recovered/concentratedby this
time by ATPS
method.
Reversemiceller systems : Reversemicelles are
stable aggregatesof surfactantmolecules and water PNEC'PITAT'ON
in organic solvents.The proteins can be extracted
Precipitation is the most commonly used
from the aqueous medium by forming reverse
technique in industry for the concentration of
micelles. In fact, the enzymes can be extracted by
m a c r o m o l e c ul e s such as proteins ano
th is p roced ure wit hout los s of biologic al ac t ivi t y .
p o l y s a c c h a r i d e s .F u r t h e r , p r e c i p i t a t i o n t e c h n i q u e
can also be employed for the removal of certain
MEMBBANE FILTNAT'ON
unwanted byproducts e.g. nucleic acids, pigments.
Membrane fijtration has become a common Neutral salts, organic solvents, high molecular
se pa ratio ntechnique in indus t r ialbiot ec hnolog y .l t weight polymers (ionic or non-ionic), besides
can be conveniently used for the separation of alteration in temperature and pH are used in
biomolecules and particles, and for the precipitation. ln addition to these non-specific
con ce ntra tion of f luids . The m em br ane f ilt r a t i o n protein precipitation reactions(i.e. the nature of the
techn iqu e b asic ally inv olv es t he us e of a s e m r - protein is unimportant), there are some protein
permeable membrane that selectively retains the specific precipitations e.g., affinity precipitation,
particles/moleculesthat are bigger than the pore ligand precipitation.
size wh ile the s m aller m olec ules pas s t hr ough t h e
Neutral salts : The most commonly used salt is
memDra ne po res .
ammonium sulfate, since it is highly soluble, non-
Memb ran es us ed in f ilt r at ion ar e m ade up o f t o x i c t o p r o t e i n s a n d l o w - p r i c e d . A m m o n i u m
po lyme ric ma ter ials s uc h as poly et her s ulf onea n d sulf aIe increases hyd rophobi c interactions between
po lyvinyl d ifluo r ide. I t is r at her dif f ic ult t o s t er i l r z e p r o t e i n m o l e c u l e s t h a t r e s u l t i n t h e i r p r e c i p i t a t i o n .
membrane filters. In recent years, microfilters and The precipitation of proteins is dependent on
ultrafilters comoosed of ceramics and steel are several factors such as protein concentration, pH
ai,a ilab le.Cle an ing and s t er iliz at ionof s uc h f ilte r s and temperature.
are easy.
Organic solvents : Ethanol, acetone and
The filtration techniques namely microfiltration, propanol are the commonly used organic solvents
u I t rafi It rati o n, and hype rfi ltrat ion (rev erse osmos i s) for protein precipitation. They reduce the dielectric
"ar,e already been described (Refer Table 20.1). constant of the medium and enhance electrostatic
274 B IOlE C HNO LO CY

interaction beiween orotein molecules that lead to

precipitatior.r Since proteins are denatured by Trnu 20.2 Ghromatographlc techniquesalong
organic solvents,the precipitation processhas to be with the principlesfor separationof proteins
carried out below OoC.
Chromatography Principle
Non-ionic polymers z Polyethylene glycol (PEC)
is a high m olec ular weight non- i o n i c p o l y m e r t h a t
(sizeexclusion)
GelJiltration Sizeandshape
can precipitate proteins. lt reduces the quantity of lon-exchange Netcharge
water available for protein solvation and
precipitates protein. PEC does not denature Chromatofocussing Netcharge
pr ot eins , bes ides being non- t ox i c . Affinity Biological
alfinityand
lonic polymers : The charged polymers such as
recognition
molecular
palyacrylic acid and polyethyleneimine are used. interaction
Hydrophobic Polarity
They fornr complexes with oppositely charged (hydrophobicity
pr ot ein m olec ulest hat c aus esc h a r g e n e u t r a l i s a t i o n ol molecules)
and precepitation.
metal-ion
lmmobilized affiniiy Metalionbinding
lncrease in temperature : The heat sensitive
proteins can be precipitated by increasing the
tenroeratLrre.
related conrpoundsfrom a mixture. Chromato-
Change in pH : Alterations in pH can also leacl graplrl, usually consistsoI a stationary phaseand
t o pr ot ein pr ec ipit at ion. mobile phase.The stationaryphaseis the porous
sol i dmatri xpackedi n a col umn(equi l i b r at ed
wit h
Affinity precipitation : Tlre affinity interaction
a sui tabl esol vent)on to w hi ch the m ixt ur e of
(e.9., betw,eenantigen arrd antibody) is exploited
compounds to be separated is loaded. The
for precipitation of proterns
compounds are el utedby a mobi l ephaseA . single
Precipitation by ligands : Ligands with specific mobi l ephasemay be usedcorrti nuously or it m ay
binding sites for proteins have been successfully be changedappropriately to facilitatethe releaseof
used for selective precipitation. desi redcornpounds. The el uatefrom the colum n
can be monitoredcontinuously (e.g.proteinelution
.rUlS.StrPrtOAt can be monitoredby ultravioletadsorptionat 280
nm), and collectedin fractionsof definitevolumes.
The biologicai products of fermentation can be
concentrated by using solid adsorbent particles. In The different types of chromatography
the early da1,s,s.11urr"d charcoal was used as tne techniquesused for separation(mainly proteins)
adsorbentrnaterial In recent years, cellulose-based alongwith the principlesaregivenin Table20.2.A
adsorbentsare employed for protein concentration. large number of matrices are commercially
And for concentration of low molecular weight availablefor purificationof proteinse.9., agarose,
c om pounds ( v it am ins , ant i b i o t i c s , p e p t i d e s ) cel l ul ose,pol yacryl ami de,porous si l i ca, cr oss-
polystyrene, methacrylate and acrylate based Iinkeddexti"an, polystyrene.
Someof the important
matrices are used. The process of adsorption can featuresof selectedchromatographic techniques
be carried out by making a bed of adsorbent are brieflydescribed.
c olum n and pas s ing t he c ult ur e b r o t h t h r o u g h i t .
Gel-filtration chromatography: This is also
The desired product, held by the adsorbent,can be
referred to as size-exclusionchromatography. ln
eluted.
this technique,the separation of moleculesis based
on the si ze, shape and mol ecul arw eight . The
PURI FI CATI O N BY CHRO M A T O G R A P H Y
sponge-like gel beadswith poresserveas molecular
The biological products of fermentation sieves for separation of smaller and bigger
( pr ot eins , phar nr ac eut ic als diag
, m olecules
A sol uti onmi xturecontai ni ng
n o s t i c c o m p o u n d s mol ecul es.
and researchrnaterials)are very effectively purified of differentsizes(e.g.differentproteins)is applied
by c hr om at ogr aphy . it is bas i c a l l y a n a n a l y t i c a l to the col umn and el uted.The smal l erm olecules
t ec hnique dealing wit h t he s e p a r a t i o n o f c l o s e l y enter the gel beadsthrough their pores and get
 Chapter2o : DoWNSTREAMPROCESSINC 279

The fresh ligand used has to be removed in the

subsequentsteps,
Small
moleculeHydrophobic interaction chromatography
(HlC) : This is based on the principle of weak
hydrophobic interactionsbetween the hycirophobic
Large
molecule ligands (alkyl, aryl side chains on matrix) and
hydrophobic amino acids of proteins. The
Fiq.20,7 : Theprincipleol gel-liftrationahromatogmphy. differences in the composition of hvdrophobic
amino acids in proteins can be used for
their separation. The elution of proteins can be
trapped.On the other hand, the largermolecules done by lowering the salt concentration,decreasing
cannotpassthroughthe poresand thereforecome t h e p o l a r i t y o f t h e m e d i u m o r r e d u c i n g t h e
(Fig. temperature.
out first with the mobile liquid 20.V. At the
indus t r iasl c a l e ,g e l -fi l tra ti o ins p a rti c u larl yuseful
t o r em o v e s a l ts a n d l o w mo l e c u l a r w ei ght F O R M U L A T I O N
compoundsfrom high molecularweight products. Formulation broadly refers to the mairrtenance
lon-exchange chromatography : lt involvesthe o f activity and stability of a biotechnological
separationof moleculesbased on their surface p r o d ucts during storage and distribuiion. -fhe
charges.lon-exchangers are of two types (cation- f o r m u l a t i o n o f l o w m o l e c u l a r w e i g h t p r o d u c t s I'
exchangerswhich have negativelychargedgroups (solvents. organic acids) can be achieved by ll

like carboxymethyland sulfonate, and anion- concentrating them with removal of most of the rl

exchangerswith positively charged groups like w a t e r . F o r c e r t a i n s m a l l m o l e c u l e s , ( a n t i b i o t i c s , Ir"

!rl
dlet . hy la m i n o e th(D y l EA E).T h e m o s t commonl y c i t r i c a c i d ) , f o r m u l a t i o n c a n b e d o n e b y i ti l
used cation-exchangers are Dowex HCR and c r y s t a l l i z a t i o nb y a d d i n g s a l t s . I

AmberlitelR, the anion-exchangers are DowexSAR liit

Proteins are highly susceptible for loss of
and A m b e rl i tel R A. l'. t
biological activity, hence their formulation
requires l :t!
In ion-exchange chromatograohy,the pH of the special care. Certain stabilizing additives are added ll
mediumis very crucial,sincethe net chargevaries to proiong the shelf life of protein. The stabilizers
with pH. ln other words, the pH determinesthe of protein formulation include sugars (sucrose,
effectivechargeon both the targetmoleculeand lactose),safts(sodium chloride, ammonium sulfate),
the ion-exchanger. The ionic bound moleculescan polymers (polyethylene glycol) and polyhydric
be elutedfrom the matrix b1,changingthe pH of alcohols (glycerol). Proteins may be formulated
the eluantor by increasingthe concentration
of salt in the form of solutions, suspensions or dry
solution. lon-exchangechromatographyis useful oowoers.
fo.r the purification of antibiotics,besides the
purificationof proteins.
Affinity chromatography : This is an elegant
method for the purificationof proteinsfrom a
complexmixture.Affinitychromatography is based chromatognphy
on an interactionof a proteinwith an immobilizeo
Ligand Type of protein
ligand. The ligand can be a specific antibody,
substrate,substrateanalogueor an inhibitor.The Antibody Antigen
im m obili z e dl i g a n d o n a s o l i d m a tri x can be Cofactor Enzyme
effectively used to fish out complementary Receptor Hormone
-itructures ln Table20.3, some examplesof ligands Hapten Antibody
-'ed ior the purificationof proteinsare given.The Inhibitor Enzyme
:ro:ein bound to the ligand can be eluted by Lectins Glycoproteins
=ei.rcinqtheir interaction. Thiscan be achievedby
Heparin Coagulation
factors
:-a:rqing the pH of the buffer,alteringthe ionic
r::^Eih or b1,usinganotherfree ligandmolecule. Metalions proteins
Metalionbinding
 2ao
Drying
Dr y ing is an es s ent ial c om p o n e n t o f p r o d u c t
f or m ulat ion. lt bas ic ally inv ol v e s t h e t r a n s f e r o f
heat to a wet product for removal of moisture.Most
of the biological products of fermentation are
sensitiveto heat, and thereforerequire gentle drying
methods.
Based on the method of heat transfer, drying
devices may be categorized as contact-,
convection-, radiation dryers. These three iypes of
dr y er s ar e c om m er c ially av aila b l e .
Spral' drying
Spr ay dr y ing is us ed f or dr y in g l a r g e v o l u m e s o f
liquids . I n s pr ay dr y ing, s m al l d r o p l e t s o f l i q u i d
c ont aining t he pr oduc t ar e pas s e dt h r o u g h a n o z z l e
directing it over a stream of hot gas. The water
evaporatesand the solid particles are left behind.
Freeze-drying
Freeze-drying or lyophilization is the most
preferred method for drying and formulation of a
wide-range of products-pharmaceuticars,
foodstuffs, diagnostics, bacteria, viruses. This is
i
B IOTE C HNO LO C\
r n a i n l y b e c a u s e f r e e z e - d r y i n g u s u a l l y do e s n o t
cause loss of biological activity of the desired
oroduct.
L y o p h i l i z a t i o n i s b a s e d o n t h e p r i nci p l e o i
s u b l i m a t i o n o f a l i o u i d f r o m a f r o z e n s t ate . In th e
a c t u a l t e c h n i q u e , t h e l i q u i d c o n t a i n i n g t h e p r o d u ct
is frozen and then dried in a freeze-dryer under
v a c u u m . T h e v a c u u m c a n n o w b e r e l ea se d a n d
t h e p r o d u c t c o n t a i n i n g v i a l s c a n b e se a l e d
e . g . , p e n i c i l l i n c a n b e f r e e z e d r i e d d i r e ctl y i n
a m p ur e s .
INTEGRATION OF
DIFFERENT PROCESSES
lt is ideal to integrate the fermentation and
downstream processing to finally get the desired
product. However, this has not been practicablefor
vanous reasons.
lntegration of certain stages in downstream
processingfor purification of product has met with
some success. For instance. protein concentration
by extraction into two phase systems combined
with clarification and purification cah be done
together.
f
n zymes ar e t he bioc at aly s t s s y nt hes i z e d b y A s per recentesti mates,
and i n sci enti fi cresearch.
L- living c ells . They ar e c om plex p r o t e r n a great majority of industrially produced enzymes
mole cu les t hat br ing about c hem ic al r e a c t i o n s are useful in processes related to foods (45"/"),
co ncern ed wit h lif e. The gener al f eat u r e s o t detergents(35'/"), textiles(10%) and leather (3"k)
en zymes ar e giv en in Chapt er 66. For detai l s on the appl i cati onsof i ndi vi dual
enzymes,Tables21.1-21.3 @iven later)must be
It is fortunat et hat enz y m es c ont inue t o f u n c t i o n
referred
(bring out catalysis)when they are separatedfrom
the cells i.e. in vitro. Basically,enzymes are non-
to xic an d b iodegr adable.They c an be pr od u c e d i n
la rge amo unt s by m ic r oor ganis m s f or in d u s t r i a l
ap plicatio ns .

Enzyme technology broadly involves

Microbial enzymeshave been utilized for many
production, isolation, purification and use of
centuries without knowing them fullv fhe first
enzymes-(in soluble or immobilized form) for the
enzyme produced industrially was takadiastase
ultima te benef it of hum ank ind. I n a d d i t i o n ,
( a f u n g a l a m y l a s e )i n 1 8 9 6 , i n U n i t e d S t a t e s .l t w a s
recomb ina nt DNA t ec hnology and p r o t e i n
used as a pharmaceutical agent to cure digestive
e ng ine erin g inv olv ed in t he pr oduc t ion o f m o r e
d isorders.
efficient and useful enzymes are also a part of
enzyme technology. The commercial production ln Europe,there existed a centuriesold practice
and use of enzymes is a major part of of softening the hides by using feces of dogs and
b iote ch no logy indus t r y . The s pec ialit i e s l i k e pigeons before tanning. A Cerman scientist (Otto
.l
microb iolo gy , c hem is t r y and pr oc es s engin e e r i n g , Rohm) demonstrated in 905 that extracts from
ire sid esb ioc hem is t r y hav e lar gely c ont r ibu t e d f o r a n i m a l o r g a n s ( p a n c r e a s e fsr o m p i g a n d c o w ) c o u l d
:re growth of enzyme technology. be used as the source of enzymes-proteases,for
leather softening.
APPLICATI O NS O F ENZYM ES
T h e u t i l i z a t i o n o f e n z y m e s ( c h i e f l y p r o t e a s e sf)o r
.l
En zvnre shav e wide r ange of applic at ion s .T h e s e laundry purposesstarted in 9.15. However, it was
- --ird e their us e in f ood or oduc t ion . f o o d n o t c o n t i n u e d d u e t o a l l e r g i c r e a c t i o n so f i m p u r i t i e s
':e:sing and pr es er v at ion, was hing po w d e r s , i n e n z y m e s . N o w s p e c i a l t e c h n i q u e s a r e a v a i l a b l e
':,i € 'rdflu f dc t ur e, leat herindus t r y ,paper in d u s t r y , f o r m a n u f a c t u r e , a n d u s e o f e n z y m e s i n w a s h i n g
--=: :a l ap plic at ions ,im pr ov em ent of env ir o n m e n t p o w d e r s ( w i t h o u t a l l e r g i c r e a c t r o n s ) .

2',1
282 B IOTE C H N OLO CY

A selected list of plant (Table 21.1) and animai

Tlru 21.1 Commerciallyproducedenzynes (Table 21 .2) enzymes with their sources atrd
fron plant sourcesand their applications a p p l i c a t i o n sa r e g i v e n .

Enzyme Source(s) Application(s) Aninral organs and tissuesare very good sources
for enzymes such as lipases, esterases and
p-Amylase Barley,
soybean Baking,preparation proteases.
The enzyme lysozyme is mostly obtained
of maltcse
syrup from hen eggs. Some plants are excellent sources
Bromelain Pinoqnnlo Baking for certain enzymes-papain (papaya), bromelain
Esterase Wheat Esterhydi'olvsis ( p i n e a p p l e ) .
Ficin Fig Meattenderiser l-imitations : There are several drawbacks
Papain Papaya Meattenderiser, associatedwith the manufacture of enzymes from
tanning,
baking a n i m a l a n d p l a n t s o u r c e s .T h e q u a n t i t i e sa r e l i mi te d
Peroxidase Horseradish Diagnostic a n d t h e r e i s a w i d e v a r i a t i o n i n t h e i r d i s t r i b u ti o n .
Urease Jackbean Diagnostic The most important limitations are the difiicuities
i n i s o l a t i n g , p u r i f y i n g t h e e n z y m e s , a n d t h e co st
factor. As regards extraction of industrial enzymes
Commercial enzymes can be produced from a
from bovine sources, there is a heavy risk of
wide range of biological sources.At present,a great
ccntaminatic,rnwith bovine spongiform encephalo-
majority (80%) of them are from microbial sources.
p a t h y ( B S Ei s p r i o n d i s e a s ec a u s e d b y i n g e s t i o no f
T he dif f er ent or ganis m s and t h e i r r e l a t i v e abnornral proteins)'.For these reasohs, nticrobial
contribution tbr the oroduction of commercial production of enzymes is preferred.
enzvmes are siven below.
Enzymes from mammalian cell cultures
Fungi - 60%
T h e r e e x i s t s a p o s s i b i l i t v o f p r o d u ci n g
Bacteria - 24"/o
c o m m e r c i a l e n z y m e s d i r e c t l y b y m a m m a l i a n ce l l
Yeast - 4% cultures. But the main constraintwill the cost facror
Streptomyces - 2o/o w h i c h w i l l b e e x t r e m e l y h i g h . H o w e v e r , c e r ta i n
Higher anim als - 6% therapeutic enzvmes such as tissue plasminogen
activator are produced by cell cultures.
Higher plants - 4"/o
A r eai br eak t hr ough f or lar ge sc a l e i n d u s t r i a l Enzymes from microbial sources
production of enzymes from rnicroorganisrns Microorganisms are the most significant and
occurred after 'l 950s. c o n v e n i e n t s o u r c e s o f c o m m e r c i a l e n z ym e s.
They,can be made to produce abundant quantities
Enzymes from animal and
o f e n z y m e s u n d e r s u i t a b l e g r o w t h c o n d i t i o n s.
plant sources
M i c r o o r g a n i s m s c a n b e c u l t i v a t e d b y u si n g
lr r t he ear ly day s , anim al and p l a n t s o u r c e s inexpensive media and production can take place
largely contributed to enzymes. Even now, for i n a s h o r t p e r i o d . I n a d d i t i o n , i t i s e a sy to
certain enzymes they are the major sources. m a n i p u l a t e m i c r o o r g a n i s m si n g e n e t i c e n g i n ee r i n g

Tnnrs21.2 Commerciallyproduced€nrymesfrom animal sourcesand their applications

Enzyme(s) Source(s) Application(s)

Amy!ase,eslerase Lamb,calf, Digestive
aids,
pepsin,
trypsin, bovine, preparation
of cheese
lipase,
rennin(chymosin), p0rctne
phospholipase,
phytase
Lysozyme Cellwallbreakagein bacteria
lql _'gg'-
Humanurine Urokinase Fordissolution
of bloodclots
Chapter21 : ENZYMETECHNOLOGY
Preserved
inoculunr
FinalpuriJication
(chromatographyetc.)
Fiq.21.1 : An outline of the flow chart for the production of enzymes by m'rcroorganisms.
te ch niq ue s to inc r eas e t he pr oduc t ion of des i r e d
enzyrnes. Recovery, isolation and purification
processesare easy with microbial enzymes than
th at with an im al or olant s our c es .
In fact, most enzymes of industrial applications
h ave b ee n s uc c es s f ully pr oduc ed by m i c r o -
organisms Various fungi, bacteria and yeasts are
emp loyed fo r t his pur pos e. A s elec t ed list o f
en zymes, mic r obial s our c es and t he applic a t i o n s
are given in Table 21 .J.
Aspergillus niger-A unique organism
for production of bulk enzymes
Among the microorganisms,A. niger (a fungus)
occupies a special position for the manufacture
of a iarge number of enzymes in good quantities.
There are well over 40 commercial enzymes
that are conveniently produced by A. niger. These
include c-amylase, cellulase, protease, lipase,
pectinase, phytase, catalase and insulinase.
THE TECHNOLOGY OF ENZYME
PRODUCTION-GENERAL
CONSIDERATIONS
ln ge ne ral, t he t ec hniques em ploy ed f o r
microbial production of enzymes are comparable
to the methods used for manufacture of other
ind ustrial p rod uc t s ( Ref erChapt er 15) . The s a l i e n t
features are briefly described.
1 . Se lection of or ganis m s
2. Formu lat ion of m edium
283
Cool
srorage
3. Production orocess
4. Recovery and purification of enzymes.
An outline of the flow chartfor enzyme production
by micrcrorganisms is depicted in Fig. 21.1.
Selection of organism
The most important criteria for selecting the
microorganism are that the organism should
produce the maximum quantitiesof desiredenzyme
in a short time while the amounts of cther
metabolite produced are minimal. Once the
organism is selected, strain improvement for
optimising the enzyme production can be done by
appropriate methods (mutagens,UV rays).From the
organism chosen, inoculum can be prepared in a
liouid medium.
Formulation of medium
The culture medium chosen should contain all
the nutrients to support adequate growth of
microorganisms, that will ultimately result in
good quantities of enzyme production. The
ingradients of the medium should be readily
available at low cost and are nutritionally safe.
Some of the commonly used substratesfor the
medium are starch hydrolysate, molasses, corn
steep liquor, yeast extract, whey, and soy bean
meal. Some cereals (wheat) and pulses (peanut)
h a v e a l s o b e e n u s e d .T h e p H o f t h e m e d i u m s h o u l d
be kept optimal for good microbial growth and
enzyme production.
2A4 BIOTECHNOLOCY

Enzyme Source(s) Application(s)

a-Amylase Aspergillus
oryzae Production
of beerandalcohol,
Aspergillus
niger of glucose
Preparation syrups,
Bacillussubtilus As a digestive
aid
Bacillus
licheniforms Removal
of starchsizes
Amyloglucosidase Aspergillus
niger Starchhydrolysis
Rhizopus
niveus
Cellulase Aspergillus
niger production
andglucose
Alcohol
Tricoderma
koningi
Glucoamylase Aspergillus
niger Production
of beerandalcohol
Bacillus
amyloliquefaciens Starchhydrolysis
Glucoseisomerase Afthrobacter
sp of highfructose
Manufacture syrups
Bacillus
sp
Glucoseoxidase Aspergillus
niger in prepared
Antioxidant foods
Invertase Saccharomyces cerevisi
ae inversion
Surcose
Preparation
of aritficial
honey,
confectionaries
Keratinase Streptomyces
lradiae of hairfromhides
Removal
Lactase Kluyveromyus
sp Lactose
hydrolysis
Sacch
aromycesIragilis Removal
of lactose
fromwhev
Lipase Candidalipolytica Preoaration
of cheese
Asperigillus
niger Flavour
oroduction
Pectinase Aspergillus
sp of fruitluices
Clarification andwines
Sclerotina
libeftina Alcoholproduction,coffeeconcentration
Penicillin acylase Escherichia
coli Production
oJ6-aminooenicillanic
acid
Penicillanase Bacillussubtilis Removal
of oenicillin
Protease.acid Aspergillus
niger Digestive
aid
for calfrennet
Substitute
Protease,
neutral Bacillusamyloliquefaciens Fishandmeattenderiser
Protease.
alkaline Aspergillus
oryzae Meattenderiser
Streptomycesgriseus Detergent
additive
Bacillus
sp Beerstabilizer
Pollulanase Klebsiella
aerogens Hydrolysis
of starch
Takadiastase Aspergillus
oryzae Supplement
lo bread,
Digestive
aid
Chaot er2 1 : EN Z YMET E C H N OL OGY
Production process
Industrial production of enzymes is mostly
carried out by submerged liquid conditions, and
to a lesser extent by solid-substrate fermentation.
In su bm er ged c ult ur e t ec hnique, t h e y i e l d s
are more and the chances of infection are less.
Hence, this is a preferred method However, solid
substrate fermentation is historically important
a nd still in us e f or t he pr oduc t ion o f f u n g a l
e nzyme s e. g. am y las es , c ellulas es , pr ot e a s e sa n d
pectrnaSes
The m edium c an be s t er lliz ed by e m p l o y i n g
ba tch o r c ont inuous s t er iliz at ion t ec hni o u e s . T h e
fe rmen tat ion is s t ar t edby inoc ulat ing t he m e d i u m .
The growth conditions (pH, temperature, O2
sup ply, n ut r ient addit ion) ar e m aint aineda t o p t i m a l
levels. The f r ot h f or m at ion c an be m in i m i s e d b y
adding antifoam agents.
fhe production of enzymes is mostly carried out
by batch fermentation, and to a lesser extent by
continuous process.The bioreactor system must be
ma inta ined s t er ile t hr oughout t he f er m e n t a t i o n
o rocess. The dur at ion of f er m ent at ion i s v a r i a o r e
around 2-7 days, in most production processes
Besides the desired enzyme(s), several other
nretabolitesare also produced. The enzyme(s)have
to be recovered and purified.
Recovery and putification of enzymes
The desired enzyme produced may be excreted
into th e c ult ur e m edium ( ex t r ac ellularen z y m e s )o r
ma y b e pr es ent wit hin t he c ells ( in t r a c e l l u l a r
e nzyme s ) . Depending on t he r equir e m e n t , t h e
comme rc ial enz y m e m ay be c r ude o r h i g h l y
p urifie d. Fur t her , it m ay be in t he s olid o r l i q u i d
form. The steps involved in downstream processing
i.e. recovery and purification steps employed will
depend on the nature of the enzyme and the degree t h e e n z y m e c a n b e o b t a i n e d b y d r y i n g . T h i s c a n b e
of p urity des ir ed.
In general, recovery of an extracellular enzyme
which is pr es ent in t he br ot h is r elat iv e l y s i m p l e r
comp are d t o an int r ac ellular enz y m e . F o r t h e
re lea seo f int r ac ellularenz y m es ,s pec ial t e c h n i q u e s
are needed for cell disruption. The physical,
chemical and enzymatic methods adopted to break
the cells and release their contents are described
elsewhere(Chapter20). The reader must invariably bodies These enzymes should be totally free from
refer them now and learn all the details, as they toxic materials, harmful microorganisms
form part of enzyme technology. Microbial cells should not cause allergic reactions.
245
can be broken down by physical means (sonication,
high pressure, glass beads). The cell walls of
bacteria can be lysed by the enzyme lysozyme. For
yeasts,the enzyme p-glucanase is used. However,
enzymatic methods are expensive.
The recovery and purification (briefly described
below) stepswill be the same for both intracellurar
and extracellular enzymes, once the cells are
d i s r u p t e d a n d i n t r a c e l l u l a r e n z y m e s a r e r e l e a s e d.
The most imoortant consideration is to minimrse
the loss of desired enzyme activity.
Removal of cell debris : Filtration or
centrifugation can be used to remove cell debris.
Removal of nucleic acids: Nucleic acids
interfere with the recovery and purification of
enzymes. They can be precipitated and removed
by adding polycations such as polyamines,
s t r e p t o m y c i na n d p o l y e t h y l e n e i m i n e .
Enzyme precipitation : Enzymes can be
precipitated by using salts (ammonium sulfate)
organic solvents (isopropanol, ethanol, acetone).
Precipitationis advantageoussince the precipitated
enzyme can be dissolved in a minimal volume to
c o n c e n t r a t et h e e n z y m e .
tiquid-liquid partition : Furtherconcentrationof
desired enzymes can be achieved by liquid-liquid
e x t r a c t i o nu s i n g p o l y e t h y l e n eg l y c o l o r p o l y a m i n e s.
Separation by chromatography : There are
several chromatographic techniques for separation
and purification of enzymes. These include
ion-exchange, size exclusion, affinity, hydrophobic
interaction and dye ligand chromatography
(Refer Chapters 20 and 64). Among these, ion-
exchange chromatography is the most commonly
used for enzyme purification.
Drying and packing : The concentrated form of
done by film evaporators or freeze dryers
( l y o p h i l i z e r s ) .T h e d r i e d e n z y m e c a n b e p a c k e d
and marketed. For certain enzymes, stability can be
achieved by keeping them in ammonium sulfate
suspensronS.
All the enzymes used in foods or medical
treatments must be of high grade purity, and must
meet the required specificationsby the regulatory
and
246
REGULATI O N O F M I CRO BI AL
ENZYME PRODUCTION
_GEN ERAL CO NSI DERATI O NS
A m ax im al pr oduc t ion of m ic r o b i a l e n z y m e s
can be achieved by optimising the fermentation
conditions (nutrients, pH, O2, temperature etc.).
For this pur pos e, a c lear under s t a n d i n g o f t h e
genetic regulation of enzyme synthesisis required.
Some of the general aspects of microbial enzyme
reg ulat ion ar e br ief ly des c r ibed.
lnd uct ion
Sev er al enz y m es ar e induc ible i . e . t h e y a r e
synth es iz edonly in t he pr es enc eof i n d u c e r s . T h e
inducer may be the substrate (sucrose, starch,
gaiactosides)or product or intermediate(fatty acid,
phenylacetate, x),lobiose). A selected list of
in du c ible enz y m es and t he r es pec ti v e i n d u c e r s i s
given in Table 21 .4.
The induc er c om pounds ar e ex pen s i v ea n d t h e r r
ha nd ling ( s t er iliz at ion, addit ion at s p e c i f i c t i m e )
also is quite difficult. ln recent years, attempts are
be ing m ade t o dev elop m ut ant s of m i c r o o r g a n i s m s
in wh ic h induc er dependenc e is elim i n a t e d .
Feedback replession
Feedbac kr egulat ionby t he end pr o d u c t ( u s u a l l y
a sm all m olec ule) s ignif ic ant ly in f l u e n c e s t h e
e nzy m e s y nt hes is . This oc c ur s wh e n t h e e n d
pro duc t ac c um ulat esin lar ge- quant it i e sL. a r g es c a l e
production of feedback regulatedenzymes is rather
difficult. However, mutants that lack feedback
repressionhave been developed to overcome this
pro blem .
Nutrient repression
The native metabolism of microorganism is so
devised that there occurs no production of
un ne c es s ar y enz y m es . I n ot her w o r d s , t h e
microorgnaismsdo not synthesizeenzymes that are
no t r equir ed by t hem , s inc e t his i s a w a s t e f u l
e xe rc is e. The inhibit ion of unwan t e d e n z y m e
production is done by nutrient repression. The
nutrients may be carbon, nitrogen, phosphate or
su lfat e s upplier s in t he gr owt h m edi u m . F o r l a r g e
scale production of enzymes, nutrient repression
must be overcome.
Glucose repression is a classical example of
n utr ie nt (more app rop ri ately catabol ite) rep ression.
B IOTE C H N OL O CY
Tlrrr 21.4 $electedexanplesof lnduclb-le
enrynesalongwith the inducers
Enzyme Inducer
Invertase Sucrose
Amylase Starch
Lipase Fattyacids
B-Galactosidase Galactosides
Penicillin
G amidase Phenylacetate
Xylanase Xylobiose
That is in the presence of glucose, the enzymes
needed for the metabolism of rest of rne
compounds are not synthesized.Clucose repression
can be overcome by feeding of carbohydrateto the
fermentation medium in such a way that the
concentration o{ glucose is almost zero at any
given time. In recent years, attempts are being
made to select nrutants that are resistant to
catabolite repressionby glucose.
For certain microorganisms, other carbon
sources such as pyruvate, lactate, citrate and
succinate also act as catabolite repressors.
Nitrogen source repression is also observed in
m i c r o o r g a n i s m s .T h i s m a y b e d u e t o a m m o n i u m
i o n s o r a m i n o a c i d s . M o s t c o m m o n l y i n e x p e n sr ve
ammonium salts are used as nitrogen sources.
The repression by ammonium salts can be
overcome by developinB mutants resistantto this
nitrogen source.
GENETIC ENGINEERING FOR
MICROBIAL ENZYME PRODUCTION
Enzymes are the functional products of genes.
Therefore, theoretically, enzymes are good
candidates for improved production through
genetic engineering
D u r i n g t h e p a s t 1 5 y e a r s , t h e a d v a n c e s i n th e
recombinant DNA technology have certainly
h e l p e d f o r i n c r e a s i n gt h e m i c r o b i a l p r o d u c t i o n o f
commercial enzymes. lt is now possible to transfer
the desired enzyme genes from one organism to
the other Once an enzyme with a potential use
i n i n d u s t r y i s i d e n t i f i e d , t h e r e l e v a n t g e n e ca n
be cloned and inserted into a suitable production
host.
 247
Chapter21 : E N Z YMET E C H N OL OC Y

Selectionof microorganism
with an enzymeof
industrialaPPlication

Purificationof ueslreo enzYIIr€ below.

.RNA Purified
1. The enzyme lipolase, found in the fungus
fat
Humicola languinosa is very effective to remove
production of
stains in fabrics. However, industrial

tn its
properties make lipolase a stronB candidate for
use fabric w a s h i n g .

\-)'\ /'/
-=-.=..--=.-_--./-----

I
tdentit*ationof
cDNAclones production.
with Probes)
(bYhYbridization
I for modification of
Protein engineering
I industrial enzymes
J
Transformationof
industrialhost organism
(e.g' A' oryzae1
I
I
J
IndustrialProduction
of desiredenzyme

Fig. 21,2: Schematicrepresentationof a cloning

lt atesvfor industrialproductionof enzymes'
e.r sr vyt ,
--
._ _- - r

Cloning strategies

A diagrammatic representationof a cloninq

is
strategyflr industrialproductionof enzymes emulsifier.
eiven-in Fig. 21 .2' This involves the development
of hastremendous
C eneti cengi neeri ng i mpacton
oi .ONn l"hraryfor the mRNA, and creation with desired
On the industrialproductionof enzymes
oligonuc leot i dpero b e sfo r th e d e s i re de n z y me'
propertiesin a cost-effectivemanner'
hr'6ridizationwith oligonucleotideprobes' the
2AA B IOTE C H N OLO CY

T r a d i ti o n a l l ye,n z y me si n fre e s o l uti ons(i .e. i n

solubleor free form) reactwith subStrates to result
in products.Such use of enzymes is wasteful,
particularlyfor industrialpurposes,sinceenzymes
are not stable,and they cannot be recoveredfor (B)
reuse.
lmmobilizationof enzymes(or cells) refersto
the technique of confining/anchoring the enzymes Fig. 21.3 : lmmobilization of enzymes by adsorption
(or cells) in or on an inert support for their (A) By van der Waals forces (E) By hydrogen
stability and functional reuse.By employing this
technique,enzymesare made more efficientand
cost-effective for their industrialuse.Someworkers
regardimmobilizationas a goosewith a goldenegg METHODS OF IMMOBILIZATION
in enzymetechnology. The commonl y empl oyed techni ques b r
lm m o b i l i z e d e n z y m e s re ta i n th ei r structural immobilization of enzymes are-adsorption,
conformationnecessaryfor catalysis.There are entrapment, covalent binding and crossJinking.
several advantagesof immobilized enzymes.
Adsorption
. Stableand more efficientin function.
A dsorpti oni nvol vesthe physi cal bi ndi ng of
. Can b e re u s e da g a i na n d a g a i n .
enzymes (or cells) on the surface of an inert
o Productsare enzyme-free. support.The supportmaterialsm?y be inorganic
(e.9.al umi na,si l i ' cagel , cal ci um phosphategel,
. ldeal for multi-enzymereactionsystems.
glass)or organic(starch,carboxymethylcellulose,
. Controlof enzymefunctionis easy. DEAE-cel Iu lose,DEAE-sephadex).
. S ui ta b l efo r i n d u s tri aal n d m e d i c a luse. Adsorptionof enzyme molecules(on the inert
support)involves weak forces such as van der
o M in i m i z ee ffl u e n td i s p o s apl ro b l e m s.
Waals forces and hydrogen bonds (Fig. 21.3).
There are however,certain disadvantagesalso Therefore,the adsorbedenzymescan be easily
as s oci a tew d i th i m m o b i l i z a ti o n . removedby minor changesin pH, ionic strengthor
This is a disadvantage
temperature. for industrial
. T he p o s s i b i l i ty
o f l o s so f b i o l o g i c aal cti vi tyof an
use of enzymes.
enz y med u ri n g i m m o b i l i z a ti o o n r w hi l e i t i s i n
us e .
Entrapment
r lm m o b i l i z a ti o ni s a n e x p e n s i v eaffai r often
E nzymescan be i mmobi l i zed by physical
requiringsophisticated equipment.
entrapmentinsidea polymeror a gel matrix.The
Immobilized enzymes are generally preferred size of the matrixporesis suchthat the enzymeis
over immobilized cells due to specificity to yield retainedwhile the substrate and oroductmolecules
the products in pure form. However, there are passthrough.In this technique,commonlyreferred
s ev er a l a d v a n ta g e s o f u s i n g i mmobi l i zed to as lattice entrapment,the enzyme(or cell) is not
multienzymesygtems suchas organelles and whole subjectedto strong binding forcesand structural
c ellso v e r i mmo b i l i z e de n z y m e sT. h e i mmobi l i zeo distortions. Somedeactivation mav however,occur
cellspossess the naturalenvironmentwith cofactor duri ng i mmobi l i zati on processdue to changesin
availability(and also its regenerationcapability) pH or temperatureor addition of solvents.The
and ar e p a rti c u l a rlsyu i ta b l efo r mu l ti pl eenzymati c matricesused for entrappingof enzymesinclude
reactions. polyacrylamide gel, collagen, gelatin, starch,
 Chapt er21 : E N Z Y M ET EC H N O L OC Y
(B)
u m i c r o e n c a p s ul a t i o n .
tr
u n
n n
Fig. 21.4 : Immobilization of enzymes by entrapment
(A) Inclusion in gels (B) lnclusion in fibres
(C) lnclusion in miuocapsules (Note : Coloured
ce llulo se , silic one and r ubber . Enz y m es ca n b e
entrapped by several ways.
1 . Enzyme inclusion in gels : This is an
entrapment of enzymes inside the gels (Fig.21.4A).
2 Enzyme inclusion in fibres : The enzymes
are trapped in a fibre format of the matrix
Fig .21 .a$ .
3 . Enzym e inc lus ion in m ic r oc aps ules : I n
:- is case, the enz y m es ar e t r apped ins i d e a
-icrocapsule matrix (Fig. 2l .4A. The hydrophobic
:rd hvdro ph ilic f or m s of t he m at r ix poly m er i s e t o
':rrn a micro c aps ulec ont aining enz y m e m ole c u l e s
-. rd e
The major limitation for entrapment of enzymes
lreir leakage from the matrix. Most workers
-
irt=:s"ology [19]
289
prefer to use the technique of entrapment for
i m m o b i l i z a t i o n o f w h o l e c e l l s . E n t r a p p e dc e l l s a r e
in use for industrial production of amino acids
(L-isoleucine,L-aspartic acid), L-malic acid ano
hydroquinone.
Microencapsulation
Microencapsulation is a type of entrapment. It
refersto the processof spherical particle formation
wherein a liquid or suspensionis enclosed in a
s e m i p e r m e a b l em e m b r a n e . T h e m e m b r a n e m a y b e
p o l y m e r i c , l i p o i d a l , l i p o p r o t e i n - b a s e do r n o n - i o n i c
in nature. There are three distinct ways of
1. Building of special membrane reactors.
2. Formationof emulsions.
3. Stabilization of e m ul s i o n s to form
m i c r o c a p s ul e s .
Microencapsulationis recently being used for
immobilization of enzymes and mammalian cells.
F o r i n s t a n c e ,p a n c r e a t i cc e l l s g r o w n i n c u l t u r e sc a n
b e i m m o b i l i z e d b y m i c r o e n c a p s u l a t i o nH . ybridoma
c e l l s h a v e a l s o b e e n i m m o b i l i z e d s u c c e s s f u l l yb y
this technioue.
Govalent binding
lmmobilization of the enzymes can be achieved
by creation of covalent bonds 'between the
chemical groups of enzymes and the chemical
groups of the support (Fig. 21 .5). This technique rs
widely used. However, covalent binding is often
associatedwith loss of some enzyme activity. The
inert support usually requires pretreatment(to form
pre-activated support) before it binds to enzyme.
The following are the common methods of covalent
binding.
Covalent
bonds
Fig. 21.5 : A general representation of immobilization
of enzymes by covalent binding (Note : coloured
290 B IOTE C H N O LO CY

1. Cyanogenbromide activation : The inert involves the risk of denaturation of the enzyme by
supportmaterials(cellulose,sepharose, sephadex) the polyfunctional reagent.
containingglyccllgroups are activatedby CNBr,
whic h th e n b i n d to e n z v m e sa n d i m mobi l i zethem CHOICE OF IMMOBILIZATION
( F ig .2 t.6 A ). TECHNIOUE

2. Diazotation: Someof the supportmaterials T h e s e l e c t i o n o l a p a r t i c u l a r m e t h o d fo r

( am i n o b e n z y lc e l l u l o s e , a mi n o d eri vati vesof immobilization of enzymes is based on a trial and
polystyrene,aminosilanizedporous glass) are error approach to choose the ideal one. Among the
subjectedto diazotationon treatmentwith NaNO, factors that decide a technique, the enzyme
and HCl. They,in turn, bind covalentlyto tyrosylor catalytic activity, stability, regenerability and cost
histidylgroupsof enzymes(Fig.2l .68). factor are important.
3. Peptidebond formation : Enzymeimmobi-
lmmobilization of L-amino acid acylase
lizationcan also be achievedby the formationof
peptide bonds between the L-Amino acid acylasewas the first enzyme to be
amino
immobilized by a group of Japanese r,r'orkers
(or carboxyl)groupsof the supportand the carboxyl
(or amino) groups of enzymes(Fig. 21.6Q. The
(Chibata and Tosa, 1969). More than 40 different
immobilization methods were attempted by this
supportmaterialis first chemicallytreatedto form
ac t i v efu n c ti o n agl ro u p s . g r o u p . O n l y t h r e e o f t h e m w e r e f o u n d b e u se fu l '
b i n d i n g t o i o d o a c e t y lc e l l u lo se ,
4. Activation by bi- or polyfunctional T h e y w e r e c o v a l e n t
t o D E A E - S e p h a d e xa n d e n t r a p m e n t
reagents : Some of the reagents such as i o n i c b i n d i n g
withi n polyacrylanride.
glutaraldehyde can be used to create bonds
between amino groups of enzymes and amino
STABILIZATION OF
gr ou p s o f s u p p o rt (e .g . a mi n o ethyl cel l ul ose,
S O L U B L E E N Z Y M ES
albu mi n , a mi n o a l k y l a te dp o ro u s gl ass).Thi s i s
depictedin Fig. 21.6D. S o m e o { t h e e n z y m e s c a n n o t b e i m m o b i li ze d
and they have to be used in soluble form e.g.
Gross-linking enzymes used in liquid detergents,some diagnostic
enzymes can be
The absenceof a solidsupportis a characteristic reagentsand food additives. Such
s t a b i l i z e d b y u s i n g c e r t a i n a d d i t i v e so r b y c h e mi ca l
feature of immobilizationof enzymesby cross-
modifications. The stabilized enzymes have longer
s re i mmobi l i zedby
. h ee n z y mem o l e c u l e a
link i n g T
them, through the half-lives, although they cannot be recycled. Some
creating cross-linksbetween
stabilization are
involvement of polyfunctional reagents.These important methods of enzyme
reagentsin fact reactwith the enzymemolecules briefly described.
and create bridgeswhich form the backboneto
hold enzyme molecules (Fig. 21.n. There Solvent stabilization
are several reagents in use for cross-linking. Certain solvents at low concentrations,stabilize
T he s e i n c l u d e g l u ta ra l d e h y d edi
, azobenzi di ne, t h e e n z y m e s , w h i l e a t h i g h c o n c e n t r a t i o n s th e
hexa me th y l e n ed i i s o c y a n a tea n d tol uene di - enzymes get denatured e.g. acetone (5%) and
isothiocyanate. ethanol (5%) can stabilize benzyl alcohol dehydro-
Glutaraldehyde is the most extensively used Eenase.
crossJinkingreagent.lt reactswith lysyl residuesof
the enzymesand forms a Schiff'sbase.The cross Substrate stabilization
links formed between the enzyme and The active site of an enzyme can be stabiiized
glutaraldehyde are irreversibleand can withstand by adding substrates e.g. starch stabilizes
extremepH and temperature. Clutaraldehyde cross- a-amylase; glucose stabilizesglucose isomerase.
link i n g h a s b e e n s u c c e s s fu l luys e dto i mmobi l i ze
severalindustrialenzymese.g. glucoseisomerase, Stabilization by polymers
pen i c i l ti na m i d a s e . Enzymes can be stabilized, particularly against
T h e te c h n i q u eo f c ro s s -l i n k i nigs qui te si mpl e increased temperature,,by addition of polymers
and cost-effective. But the disadvantaee is that it s u c h a s g e l a t i n , a l b u m i n a n d p o l . v e t h y l e n eg l yco l .
Chaot er21 : E N Z Y M ET EC H N O L OC Y

Support Reactive lmmobilizedenzyme

HCI

* fnzir pq.
=N:N ------l

\-r

Oligogluta-
raldehyde

Fig. 21.6 : lmmobilization of enzymes by covalent binding (A) Cyanogen bromide activation, (B) Diazotation,
(C) Peptide bond formation, (D) Activation by bifunctional agent.
 292 B IOTE CHNO LO CY

o Acylation of enzymesby adding groupssuch as

acetyl,propionyland succinyl.

Stabilization by rebuilding
the stabilityof the enzymesis due
Theoretically,
F to hydrophobicinteractionsin the core of the
l1 enzyme.lt is therefore,proposedthat enzymescan
frr be stabi l i zed by enhanci ng hydr ophobic
interactions.For this purpose,the enzyme is first
Fig, 21,7 : Immobilization of enzyme molecules by unfold and then rebuilt in one of the following
cross linking. ways (Fig. 21.8).
o The enzymecan be chemicallytreated(e.9.urea
and a disulfide)and then refolded.
Stabilization by salts
. The refoldingcan be done in the presence
of low
Stabilityof metalloenzymes can be achievedby mol ecul arw ei ghtl i gands.
adding saltssuch as Ca, Fe, Mn, Cu and Zn e.g.
r For certain enzymes, refolding at higher
proteasescan be stabilizedby addingcalcium.
temperatures (around50"C)stabilizesthem.

Stabilization by chemical modifications

Stabilization by site.directed
Enzymescan be stabilizedby suitablechemical mutagenesis
modificationswithout loss of biological activity. mutagenesis
Site-directed has been successfully
Thereare severaltypesof chemicalmodifications. usedto producemore stableand functionallymore
. Addition of polyamino side chains e.B. efficientenzymese.g.subtilisinE. For moredetails,
I
polytyrosine,polyglycine. ReferChapter10.
I

*gP
Refoldingin
the presence
Unfold of a ligand

Native enzyme

Fig. 21.8 : Stabilization of an enzyme by refolding.

Chaot er21 : EN Z YMET E C H N OL OC Y 293

' ' IAEIE 11,5'tercCtG{ eXAmp|gS Or rmmGDrFZ€d Ce{ ,

(to bring out one or two enryme reactionsl In industrial applications

I mmobilized microorgantsm Application(s)

(microbial biocatalvst)

Escherichia
coli Forthesynthesis acidfromfumaric
of L-aspartic acidandNH,
Escherichia
coli Forthe production
of L-tryptophan
fromindoleandserine
Pseudomonas
sp Production fromglycineandmethanol
of L-serine
Sacch ae
aronycescerevisi Hydrolysis
of sucrose
sp
Saccharomyces Largescaieproduction
of alcohol
Zymononas
mobilis Synthesis andgluconic
of sorbitol acidlromglucose
andfructose
Anthrobacter
sinplex of prednisolone
Synthesis fromhydrocortisone
Pseudononas
chlororaphis Production
of acrylamide
fromacrylonitrile
Hunicolasp Fortheconversion
of rifamycin
B to rifamycin
S
andyeasts(several
Bacteria sp) In biosensors

IMMOBILIZATI O N O F CELLS lmmobilized non-viable cells

lmmo bi liz ed indiv idual enz y m es c a n b e I n m a n y i n s t a n c e s ,i m m o b i l i z e d n o n - v i a b l ec e l l s

successiully used for single-step reactions They
are, however, not suitable for multienzyme
reactions and for the reactions requiring cofactors.
Th e wh ole c ells or c ellular or ganelles c a n b e
immobilized to serve as multienzyme systems. In
ad ditio n, i m m obiliz ed c ells r at her t han e n z y m e s
are sometimes preferred even for single reactions,
due to cost factor in isolating enzymes. For
th e e nzym es wh ic h depend on t he s p a c i a l
arra ng eme ntof t he m em br ane, c ell im nr ob i l i z a t i o n
is preferred.
Immobilized cblls have been traditionally used
for the treatment of sewage.
The tec hniquesem ploy ed f or im m obiliza t i o n o f
cells a re alm os t t he s am e as t hat u s e d f o r
immob iliz at ion of enz y m es ( dis c us s edalr ea d y )w i t h
appropriate modifications. Entrapment and surface
attachment techniques are commonly used.
Gels, and to some extent membranes, are also
e mplo ye d.
lmmobilized viable cells
are preferred over the enzymes or even the viable
c e l l s . T h i s i s m a i n l y b e c a u s eo f t h e c o s t l y i s o l a t i o n
and purification processes.The best example is the
immobilization of cells containing glucose
isomerase for the industrial production of high
fructose syrup. Other important examples of
m i c r o b i a l b i o c a t a l y s t sa n d t h e i r a p p l i c a t i o n s a r e
given in Table 21 .5.
Limitations of immobilizing
eukaryotic cells
Prokaryotic cells (particularly bacterial) are
mainly used for immobilization. lt is also possible
to immobilize eukaryotic plant and animal cells.
Due to the presence o{ cellular organelles,the
metabolism of eukaryotic cells is slow. Thus, for
the industrial production of biochemicals,
prokaryotic cells are preferred. However, for the
production of complex proteins (e.g. immuno-
globulins) and for the proteins that undergo post-
translational modifications, eukaryotic cells may
be used.

The viability of the cells can be preserved by

mild immobiliz at ion. Suc h im m obiliz ed ce l l s a r e
particularly useful for fermentations. Sometimes
mamma lian c ell c ult ur es ar e m ade t o f un c t i o n a s
immo bilized v iable c ells .
EFFECT OF IMMOBILIZATION
ON ENZYME PROPERTIES
Enzyme immobilization is frequently associated
with alterations in enzyme properties, particularly
294 BIOTECI-JNOLOCY

the kinetic propertiesof enzymes.Some of them

are listedbelow.
1. Thereisa substantial
decreasein the enzyme
oo specificity.This may be due to conformational
!c oo lmmobilized changes that occur when the enzyme gets
CO oo o oo enzymes/cells i mmobi l i zed.
2. The kinetic constantsKn,,and Vn'"" of an
immobilizedenzymedifferfrom that of the native
enzyme.This is becalrse
the conformationalchange
of the enzyme will affect the affinity between
enzymeand substrate.

IMMOB ILIZE D E N ZY ME R E A GTOFS

The i mmobi l i zedenzymescel l s are uti lizedin

the industrialprocessesin the form of enzyme
reactors They are broadly of two types-batch
reactorsand continuousreactors.The frequently
usedenzymereactorsare shown in Fig. 21.9.
(B)
Batch reactors

------+ Product In batchreactors,

the immobilizedenzymesand
substrates are placed,and the reactionis alloweo
to takeplaceunderconstantstirring.As the reaction
is completed,the product is separatedfrom the
enzyme(usual l yby denaturati on).
Fluidized Solubleenzymesare commonly used in batch
bed of immobilized
enzymes/cells reactors.lt is ratherdifficultto seoarate
the soluble
enzymes from the products, hence there is a
limitation of their reuse. However, special
techniqueshave been developedfor recoveryof
sol ubl eenzymes, al thoughthi smay resul it n lossof
enzymeactivity.
(c) Stirred tank reactors : The simplest form
Substrate of batch reactor is the stirred tank reactor
(Fig.2l .9A). lt is composedof a reactorfitted with
a stirrerthat allows good mixing, and appropriate
temperatureand pH control.However,there may
oo o occur lossof someenzymeactivity.A modification
o1o o lo lmmobilized
enzymes/cells of stirred tank reactor is basket reactor. In this
o <--)F--> cc system,the enzyme is retainedover the impeller
o o loo blades.Bothstirredtank reactorand basketreactor
6 eLJ(-) o o have a well mixed flow pattern.

Plug flow type reactors : These reactorsare

Fig. 21.9 : lmmobilized enzyme (cell) reactors
(A) Batch stirred tank reactor, (B) Packed bed reactor,
(C) Fluidized bed reactor, (D) Continuous
stirred tank rcactor.
alternativesto flow pattern type of reactors.The
flow rateof fluidscontrolledby a plug system.The
plug flow type reactorsmay be in the form of
packed bed or fluidized bed (Fig. 21.98 and
21.9O. Thesereactorsare particularlyusefulwhen
Chaot er2 1 : EN Z YMET EC H N O L OC Y
(A)
Magnetic
stirrer
Fig. 21.10 : Membrane reactors (A) Batch membrane
reactor, (B) Continuous membrane reactor,
(C) Recycle membrane reactor (Coloured lines
indicate membranes).
there occurs inadequate product formation in flow
type reactors. Further, plug flow reactors are also
useful for obtaining kinetic data on the reaction
systems.
Continuous reactors
In coniinuous enzyme reactors,the substrateis
a dd ed cont inuous ly while t he pr oduc t is r e m o v e d
simulta neous ly .I m m obiliz ed enz y m es c a n a l s o b e
used for continuous operation. Continuous reactors
have certain advantagesover batch reactors.These
includ e c ont r ol ov er t he pr oduc t f o r m a t i o n ,
convenient operation of the system and easy
automation of the entire process.There are mainly
two tvoes of continuous reaclors-continuous stirred
295
tank reactor (CSIR) and plug reactor (PR). A
diagrammatic representationof CSTR is depicted
in Fig. 21 .9D. CSTR is ideal for good product
formation.
Membrane reactors
Severalmembraneswith a variety of chenrical
compositions can be used. The commonly used
membrane materials include poivsulfong,
polyamide and cellulose acetate The biocatalysts
(enzvmes or cclls) are normally retained on the
membranes of the reactor. The substrate is
introduced into reactor while the product passes
o u t . C o o d r n i x i n g i n t h e r e a c t o rc a n b e a c h i e v e d b y
using stirrer (Fig. 21.10A\. In a continuous
membrane reactor, the biocatalysts are held over
membrane lavers on to w,hich sr:bstratemolecules
are passed (Fig. 21.108\.
ln a recycle model membrane reactor, the
conlents (i.e. the solution containini4 enzymes,
cofactors,and substratesalong with freshly released
product are re,:yclcd bv using a pump
( F i g . 2 1. l 0 a f h e p r o r l u c t p a s s e so u t w h i c h c a n b e
recovereo.
APPLICATIONS OF IMMOBILIZED
ENZYMES AND CELLS
lmmobilized enzymesand cells are very widely
used for industrial, analytical and therapeutic
purpose, besides their involvement in food
production and exploring the knowledge of
biochemistry, microbiology and other allied
specialities. A brief account of the industrial
applications of immobilized cells is given in
Table 21.5.
MANUFACTURE OF COMMENCTAL
PRODUCTS
A selected list of important immobilized
enzymes and their industrialapplications is given
in Table 21 .6. Some details on the manufacture of
L-amino acids and high fructose syrup are given
hereunder.
Production of L.amino aeids
L - A m i n o a c i d s ( a n d n o t D - a m i n o a c i d s )a r e v e r y
important for use in food and feed supplementsand
medical purposes. The chernical methods
employed for their production result in a racemic
mixture of D- and L-amino acids. Thev can be
296 B IOTE C H N OLO CY

lmmobilized enzyme Application(s)

Aminoacylase Production
of L-aminoacidsfromD, L-acylaminoacids
isomerase
Glucose syrupfromglucose
of highfructose
Production (orstarch)
Amylase of glucose
Production fromstarch
Invertase of sucrose
Splitting to glucose
andfructose
B-Galactosidase to glucose
of lactose
Splitting andgalactose
Penicillin
acylase production
Commercial penicillins
of semi-synthetic
Aspartase Production acidfromfumaric
of aspartic acid
Fumarase of malicacidfromfumaric
Synthesis acid
Histidine lyase
ammonia Production acidfromhistidine
of urocanic
Ribonuclease Synthesis fromRNA
of nucleotides
Nitrilase Production fromacrylonitrile
of acrylamide

acvlated to form D, L-acyl amino acids. The High fructose syrup can be produced from
immobilized enzyme aminoacylase (frequently glucose by employing an immohilized enzyme
imm obiliz ed on DEAE s ephadex ) c a n s e l e c t i v e l y glucose isomerase. The starch containing raw
hydrolyse D, L-acyl amino acids to produce materials(wheat, potato, corn) are subjectedto
L-a m ino ac ids . hydrolysisto produceglucose.Glucoseisomerase
then isomerises glucoseto fructose(Fig.21.11\.fhe
Aminoacylase
D, L-Acyl > L-Amino acids + product formed i s H FS contai ni ng about
a m ino ac ids D, L- Ac v l a m i n o a c i d s 50ozb fructose.(Note : Someauthorsuse the term
high fructose corn syrup i.e. HFCSin place of HFS)
The free L-amino acids can separatedfrom rne
unhydrolysed D-acyl amino acids. The latter can Gl ucose i somerase: Thi s i s an i ntracellur ar
be recemized to D, L-acyl amino acids and enzymeproducedby a numberof microorganisms.
recycled through the enzyme reactor containing The species of Arthrobacter, Bacillus and
imm obiliz ed am inoac y las e. Huge q u a n t i t i e s o f Streptomycesare the preferredsources.Being an
L -met hionine, L- pheny lalanine L- t r y p t o p h a n a n d i ntracel l ul arenzyme, the i sol ati on of gl ucose
L -va linear e pr oduc ed wor ldwide by t h i s a p p r o a c h . isomerasewithout loss of biological activity
requi resspeci aland costl y techni ques.Man y a
Production of high fructose syrup ti mes, w hol e cel l s or partl y broken cel l s ar e
i mmobi i i zedand used.
Fructose is the sweetest among the
monosaccharides,and has twice the sweetening
strengthof sucrose.Clucose is about 757o as sweet ENZYMES AND CELLS.
as sucrose. Therefore,glucose (the most abundant 'MMOB'LTZED APPL'CAT'ONS
ANALYT'CAL
monosaccharide)cannot be a good substitute for In biochemical analysis
sucrose for sweetening. Thus, there is a great
demand for fructosewhich is very sweet, but has the
l mmobi l i zedenzymes(or cel l s)can be usedf or
same calorific value as that of glucose or sucrose
the developmentof preciseand specificanalytical
techniques for the estimation
of severalbiochemical
High fructose syrup (HFS) contains compounds.The pri nci pl e of anal yti cala ssay
approximately equivalent amounts of glucose and pri mari l yi nvol vesthe acti on of the i mmob ilized
fructose. HFS is almost sirnilar to sucrose from enzymeon the substrate. A decrease in the substrate
nutritional point of view. HFS is a good substitute concentration or an increasein the productlevelor
for sugar in the preparationof soft drinks, processed an alterationin the cofactorconcentration can be
foods and bakine. used for the assay.A selectedlist of examplesof
Chapt er21 : EN Z YMET E C H N OL OC Y 297

Starch
(wheat,potatoes,corn)

| ,r-Rmvlase
I and glucoamylase
+
Glucose
Thermistorlmmobilized
u**"*
I isomerase enzyme

J
High fructosesyrup (B)
(fructose/glucose" 50/50)
Electrode
Fig. 21.11 : Production of high fructose syrup from
statch (glucose isomeraso is the immobifized
enzyffie).

{-lmmobilized
immobilizedenzymesused in the assayof some enzyme
substancesis given in Table 2I .7. fwo types of
detectorsystemsare commonlyemployed.
Glass
Thermistorsare heat measuringdeviceswhich (c) electrode
can record the heat generatedin an enzyme
catalysedreaction.Electrodedevicesare used for
measuringpotential differencesin the reaction
system.ln the Fig. 21.12, an enzyme thermistor
and an enzyme electrode, along with a specific
ureaseelectrodeare depicted.

In affinity chromatography and

purification Fig. 21.12 : lmmobilized enzymes ot cells in
lm m obi l i z e de n z y me sc a n b e u s e d i n affi ni ty analytical biochemistry (A) Enzynp thermistor,
chromatography.Basedon the propertyof affinity, (B) Enzyme electrode, (C) lJrease electrode.

it is possible to purify several compounds e-g.

antigens, antibodies, cofactors.

lmmobilized enzyme Substance assayed

Glucoseoxidase Glucose
Urease Urea
A biosensoris an analyticaldevice containing
Cholesterol
oxidase Cholesterol
an immobilized biological material (enzyme,
Lactate
dehydrogenase Lactate anti body,nucl ei c aci d, hormone, organel l eor
Alcohol
oxidase Alcohol whole cell) which can specificallyinteract with
Hexokinase ATP an analyte and produce physical, chemical
Galactoseoxidase Galactose or electrical signals that can be measured.
Penicillinase Penicillin An analyte is a compound (e.9. glucose,urea,
Ascorbic
acidoxidase Ascorbicacid drug, pesticide) whose concentration has to
be measurecl.Biosensorsbasically involve the
L-Aminoacidoxidase L-Aminoacids
quantitative analysis of various substancesby
Cephalosporinase Cephalosporin
convertingtheir biologicalactionsinto measurable
Monoamine oxidase Monoamine s.
si gnal
29', B IOTE C HNO LO CY

-----)l Processor
I
l)
z)

-4
>.i
(
Analyte Opticallyo!'electronicallY
aclrvesunace
Bound
analyte

Biological
component
(e.9.enzyme)

Fig. 21.13 : A diagrammatic representation of a biosensor.

A great majority of biosensorshave immobilized material to form a bouno anal ytew hi ch in t ur n

enzymes. The performance of the biosensors is produces the electronic responsethat can be
mostly dependent on the specificity and sensitivity measured.
of the biological reaction, besides the stabllity of
the enzyme. Biologicalmaterial + AnalYte

General features of biosensoYs

A biosensor has two distinct components

BoundanalYte
( Fig. 21. 13) .
I
1. Biologic al c om ponent - en z y m e , c e l l e t c . J
resPonse
Biological
2. Physical component-transducer, amplifier
etc. +
I
The biologic al c om ponent r e c o g n i s e s a n d Electronic resPonse
interacts with the analyte to produce a I
physical change ('a signal) that can be detected,
+
Measurement
by the transducer. In practice, the biological
material is appropriately immobilized on to
the transducer and the so prepared biosensors ln some instances, the analyte is converted to a
can be repeatedly used several times (may be product which may be associatedwith the release
ar ound' ' 10, 000 t im es ) f or a l o n g p e r i o d ( m a n y of heat, gas (oxygen), electrons or hydrogen ions.
m ont hs ) . The transducet can'convert the product linked
c h a n g e s i n t o e l e c t r i c a l s i g n a l s w h i c h ca n b e
Principle of a biosensor amplified and measured.

The des ir ed biologic al m a t e r i a l ( u s u a l l y a

TYPES OF BIOSENSORS
s pec if ic enz y m e) is im m obiliz e d b y c o n v e n t i o n a l
methods (physical or membrane entrapment, non- There are several types of biosensorsbased on
c ov alent or c ov alent binding) . T h i s i m m o b i l i z e d the sensor devices and the type of biological
biological material is in intimate contact with the materials used. A selected few of them are
transducer. The analyte binds to the biological discussedbelow.
Chaot er21 : E N Z Y M ET EC H N O L OGY
Tt
-tl _
T-
TT
A A
Substrate
Enzyme
(immclbilized
on
membrane)
Fig. 21.14 : A diagrammatic representation of an ampercmetric biosensor
E L E C T R O C H E M I CA I- B'OSEA'.SOFS
Electrochem ic albios ens or sar e s im ple d e v i c e s
based on the measurements of electric current,
io nic or conduc t anc e c hanges c ar r ied c u t b y
biaelectrodes.
Amperometric biosensors
These biosensors are based on the movement of
electrons (i.e. determination of electric current) as
a result of enzynre-catalysed redox reactions.
Normally, a constant voltage passes between the
ele ctro de s w hic h c an be det er m ined. I n a n
enzymatic reaction that occurs, the substrate or
oroduct can transfer an electron with the electrooe
surfaceto be oridised or reduced (Fig. 2I .14).lhis
results ih an altered current flow that can De
measured. The magnitude of the current is
proportional to the substrateconcentration. Clark
oxygen electrode which determines reduction of
Or, is the simplestform of amperometric biosensor.
Determination of glucose by glucose oxidase is a
good exanrple.
In the first generation amperometric biosensors
,described above), there is a direct transfer of the
electronsreleasedto the electrode which mav oose
some practical difficulties. A second generation
amperometric biosensors have been deveioped
rrherein a mediator (e.g. ferrocenes)takes up the
electrons and then transfers them to electrorie.
T:rese biosensors however. are vet to become
co pu rar.
299
A
A
A
A A Current
tI
I
Product
ll
Electrode
Blood-glucosebiosensor : lt is a good example
of amperometric biosensors, rvidely used
t h r o u g h o u t t h e w o r l d b y d i a b e t i c p a t i e r r t s .B l o o d -
glucose biosensor looks like a watch pen and has
a s i n g l e u s e d i s p o s a b l e e l e c t r o d e ( c o n s i s t i n go f a
Ag/AgCl referenceelectrode and a carbon working
e l e c t r o d e )w i t h g l u c o s e o x i d a s e a n d a d e r i v a t i v eo f
ferrocene (as a mediator). The electrodes are
covered with hydrophilic mesh guaze for even
spreading of a blood drop. The disposable test
s t r i p s . s e a l e d i n a l u m i n i u m f o i l h a v e a s h e l f - l i f eo f
arourrd six months.
An amperometric biosensor' for assessing the
freshness of fish has been developed. The
accumulation of ionosine and hypoxanthine in
r e l a t i o n t o t h e o t h e r n u c l e o t i d e si n d i c a t e sf r e s h n e s s
of fish-how long dead and stored. A biosensor
utilizing immobilized nucleoside phosphorylase
and xanthine oxidase over an electrode has been
developed for this purpose.
Potentiometric biosensors
In these biosensors, changes in iontc
concelrtrations are determined by use of ion-
selective electrodes (Fig, 21 .15\. pH electrode is
the most commonly used ion-selective electrode,
since many enzymatic reactionsinvolve the release
or absorptionof hydrogen ions. The other important
electrodes are ammonia-selective and COz
selective electrodes. The ootential difference
obtained between the potentiometricelectrode and
ia 300 B IOTE C H N OLO CY
t
I

I
I

T TT A
A
trI A ,,, \ H+
------------f
Potential

AA
T
Substrate
I
Product Potentiometric
electrode
Enzyme

the reference electrode can be measured. lt is
proportionai to the concentration of the substrate.
The major limitation of potentiometric biosensorsis
the sensitivitv of enzvmes to ionic concentrations
such as H+ and NHl.
Ion-selective field effect transistors (ISFET) are
the low cost devices that can be used for
miniaturization of ootentiometric biosensors. A
good example is an ISFETbiosensorused to monitor
intramyocardial pH during open-heart surgery.
thermometric biosensors.They are more commonly
referred to as thermal biosensors or calorimetric
biosensors. A diagrammatic representation of a
thermal biosensor is depicted in Fig. 21 ,16. lt
consists of a heat insulated box fitted with heat
e x c h a n g e r( a l u m i n i u m c y l i n d e r ) .T h e r e a c t i o n ta ke s
place in a small enzyme packed bed reactor.As the
substrate enters the bed, it gets converted to a
product and heat is generated The difference in the
temperature between the substrateand product is
measured by thermistors. Even a small change in

Gonductimetric biosensots

There are several reactions in the biological Substrate

systems that bring about changes in the ionic
species. These ionic species alter the electrical
conductivity which can be measured. A good lnsulated
example of conductimetric biosensor is the urea box
biosensor utilizing immobilized urease. Urease Heat
catalysesthe following reaction. exchanger
(aluminium
HzN.. cylinder)
urease
+ 3H2o | 2NHj+ HCoJ+ oH- Matched
,/)c=o
Ha N' thermistors

The above reaction is associated with drastrc

alteration in ionic concentrationwhich can be useo
for monitoring urea concentration. In fact, urea
biosensorsare very successfullyused during dialysis
and renal surgery.
lmmobilized
enzymebed
T H E N M OM ET B' C B TO SE'VSORS
Fig. 21,16 : A diagrammatic representation of
Several biological reactions are associatedwith
the production of heat and this forms the basis of
Ch a pt er21 : E NZ Y MET EC H N O L OC Y 301

the temperature can be detected by thermal

Excitation
b iosen so rs. light
T he rmome tric bio s ens or s ar e in us e f or t he
estimation of serum cholesterol When cholesterol
get s o xid ize d b y the enz y m e c holes t er ol ox idas e,
heat is generatedwhich can be measured.Liker,vise,
estimations of glucose (enzyme-glucose oxidase),
urea (enzyme-urease),uric acid (enzyme-uricase)
and penicillin G (enzyme-BIactamase)can be done
by the se bio se nsor s . I n gener al, t heir ut ilit y is - Polymethyl-
however, limited . methaacrylate
Thermometric biosensorscan be used as a part Fluorescentdye
of e nzyme -linked im m unoas s ay ( ELI SA) and t he in silicone
new technique is referred to as thermometric ELISA Lactatemono-
(TELtSA) oxygenase
memorane

OPTICAL B'OSE'VSOBS C O 2 +A c e t a t e +H 2 O

Ootical b iosen so r sar e t he dev ic es t hat ut iliz e

the principle ol optical measurements(absorbance,
fluorescence,chemiluminescenceetc.) They employ
the use of fibre optics and optoelectronic
transducers The word optrode, representing a
condensationof the words optical and electrode, is
common lv u se d. O piic al bios ens or s pr im ar ily
involve en zymes a nd ant ibodiesas t he t r ans duc ing
elemen ts.
Optical biosensors allow a safe non-electrical m e a s u r e o .
remote sensing of materials. Another advantage is
that th ese b iosen s or s us ually do not r equlr e
referencesensors,as the comparative signal can be
generate d using th e s am e s our c e of light as t he
sam plin g se nsor. Som e of t he im por t ant opt ic al monitoring of diabetes. A simple technique
biosensorsare briefly described hereunder.
Fibre optic lactate biosensor
Fig. 21 .17 represents the fibre optic lactate
biosensor.lts working is based on the measurement
of ch an ge s in mo lec ular O , c onc ent r at ion
determining the quenching effect of O, on a
fluorescent dye. The following reaction is catalysed
bv the enzvme lactate monooxygenase
Lactatemonooxygenase
CO2 +Acetat e+ H2O
The amount of fluorescence generated by the
dved film is d ep en dent on t he 02. This is bec aus e
O . ha s a qu en ch ing ( r educ ing) ef f ec t on t he
Fig. 21.17 : Diagrammatic representation of a fibre
optic lactate biosensar.
fluorescence.As the concentration of lactate in the
reaction mixture increases, O, is utilized, and
consequently there is a proportionate decrease ln
the quenching effect. The result is that there is an
increase in the fluorescentoutput which can be
for blood glucose
Optical biosensors
Estimationof blood glucose is very important for
involving paper strips impregnatedwith reagentsis
used for this purpose. The strips contain glucose
oxidase, horse radish peroxidaseand a chromogen
( e . g . t o l u i d i n e ) . T h e f o l l o w i n g r e a c t i o n so c c u r '
C Iucose
Clucose -------=- Cluconic acid + HrOt
oYtoas e
by
Peroxidase,
chromogen + 2Hro, colour dye
+ 2HrO
.
The intensitv of the colour of the dye can be
measured by using a portable reflectance meter.
Clucose strip production is a very big industry
worldwide.
Colorimetric test strips of cellulose coated
with appropriate enzymes and reagentsare in use
for the estimation of several blood and urine
oarameters.
302 B IOTE C H N OL O CY

Trru 21.8 A selected list of organisms along with the analytes and the types of biosensors

0rganism Analyte Type of biosensor

Escherlchia
coli Glutamate (C02)
Potentiometric
flava
Sarcina Glulamine (NHr)
Potentiometric
Proteus
morganii Cysteine (H2s)
Potentiometric
Nitrosonanas
sp Ammonia (02)
Amperometric
Lactabacillus
fermenti Thiamine (mediated)
Amperometric
Lactobacillus
arabinosus Nicotinic
acid (H+)
Potentiometric
Desulfovibrio
desulfuricans Sulfate (S03)
Potentiometric
Cyanobacteria Herbicides (mediated)
Amperometric
N4anyorganisms Biological
oxygen (02)
Amperornetric
(BOD)
demand

Luminescent hEosensors to detect gaseoLrssubstratesor inhibitors can also be

urinary infections attachedto thesecrystals.
Th e m ic r oor ganis m s in t he ur i n e , c a u s i n g A piezoelectric
biosensorfor organophosphorus
urinary tract infecticns, can be detected by i nsecti ci dehas been devel oped i ncorporat ing
e mpl oy ing lum ines c ent bios ens o r s . F o r t h i s acetylcholineesterase.Likewise,a biosensorfor
purpose, the immobilized (or even free) enzyme tormaldehyde hasbeendevelopedby incorporating
namely luciferase is used. The microorganisms,on formaldehydedehydrogenase.A biosensor for
lysis release ATP which can be detected by the cocainein gasphasehasbeencreatedby attaching
fo llow ing r eac t ion.The quant it y of lig h t o u t p u t c a n cocaineantibodiesto the surfaceof piezoelectric
be measured by electronic devices. crystal.
L eu c if er in+ ATP+ 02
Lirnitations of piezoelectric biosensors
II LUCrrerase
It is very difficult to use'thesebiosensorsto
J
determinesubstances in solution.This is because
Oxyluciferin+ CO2 + AMP + Pyrophosphaie+ Light
the crystalsmay ceaseto oscillatecompletelyin
vi scousl i qui ds.
Other optieal biosensors

Optical fibre sensing devices are in use for
mea s ur ingpH, pCO , and pO , in c r iti c a l c a r e , a n d
su rgi c al m onit or ing.
P'EZOELECTR'C BIASEfi'SOBS
Piezoelectric biosensors are based on the
principle of acoustics (sound vibrations), hence
they are also called as acoustic biosensors.
Piezoelectric crystals form the basis of these
biosensors.The crystalsr,vithpositive and negative
charges vibrate with characteristic frequencies.
Adsorption of certain molecules on the crystal
surface alters the resonancefreouencieswhich can
be measured by electronic devices. Enzymeswith
WT'OLE CELT- B'OSEN'SOFS
Whole cell biosensors are particularlyusefulfor
multi-stepor cofactor requiringreactions.These
biosensors may employ live or dead microbial
cells.A selectedlist of someorganismsalongwith
the analytesand the types of biosensorsused is
given in Table21.8.
Adtramtages 0f m;crobial eell
blosensErs
The mi crobi alcel l s are cheaperw i th l onger
half-lives. Further, they are less sensitive to
variationsin pH and temperaturecompared to
isolatedenzymes.
9BPl"'?-1E N Z Y M ET EC H N O L OC Y 303

/\.:,',,,
.r'"', , \
-)
'.--t,/t'..
ni'

(B)
ii'",
,-, o^ on
Z,o,-"
Ag-Ab

(c) a@ @ 00r@
+A A A
+/\
,M' ,t__\
A +
.&-,
Ag-Enz + Ag
(D)

Fig. 21.18 : Diagrammatic representation of selected immunobiosensors (A) Direct binding of antigen to
immobilized antibody, (B) Antigen-antibody sandwiches (immobilized antigen binds to antibody and then to a
second antigen), (C) Antibody binds to immobilized antigen which gets partially released by a competitive free
antigen, (D) lmmobilized antibody binds to free antigen and enzyme labeled antigen (in competilion).

l=iin itatip ns of r nic r obia! c ell hios ens or s based on amperometricor potentiomeiricbio-
sensors.There are severalpossibleconfigurations
The who le c ells , in gener al, r equir e lon g e r
for i mmunobi osensors and some of them are
p erio dsfo r cata ly s is .I n addit ion, t he s pec if ic it ya n d
depi cted i n Fi g. 21.18, and bri efl y descri bed
sensitivity of whole cell biosensorsmay be lower
hereunder.
compared to that of enzymes
1. A n i mmobi l i zedanti bodyto w hi ch anti gen
il,tMUNaSxosE^tsoBs can di rectl ybi nd (Fi g.21.18A ).
lmmunobiosensors
or immunochemical
bio- 2. A n i mmobi l i zed anti gen that bi nds to
sen so rs wo rk on t he pr inc iple of im m unologi c a l anti bodyw hi ch i n turn can bi nd to a free secono
'cecif
icitv, co upled wit h m eas ur em ent ( m os t l y ) anti gen(Fi g.21.18A .
 304 B IOTE C H NO LO CY
tt
it
3 . A n a n ti b o d yb o u n d to i mmobi l i zedanti gen
wh i c h c a n b e p a rti a l l yre l e a s e b
d y competi ngw i th
free antigen(Fig.21.18Q. biosensorapplications

4 . An i mmo b i l i z e d a n ti b o dy bi ndi ng free Area of application Market share (7.)

antigenand enzymelabeledantigenin competition
Medicalandhealth 60%
( Fi g .2 1 .t8 D ).
Industry 1lYo
.l
For the biosensors -3, piezoelectricdevices andveterinary
Agriculture lfo
c a nb e u s e dT. h e i m m u n o b i o s e n sors
usi ngenzymes
Defence 7%
(4 above, Fig. 21.lBD) are the most commonly
used. These biosensorsemploy thermonretricor Environmental 6To
amperometric devices.The activityof the enzymes Research 4%
bound to immunobiosensors is dependenton the Bobotics 3%
relativeconcentrations of the labeledand unlabeled Others 2%
antigens. The concentrationof the unlabeled
antigencan be determinedby assaying the enzyme
activity. can be detected by using ATPase. One
pharmaceutical company has developed
APPLICATIONS OF BIOSENSORS i m m o b i l i z e d c h o l e s t e r o l o x i d a s e s y s t e m fo r
measurementof cholesterol concentration in fooos
have becomevery popularin recent (e.9.
Biosensors
butter).
y e a rs .T h e y a re w i d e l y u s e d i n vari ous fi el ds.
B io s e n s o rs
a re s ma l l i n s i z e a n d can be easi l y
Applications in pollution control
ha n d l e d .T h e y a re s p e c i fi c a n d sensi ti ve,and
work in a cost-effectivemanner. The tentatrve B i o s e n s o r s a r e v e r y h e l p f u l t o m o n i to r
marketshareof biosensorapplicationsis given in' environmental (air, water) pollution. Th e
Table21.9. Someof the importantapplicationsof concentrations of pesticides and the biological
biosensors are broadlydescribedhereunder. oxygen demand (BOD) can be measured by
biosensors. Several environmental pollutants can
Applications in medicine and health b e e v a l u a t e d f o r t h e i r m u t a g e n i c i t y b y e m p l o yi n g
biosensors. For more details or biosensors ro
Biosensorsare successfully used for the monitor environment, refer Chapter 54.
quantitative estimation of several biologically
importantsubstances in body fluids e.g. glucose, Applications in military
cholesterol,urea. Glucosebiosensoris a boon for Biosensorshave been developed to detect the
diabetic patientsfor regularmonitoringof blood' toxic gasesand other chemical agents used during
glucose.Blood gasmonitoring for pH, pCO, and war.
pO, i s c a rri e do u t d u ri n gc ri ti c a lc areand surgi cal
monitoring of patients.Mutagenicityof several
c he mi c a l sc a n b e d e te rmi n e d b y u si ngbi osensors.
Severaltoxic compoundsproducedin the body can
also be detected.

Applications in industry The industrial and analytical applications of

Biosensorscan be used for monitoring of
fermentation products and estimation of various
ions. Thus, biosensorshelp for improving the
fermentationconditionsfor a bettervield.
Now a days, biosensorsare employed to
measure the odour and freshnessof foods. For
instance,freshness
of storedfish can be detectedby
ATPase.ATP is not found in spoiledfish and this
immobilized enzymes have been described in the
preceeding pages.The therapeutic applications are
dealt with here.
There are several limitations for the direct use of
enzymes for therapeutic purposes. These include
the poor availability of the enzyme at the site of
action, sensitivityto natural inhibitors, degradation
by endogenous proteasesand immunogenicity of
certain enzymes.
 C h a pt er21 : E NZ Y M ET EC H N O L OC Y 305

Some other importanttherapeuticapplications

Gelcontaining of immobilized enzymes and cells are briefly
immobilizedureasedescribed.

Drugin a pH lmproved drug delivery

polymer
sensitive
Fig. 21.19 : A diagrammatic representation of
immobilized urease for drug delivery.
Someof theselimitationscan be overcomeby polymer
e mp loy ingim m obi l i z e de n z y m e sfo r th e ra p e u ti c (Fi g.
applications. A few examplesare listedbelow.
1. lmmobilizedstreptokinase and urokinase(on
Sephadex)can be used for the treatment of
thromboses.
2. Some successhas been reported in the modulated by immobilized glucose oxidase
by using immobilized enzymes
Urea-ureasemodulated system : By using
i mmobi l i zed urease enzyme al ong w i th the
substrate urea,the drug deliverycan be enhanced.
As ureasesplitsurea,thereis an increasein the pH
due to the formation of ammonium hydroxide.
The drug l ocatedi n a pH sensi ti vebi oerodi bl e
can be effectivelyreleasedfor its action
21.19.
Glucose oxidase-glucosemodulated system :
For i nsul i n del i very to the human body, a
bi oerodi bl epol ymeri csystemcontai ni ngi nsul i n
hasbeendeveloped.The deliveryof insulincan be
It,

treatment of inborn errors by employing enzyme (Fig. 21.20). As glucoseoxidaseacts on tl

i mmobiliz ed enz y m e s e .g . p h e n y l a l a n i n e gl ucose,gl uconi caci d i s producedw hi ch l ow ers tl

,I
hydroxylaseto treat phenylketonuria,lysosomal the pH. The low pH in turn, causesthe releaseof
a 1, 4-glucosidaseto correct type ll glycogen i nsul i nfrom the bi oerodi bl epol ymeri csystem. :!l'
it '

storagedisease(Pompe'sdisease).
lmmobilization of artificial cells
;;
A n arti fi ci alcel l pri mari l yconsi stsof a spheri cal ii:
lmmobilized semipermeable membrane with , comparable lr:
glucoseoxidase
di mensi ons of a l i vi ngcel l .The bi ol ogi calmateri al s
suchas enzymesenclosedwithin the artificialcells
can be i mmobi l i zed. The so i mmobi l i zedcompact
in pH
Insulin arti fi ci alcel l scan functi onas arti fi ci alorgans.The
sensitive
polymer important artifical organs constructed include
artificial kidney, artificial liver, blood detoxifiers
and immunosorbents. Theirfunctioningis however,
very l i mi ted.
systemcan be i mmobi l i zedi n the
Mul ti enzyme
form of artificial cells for the conversion of
Fig. 21.20: A diagrammaticrepresentationof
a substrateto a product, through a series of
immobilized glucose oxidase for insulin delivenJ.
reactions.

Biotechnology [20]
 f \ ; , | ic r oor ganis m s pos s es s t h e c a p a b i l i t y t o
i V 6 enz y m at ic allym odif y a wid e r a n g eo f o r g l n i c
cornpourrds. Biotransfornrations(biconversions or
microbial transformations) broadly refer to the
processes in u,hich nticroorganisms convert
organic compounds into structurally related
products. In other words, biotransformationdeals
wit h m ic r obial ( enz y m at ic ) c : o n v e r s i o n o f a
s ubs t r at eint o a pr oduc t wit h a li m i t e d n u m b e r ( o n e
or a f ew) enz y m at ic r eac t ions .T h i s i s i n c o n t r a s tt o
fermentation which involves a large number
reactions (often complex in nature).
Alt hough t her e ar e hu n d r e d s o f bio-
t r ans f or m at ions k nown, only a s e l e c t e d f e w o f
t hem ar e us ef ul f or t he s y nt hes i so f c o m m e r c i a l l y
im por t ant pr oduc t s . The significance of
bioc onv er s ion r eac t ions b e c o m e s obvious
when t he pr oduc t ion of a pa r t i c u l a r c o m p o u n d
is eit her dif f ic ult or c os tl y b y c h e m i c a l
m et hods . Fur t her , biot r an s f o r m a t i o n s a r e
gener ally pr ef er r ed t o c hem ic al r e a c t i o n s b e c a u s e
of s ubs t r at e s pec if ic it y , s t e r e o s p e c i f i c i t y a n d
m ix ed r eac t ion c ondr t ior r s ( p H , t e n r p e r a t u r e ,
pr es s ur e) . The env ir onm ent a l l t o l l u t i o n c i u e
t o biot r ans f or m at ion is alnr os t i n s i q n i f i c a n t o r
negligible. ln addit ion, it i s e a s y k ) a p p l y
r ec onr binant DNA t ec hnology t o m a k e d e : s i r e d
im pr ov em ent s in biot r ans f o r m a t i o n s . A n o t h e r
pr ac t ic al adv ant ageof biot r ansf o r m a t i o n si s t h a t r t
is eas y t o s c ale- up t he pr oc es s e sd u e t o l i m i t e d
num ber of r eac t ions .
306
h "i,s *5 $jr!'' S,i 1; ["i i.i{ !'J$ ir$ (ritl! & F E# Hd
t;li':+{:"i'[ #:l Ii,
M a n y t y p e s o f c h e m i c a l r e a c t i o n s occu r i n
-fhese
biotransformations. include oxidatian.
reduction, hydrolysis, condensation, isomerization,
formation of new C-C bonds, synthesis of chiral
compounds and reversal of hydrolytic reactions.
A m o n g t h e s e , o x i d a t i o n , i s o m e r i z a ti o n a n d
h y d r o l y s i s r e a c t i o n sa r e n r o r e c o m m o n l y oi r se n ve d
in biotransforrnatior-rs Many a times bro-
t r a n s f o r m a t i o n s i n v o l v e m o r e t h a n o n e tvo e o f
r e a c t i o r . r . A s e l e c t e d l i s t o f i m p o r t an t b i o -
t r a n s f o r m a t i o n r e a c t i o n s a l o n g w i t h t h e m i cr o -
o r g a n i s m si n v o l v e d i s g i v e n i n T a b l e 2 2 . 1
T h e c o n v e r s i o n t i m e r e o u i r e d fo r b i o -
transfornratior-r rs related to the tvoe of reaction, the
s u b s t r a t e c o n c e n t r a t i o n a n d t h e m i c r o o r e a n i sn - r
r u s e d .I n g e n e r a l , o x i d . . r t i o n ,h y d r o l y s i s a n d d e h y-
d r a t i o n r e a c t i o n sa r e c o m p l e t e d i n a f e w h o u r s
SOUROES OF BIOCATA!-Y$T$
A f {P T E G F I N I G U E S F O R
B I O T R A h {S F O R M A T 9 C I F {
A r v i d e v a r i e t y o f b i o l o g i c a l c a t a l y s ts ca n b e
t u s e df o r b i o t r a n s f o r m a t i o nr e a c t i o n s T h e se i n cl u d e
growing cells, resting cells, killed cells,
immobilized cells, cell-free e\tr.rcts, enzymes .rnd
immobilized enzymes. The most important sources
of biocatalysts and the procedures employed for
b i o t r a n s f o r r n a t i o na r e b r i e f i v d e s c r i b e d .
 : B IO T R AN SF OR M AIION S

Ttr;tt 22.1 A selected list of important biotransfornation reactions

Type of reaction Example Commonly used microorganism

Oridation Tryptophan
------+5-Hydroxytryptophan Bacillussubtitis
Naphthalene
----+ Salicylic
acid Corynebacterium
sp
Reduction Benzaldehyde
-----+Benzylalcohol Saccharomyces
cerevisiae

flPryP:l9igrf:r_- Pentachroroanirine
st!| l:1,Y,u ?,:i:11:!
::_'
Hydrolysis ------+f.tr.ly.lir.
Anhydrotetracycline Streptomyces
aureofaciens
Menthyl ---+ Menthol
laureate Mycobacteriun
phlei
Condensation ------+Streptomycin-phosphate
Streptomycin griseus
Streptonyces

Growing cells reactions are in fact carried out by using

Th e d es ir ed c ells ar e c ult iv at ed in a s u i t a b l e
med ium. As t he gr owt h of t he c ells oc c ur s ( 6 2 4
hours), a concentrated substrate is added to tne
cultu re. So m et im es ,addit ion of em uls if ier s( T w e e n ,
o rga nic sol v ent s )is r equir edt o s olubiliz e s u b s t r a t e s
and/or products e.g. steroid biotransforrnation.fhe
substrateconversion to product can be monitoreo
by spectroscopic or chromatographic techniques
Biotransformatiorr can be terminated when the
p rod uct for m at ion is opt im um .
Non-growing cells
The non-growing cells are preferred ior
bio tran sfo r m at ion r eac t ions f or t he f ol l o w r n p
reasons.
. Very high concentration of substratecan be used
(with hig h s ubs t r at ec onc ent r at ion,gr owin g c e l l s
stop their gror,r,th).
. Cells ca n be was hed and us ed and t hus t he r e w i l l
b e n o con t am inat ing s ubs t anc es .
. Conversion efficiency of substrate to product is
high.
imnrobilized cells e.g. commercial production of L-
alanine and malic acid (for detailsReferChapter2 j ).
lmmobilized enzymes
Cell-free enzyme systems in the form of
inrmobilized enzymes are most commonly used
in biotransformations, due to the follor,r,rng
advantages.
. No occurrence of undesirable side reactions.
. The desired products are not degraded.
. There is no transport barrier across the cell
mernbrane for the substrateor orclduct
. The isolation and recoverv of the product is
simpler and easier.
S e v e r a li m m o b i l i z e d e n z v m e s v s t e m sh a v e b e e n
developed for biotransformations e.g. glucose
isomerase, penicillin acylase (For details, Refer
C h a p t e r 2 . 1) .
PRODUCT RECOVERY IN
BIOTRANSFORMATIONS

o Biotransformationcan be optimized by creating In most biotransformationreactions,the desired

suita ble env ir onm ent al c ondit ions (pH, end products are extracelliilar.'fhe product may be
temperature etc ). either in a soluble or suspendedstate.When whore
o Product isolation and its reco\/erv are easv. cells are used, they have to be separated and
repeatedly washed (with water or organic solvent)
as required. -The extracted product can be
lmmobilized cells
recovered by employing the commonly used
Biotransformations can be carried out t e c h n i q u e s - p r e c i p i t a t i o nb y s a l t s , e x t r a c t i o n w i t h
co ntin uo uslv by em ploy ing im m obiliz ed c e l s . solvents, adsorption to ion-exchangers etc. The
Fu rthe r,the sam e c ells c an be us ed again and a g a i n . v o l a t i l e p o d u c t s c a n b e r e c o v e r e d b y d i r e c t
Se ve ral bio c onv er s ions wit h s ingle or m ul t i s t a s e d i s t i l l a t r o n f r o m t h e m e d i u m .
 308 B IOTE C H N OLO CY

tl
I Stigmasterol
i Chemical
i reactions
Progesterone
Diosgenin
l_ nigricans
I Rhizopus
+
11 o-Hydroxyprogesterone

i Chemical
i reactions
Y
Corynebacterium simplex
Reichstein's (cortisol)
Hydrocortisone Prednisolone
S
substance
(11-deoxycortisol) i Chemical
i reactions
Corynebacterium simplex
Coftisone Prednisone

Fig. 22.1 : Biotransformationof commercially important steroids.

EXAMPLES OF BIOTBANSFORMATIONS Commercial production of steroids is very

important. Earlier, cortisone was chemically
A large number of biotransformations are
synthesized, and this processinvolvedas many as
descrlbedin literature.Of these,only a selected
37 reactions.The cost of the so obtainedproduct
few are imoortantfor industrialand commercial
was around$200/g(in 1950).With the introduction
purposes.The major limitationswith some of the
of biotransformation the numberof steps
reactions,
are that eitheryieldsare low or (microbial
biotransformations
and chemicalput together)was re.duced
the processis expensiveor the marketitselfis very
to ll, and cost of the productwas reducedto just
limi te d .
$1/g i n 1980! The credi t obvi ousl ygoes t o t he
developments in biotransformation.

Types of reactions in biotransformation

of steroids

As alreadystated,amongthe largenumberand The microbialtransformationof steroidsbroadly

a wide rangeof biotransformations,
only a selected involvesoxidation (introduction
of hydroxylSroups/
few are commerciallyimportant.Someof theseare splittingof sidechains,productionof epoxidesetc.)
brieflydescribedhereunder. reduction (conversionof aldehydesor ketonesto
alcohols, hydration of double bonds), hydrolysis
BIOTRANSFORMATION OF STEROIDS and ester formation.

All the sferoidspossessthe basicstructurenamely

Production process of steroids
cyclopentanoperhydrophenanthrene. Steroids as
hormones (glucocorticoids, mineralocorticoios, The production of steroids,entirely by bio-
androgens,estrogens)perform a wide range of transformation reactions is not practicable.
functions.They are very usefultherapeutically. For Therefore,microbial transformationalong with
instance,cortisone,due to its anti-inflammatorychemicalreactionsis carriedout. The major steps
actionis usedin the treatment of rheumatoid of steroidsare
arthritis involved in the biotransformation
and skin diseases;derivativesof progesterone and depicted in Fig. 22.1. Stigmasterolextracted from
estrogens are employedas contraceptives. Certain soybeansor diosgeninisolatedfrom the rootsof the
derivativesof cortisone(e.g.pred.nisolone)are more Mexican barbascoplant can serveas the starting
effectivein theirtheraoeuticaction. can be chemicallyconverted
material.Stigmasterol
Chaoter22 : BIOTRANSFORMATIONS 309

to progesterone which is subjected to bio- Cholesterol

transformationto form 11a-hydroxyprogesteroneby II
the microorganism, Rhizopus nigricans. Cortisol
(hydrocortisone), produced from 11cx-hydroxy- t
progesterone by chemical reactions, undergoes 3-Hydroxy5-
(organism-Coryne- androstene17-one
microbial transformation
bacterium simplex\ to form prednisolone. Further, I
cortisone formed from cortisol can be subjected to
biotransformation by Corynebacterium simplex to
I
Androstendione
p ro du ce pre dn iso ne,W hen dios genin is us ed as t h e
starting compound, substance S can be produced
II
by chemical reactions which can be converted to J
cortisol by biotransformationwith the help of tne Androstadiendione
m icroorganism Cu rvuIaria I u nata. II
Biotransformation of steroids is usually carried t
out by hatch fermentation. tJse of immobilized 9cr-Hydroxy-
androstadiendione
c e lls o r immob iliz ed enz y m esis gaining im por t an c e
in recent years. This is advantageous since the Fig. 22.2 : Biotransformationof cholesterol by
biotransformation is more efficient with high mycobacteria to commercial products.
substrateconcentration, short conversion time and
good product recovery. Since the steroids are not
water soluble, the microbial transformation reactions involved in the microbial transformation
reactions have to be carried out in organic solvent o f a n t i b i o t i c s .
(water-immiscible) system. However, the organic
solventsare toxic to micro-organismsor enzyntes.lt Biotransformation of penicillin G : Microbial
is ideal to use an aqueous two phase system for t r a n sformation, in association with chemical
biotransformationof steroids. synthesis, is routinely used for the commercial
production of semisynthetic penicillins and
cephalosporins. The enzymatic cleavage of
Biotransformation of cholesterol
penicillin by penicillin acylase into 6-amino-
Certain commercially important steroids (e.g. penicillanic acid is a very important reaction
androstendione, androstadiendione) can be ( F i g . 2 2 . 3 ) . P e n i c i l l i n C g e t s i n a c t i v a t e d b y i t s
produced directly from cholesterol by biotransfor- c o n v e r s i o nt o b e n z y l p e n i c i l l o i ca c i d b y t h e e n z y m e
mation (Fig. 22.2). peni cillinase (p-|actamase).

BIOTRANSFORMATION
OF ANTIBIOTIC S G
Penicillin
Production of new antibiotics or modifications
in the existing ones for more effective treatment Phenyr,aceric
of the diseases is always on the priority of
""'o7
W:-
t he p ha rmaceu t ic al indus t r y . Fur t her , ant ibiot i c s
with wide r an t im ic r obial s pec t r um , r educe d
toxicity, low allergic reactions and decreased 6-Aminopenicillan
ic
\
Benzylpenicilloic
acid acid
resistance are highly advantageous. Biotrans-
formation reactions significantly contribute for I cr'eri.at
improving the pharmaceutical products. I reacvlation
J
Semisynthetic
Direct biotransformation penicillin
Acyla tion a nd deac y lat ion, phos phor y lat io n , Fig. 22.3 : Biotransformationof penicillin G.
ade nylatio n a nd hy dr oly s is ar e s om e of t h e
310 B IOTE CHNO LO CY

Biotrans{ormation of narbomycin : Hydroxylation Tryptophan

of nar bom y c in t o pic r om y c i n ( b r o u g h t o u t b y t-
| ryplopnanase
Streptomyces sp) is another good example of
J
microbial transformation. lndole
Biotransformation of macrolides : The
I Naphthalene
m ac r olide ant ibiot ic s on deacy l a t i o n w i l l g i v e l e s s (cloned)
active products.These products can be used for the fdioxygenase
Indole2, 3-dihydrodiol
production of more active semisynthetic
m ac r olides .
I Spontaneous
+
lndirect biotransformation Indoxyl
The bioeyntheticprocessesof antibiotics can be
I o",ou.on
c ont r olled by t he addit ion c e r t a i n i n h i b i t o r s o r +
modified substratesto the medium. In other words, Indigo
t he bios y nt hes isof ant ibiot ic so c c u r s i n a c o n t r o l l e d
f as hion in t he indir ec t biot r an s f o r m a t i o n . Fig. 22.4 : Microbial production of indigo.
Biotransformation of actinomycins : The
microorganism Streptomyces parvulus produces
new actinomycir-rsin the presence of 4-methyl- BIOTRANSFORMATION FOR THE
pr oline ( pr oline analog) in t h e r n e d i u m . T h e n e u , PRODUCTION OF ASCORBIC ACID
ant ibiot ic s will h: r v e 4- m et lr y l p r o l i n e i r r p l a c e o f
A s c o r b i c a c i d ( v i t a m i n C ) c a n b e c o m m e r ci a l l y
pr oline and t hes e ac t inom y ci n s a r e m o r e e f f i c i e n t
produced by a combination of chemical and
in t heir f unc t ion.
microbial transformationprocesses.For full details,
Biot r ans f or m at ion of r ib o s t a m y c i n : In the the reader must refer Chapter 24.
bios y nt hes is of neom y c in, r i b o s t a m y c i n i s a n
int er m ediat e. By em ploy ing m u t a n t s t r a i n s o f BIOTRANSFORMATION OF GLYCEROL
Streptomyces fradiae, ribostamycin can be TO DIHYDROXYACETONE
pr oduc ed in lar ge quant it ies.
Dihydroxyacetone is used in cosmotics and
Several other rnutant strains of microorganisms
suntan lotions. Certain acetic acid bacteria can
have been created by recombinant DNA
convert glycerol to dihydroxyacetone through the
technology for the production of modified
orocess of biotransformation.
ant ibiot ic s of am inogly c os ide sa n d r i f a m y c i n s .
Glycerol
BIOTRANSFORMATION OF
ARACHIDONIC ACID TO Acetobactersuboxydansor
PROSTAGLANDINS A. xylinum or
GIuconobacter melanogen us
Prostaglandins(PC) have a wide spectrum of
biological functions. They are important for
Dihydroxyacetone
pharmaceutical and therapeutic purposes. For
instance, PGE.,servesas a contraceptive; PCC, rs Cood oxygen supply, temperature 26-28"C and
used in the treatment of congenital heart failure; pH 6.0 are ideal for the optimal biotransformation.
PCC, f or r eliev ing labour pa i n s .
BIOTRANSFORMATION FOR THE
The unsaturated fatty acid arachidonic acid is
PRODUCTION OF INDIGO
the precursorfor the biosynthesisof prostaglandins.
Some success has been reported in the l n d i g o c a n b e s y n t h e s i z e d b y m i cr o b i a l
biot r ans f or m at ion of ar ac hid o n i c a c i d t o P G E . , transformation. This has been made possible by
PCE2,PCFl and PCF, by us in g f u n g i . l t i s e x p e c t e d cloning a single Pseudomonasgene that encodes
t hat in t he c om ing y ear s , p r o s t a g l a n d i n s w i t h naphthalenedioxygenasein the creation of E. coli.
improved efficiency will be produced by The relevant reactions of biotransformationfor tne
biotranSformations. production of indigo are depicted in Fig. 22.4.
 h e comme rc ially im por t ant or ganic s olv ent sa r e
J
I ethanol, acetone, butanol and glycerol. These
compounds were mostly being produced in
oetrochemical industries and little attention was
p aid fo r th eir m ic r obial pr oduc t ion. lt was ir r m i d
1 97 0, du e to ec onom ic and polit ic al r eas o n s ,
petroleum and natural gas became scarce. Since
then, scientists have started looking for suitable
alternativesfor commercial production of organic
soiven ts.Microbial pr oduc t ion of or ganic s olve n t s
u tilizin g lo w c os t r aw m at er ials ( e. B wo o d ,
cellu lose, starc h) , is c ons ider ed t o be an id e a l
alternative.
Alcoh ol, ch em ic ally et hanol ( C2H50H) has b e e n
produced by fermentation for thousands of years.
However, this was mostly associatedwith brewer
and distiller industries.At present, most developed
cou ntrie s pro du c e indus t r ial alc ohol by c hem i c a l
means. The petrochemical ethanol is manufactured
by hydration of ethylene (C2H4).
In the dev eloping c ount r ies , m ic r o b i a l
fermentation processes are preferred for the
p rod uction of alc ohol. This is m ainly bec aus e o f
the che ao raw m at er ials av ailable. At t he c ur r e n t
rate of oil price, it is cheaper to produce alcohol by
petrochemical means rather than fermentation.
However, large scale production of alcohol in a
g ive n co un try depends on t he polit ic al a n d
e c o n o m i c c o n s i d e r a t i o n sb, e s i d e st h e a v a i i a b i l i t yo t
r a w m a t e r i a l s . Wi t h i n c r e a s i n g o i l p r i c e s , m a r r y
countries now realise the ootential of alcohol
production by fermentatron.
Ethanol as a motor fuel
Prior to Seconci World War, ethannl \ /as
extensively used as a motor fuel. in fact, some
m o t o r c o m p a n i e s ( e . 9 . H e n r v F o r d ) d e s i g n e ds o n r e
v e h i c l e s t o r u n o n a l c o h o l o r p e t r o l o r a n r i x t u r eo f
both.
Brazil was the first country to produce ethanol
in large scale by yeast fermentatinn, utilizing
sugarcaneand cassava.This alcohol used as motor
fuel is referred to as green petroL Brazil started
growing alcohol-producing plants in a vast area,
and installed several fermentationand distillation
plants. By 1988, about 90"h of the new cars in
Brazil had enginesthat run on alcohol as a fuel.
Excited by the success of Brazil, nrany other
countries have also started large scale production
of alcohol by fermentation.
Advantagesof ethanol as motor fuel : There are
distinct acJvantagesof using ethanol in place of
petrol for motor vehicles. Ethanol powered engines
cause less environmentalpollution. They produce
60% less carbon dioxide, 65% less hirdrocarbons
and 15"k lessnitric oxide when compared to petrol-
run vehicles. Thus, vehicles rul-r on ethanol or
e t h a n o l - p e t r o lm i x c a u s e c o m p a r a t i v e l yl e s s g l o b a l
warming, and are consideredro be environmental-
3tt
312 B IOTE C H N OLO CY

friendly. Another advantageof ethanol use as a

motor fuel is the safefy.The flash point (i.e. the
temperature at which a substanceignites)of ethanol
is three times higherthan that of petrol (45"C for
Microorganism Source of carbohydrate
ethanolcomparedto 13'C for petrol).
Disadvantagesof ethanol as motor fuel : At Yeasts
present,alcohol is about twice costilier than petrol Saccharomycescerev isiae Starch,
sugar
in most countries(This may not however,be a S. ellipsoideus Starch,
sugar
lim i ta ti o ni n th e l o n g ru n w i th th e avai l abi l i tyof Kuyveromyces fragilis Starch,
sugar
c he a p s u b s tra te sa n d l a rg e s cal e i ndustri al
pr od u c ti o no f a l c o h o l ).S i n c eth e fl ash poi nt for Bacteria
o f e n g i n e si n col d w i l l oe
et ha n o il s h i g h e r,s ta rti n g Zymomonas mobilis Starch
l y re a c tw i th a l loyscontai ni ng
dif f i c u l t.Eth a n oma Candidapseudotropicalis Lactose,whey
aluminiumand magnesiumand may damagethe C. utilis Sulfitewasteliquor
containers.The alcohol used in motor vehicres
shouldbe of high purity,and shouldnot be allowed
starchy materials (wheat, rice, maize, potato) and
to pick up waterfrom the air.A mixtureof ethanol
cellulosic materials (wood, agricultural wastes).
with waterdoesnot burn readily,and furtherit also
Some more examples of potential raw materialsare
causescorrosionof enginesand tanks.
given in Table 23.2. The biomass (starchy and
Gasohol as a fuel : A mixture of about 20"/" c e l l u l o s i c m a t e r i a l s )s i g n i f i c a n t l yc o n t r i b u t e sf o r th e
ethanol with Bo% petrol, known as gasoholis now p r o d u c t i o n o f a l c o h o l . F o r m o r e d e t a i l so n b i o m a ss,
being used as a motor fuel some countrieslike refer Chapter 31.
USA. The ethanolusedfor this purposeshouldof
100% purity,otherwisepetrol and ethanoldo not Pretreatment of raw materials : Most of the raw
form a uniformmixture. materials of alcohol fermentation require some
degree of pretreatment.The actual processdepends
PRODUCTION OF ETHANOL BY o n t h e c h e m i c a l c o m p o s i t i o n o f t h e r a w m a t e r i a t.
FERMENTATION I n g e n e r a l ,t h e s u g a r y r a w m a t e r i a l sr e q u i r e m i ld o r
no pretreatment while the cellulosic materials need
As alreadystated,in recentyearsmanycountries
havestartedproductionof ethanolby fermentation
process.The organisms and the raw materialsuseo,
along with the biosynthetic pathway, the
productionand recoveryprocesses for alcohol are
br ie fl y ' d e s c ri b e d .
Sugary Starchy Cellulosic
materials materials materials
Microorganisms
Molasses Cereals Wood
Certain yeastsand bacteria are employed for
Sugarcane Wheat Sawdust
alcohol fermentation.The type of the organism
chosen mostly depends on the nature of the Sugarbeet Maize Agricultural
wastes
substrate used (Iable 23.1). Among the yeasts, Sweetpotato Barley Paperwastes
Saccharomycescerevisiaeis the most commonly Sweetsorghum Sorghum Municipal wastes
solid
used, while among the bacteria, Zymomonas Whey Corn
mobilisis the mostfrequentlyemployedfor alcohol Sulfitewaste Rice
pr o d u c ti o n . Starchy roots
Sucrose
Laclose Potato
Raw materials
Glucose Tapioca
Thereare a largenumberof raw materialsthat Milled products
can serve as substrates
for alcohol fermentation. Wheatflour
They may be broadly categorizedas sugary
Cornfeed
materials(e.g. molasses,
whey, glucose,sucrose),
Chapter23 : MICROBIALPRODUCTIONOF ORCANICSOLVENTS 313

STARCH concentrationof alcohol (up to 13% by some

! strainsagainst5% by yeasts).
intH,l,trfrEl
j Glucoseto alcohol-conversion profile : Theoreti-
Glucose cally, one gram of glucosecan be convertedto
0.511gramsof ethanol.ln fact,a conversion yieldof
(glucose,
i r$.#r,y# 95% was observedwhen pure substrates
lactose,sucrose) are used.For industrialgraderaw
eyrJvate materials(cornstarch)the yield is around90%. lt is
estimated that for 100 g pure glucose, 48.5 g of
I Pyruvate
decarboxylase ethanolis produced,along with 46.5 g of CO2,3.3
CO2(lThiamine pyrophosphate,
Mgz+ g glyceroland 1.3 g of biomass(yeastcells).
+
Acetaldehyde
t.,,,, Production process of ethanol
Atcoholdehydrogenase
I Ethanoloroductioncan be carriedout in three
I NADH+ H+
J stages-preparationof nutrient solution and
Ethanol inoculum, fermentationproper and recovery.A
flow diagramfor alcoholproductionis depictedin
Fig. 23.1 : Biosynthesis of ethanol. Fig. 23.2.

extensive pretreatment This is because the RAW MATERIALS

haveto be subjectedto acidic
cellulosicsubstances (starch,cellulose,molasses)
or enzyme hydrolysisto releasemonosaccharide I
unitsthat are neededfor alcohol oroduction. J
PRETREATMENT
Biosynthesis of ethanol (hydrolysis, filtration)
clarification,
The pathwayfor the biosynthesis of ethanolis I
depictedin Fig. 23,1. Clucosegetsbrokendown to
STEBILIZATION
pyruvateby glycolysis. Underanaerobicconditions
(i.e. in the absenceof 02), pyruvateis converted I
to acetaldehyde by the enzyme pyruvate PRECULTURE----+ FERMENTATION{---l
+
decarboxylase. Acetaldehydeis then reducedby
alcohol dehydrogenase to form ethanol. | | Recycle
+l
It is observed -
SEPARATION CELL
'that under aerobic conditions MATERTAL
(adequate 02 supply) with excess glucose I
contentin the medium,the microorganisms(e.g.5. J
DISTILLATION
cerevisiae)grow well without producingalcohol.
However, under anaerobic conditions, growth I
+
slows down and alcohol production occurs.
-}ABSOLUTE
DEHYDRATION
Regulation of synthesis z Ethanol at high I ETHANOL
concentration in the medium inhibits its own J
biosynthesis. This is particularlyobservedwhen DENATURATION
yeastsarethe fermentation organisms.lt is generally
seen that growth of yeastsceasesat 5% ethanol
I
+
concentration(volume/volumein water). lt is STTLLAGE_______+
FUEL
strikingto notethatyeastsaresensitiveto inhibition (conaentration)
reIrlEPen
by endogenouslysynthesized ethanoland not to
the ethanol added to the medium. Some
Fig. 23.2 : A schematic representation of ethanol
biotechnologistsprefer to use the bacterium production by fermentation.
Zymononasmobilis, since it can tolerate a high
314
Preparation of nutrient solution (medial
The various raw materialsfor alcohol productron
have already been described (Refer Table 23.1).
The m os t c om m only us ed r a w m a t e r i a l s a r e
molasses,whe.v,grains, potatoesand u,ood wastes.
Wherr molasses is used for fermentation, it is
dilut ed wit h wat er s o t hat t he s u g a r c o n c e n t r a t r o n
is in tht-,r-angeof 1O-18"/,'.A concentration lrigher
than this is cietrimentalto the yeast. Wherr starchy
materials (corn, barely) are used, they have to be
first hydrolysedbv pretreatmentfor use as nutrients.
' I his
m ay be done by bar ley m a l t , c l i l u t e a c i d s o r
fungal amylases (e.g. Aspergi!lus sp, Rhizotrtussp).
M os t f r equent ly m as hing,wit h b a r e l y m a l t i s u s e d
Molasses contains most of the nutrients required
for alcohol fermentation. However, ammonium
s alt s s uc h as am m onium s ulf a t e o r p h o s p h a t e a r e
added t o t he nut r ient s olut io n t o s u p p l y n i t r o g e n
and phos phor us The pH of t h e m e d i u m i s a d j u s t e d
t o 4- 5, by adding s ulf ur ic ac id o r l a c t i c a c i d .
Preparation of inoculum
After selection of the clesiredorganism (yeastor
bacteria) and its isolation in pure fornr, the
r noc ulum is pr epar ed under a s e p t i cc o n d i t i o n s . F o r
ihis pur pos e, t he or ganis m s a r e f i r s t c u l t u r e d i n
f las k s r r nderaer obic c ondit ion s t o i n c r e a s et h e s r z e
of t he inoc ulunr whic h c an be u s e d f o r i n o c u l a t i o n .
Fermentation proper
Batch fermentation process was originally
adopted in Brazil. Now, at most places continuous
fermentation is used. There are several advantages
of c ont inuous f er m ent at ion . T h e s e i n c l u d e t h e
r et ent ion of t he f er m ent in g o r g a n i s n r s i n t h e
bioreactor"by separation and recycling, and the
continuous evaporation of fermentation broth. lt
has been pos s ible t o inc r eas e a l c o h o l p r o d u c t i o n
bv 1Q - 12 iold bv c ont i n u o u s f e r m e n t a t i o n
comoared to conventional batch fermentation
I nc ius t r ialor or iuc t ion of alc o h o l i s c a r r i e d o u t r n
huge ier m ent er s up t o a s iz e o f 1 2 5 , 0 0 0 g a l l o n s .
The voltrnre of incculum is usually around 4oh o'[
ihe fermr:nter fhe ideal pH is around 4.0-4.5.fhe
initial fenrperature is kept between 21-26"C. As
the fernrentation proceeds, the temperature raises
t o ar ound : 10' C. lt is nec es s a r y t o u s e c o o l i n g
devices and bring down the ternperature to less
ihan 27"C. Ethanol gets evaporated at temperature
B IOTE CHNO LO CY
a b o v e 2 7 " C . A e r a t i o n ( O , s u p p l y ) i s i n i ti a l l y
required for good growth of the organisms. Later,
anaerobic conditions are created by withdrawl of
O, coupled with production of COr. lt takes about
2-3 days for the fermentationto be completed. The
actual time period depends on the raw materials
used and the temperature of incubation.
As tlie fermentation is complete. the
fermentation broth contains ethanol in the range of
6-9oh by volume. 1'his representsabout 90-95"/0
conversion of substrate to ethanol. The overall
empirical fornrula for ethanol production is given
below.
FermentatioS
c6H1206 2c2H5oH + Zco,
Glucose Ethanol
Recovery of ethanol
The cell mass is separatedby centrifr-rgation or
sedimentation.Ethanol from the fermentation broth
can be recoveredby successivedistillations.By this
processes,it is easy to obtain ethanol of around
95"h. For a concentration above 957o, special
techniques of distillation have to be adopted. For
p r e p a r a t i o n o f a b s o l u t e ( 1 0 0 %) a lco h o l , a n
azeotropic mixture of benzene, n,ater and alcohol
i s f i r s t p r e p a r e d . T h i s m i x t u r e i s t h e n d i sti l l e d b y
g r a d u a l l y i n c r e a s i n g t h e t e m p e r a t u r e . By th i s
technique, it is possible to first remove benzene-
etharrol-w'atermixture, and then ethanol-benzene
mixture. Thus, absolute alcohol is left out.
Stillages in alcohol production
Large volumes of wastes which are technically
referredto stillagesare formed during the course of
alcohol fermentation. Attempts are made to
f r u i t f u l l y u t i l i z e s t i l l a g e sf o r v a r i o u s p u r p o se s.
o To use as feed or fertilizers.
. For converting to single-cell protein.
. To use a fuel.
. For production of rnethanol.
Genetic englneering for improved
alcohol production
With the advent of recombinant DNA
technology, attempts are being made worldwide to
develop new strains of organisms for increasing
alcohol oroduction. Some success has been
achieved in creating more efficient microorganisms
with the desired characteristics.
Chapter23 : MICROBIALPRODUCTIONOF ORCANICSOLVENTS 315

Glucose
i Glycolysis

+ Acetoacetic
acid
J
Aceticacid CrotonylCoA I
l+
'| CH3-CO-CH3
ButyrylCoA Acetone
I
+l +
Butyricacid
'j lsoProPanol

cH3-cH2-cH2-cH2-oH
Butanol

Fig. 23,3 : Biosynthesis of acetone and butanol (Note : Acetic acid and butyric acid
can also be produced by this pathway)

. lmproved production oi alcohol. available from the byproducts of the petroleum

o Resistanceto high alcohol concentration.
o Resistanceto high temperature.
. Rapid ccnversion of substrateto alcohol.
It has also been possible to inrproVe alcohol
p rod uction b y us ing im m obiliz ed enz y m es i n
reactor systems.
industry.The production of acetone and butanol by
fermentation is almost discontinued in many
countries. lt is however, said that being a very
simple process, developing countries (with cheap
raw materials)may soon opt for fermentation rather
than petroleum-based products due to economic
reasons.

Production of acetone and butanol

Certain bacteria of Clostridium species are

Both acetone and butanol are good organic
solvents. Decades ago, acetone was used as a
g ela tinizing a gent f or nit r oc ellulos e in t h e
manufactureof explosives.lt is also used in dyestuff
industry. Butanol is required for the synthesis of
butadiene to produce synthetic rubber. At present,
b uta no l is extens iv ely us ed in br eak f luids , f o r
antibiotic recovery,as amines for gasolineadditives
and as ester in protective-coatingindustry.
The production of acetone and butanol by
anaerobic fermentation is almost a century old.
-r is process was carried out by using starch or
capable of producing acetone, butanol, butyric
acid and isopropanol through fermentation of
molasses,wood hydrolysates,starch, sucrose and
pentoses.Depending on the strain of the bacterium
and the fermentation conditions employed,
the relative production of the four compounds
vanes.
For C Iostr i d i u m acetobutyIi cu m, the fermentation
oroducts are acetone and butanol Butanol-
isopropanol or butyric acid-acetic acid can be
produced by Clostridium butyricum.
Biosynthesis of acetone and butanol

.-.arrich raw materials (potato, molasses)and the The sequence of reactions leading to the
-'-anism Clostridium acetobutylicum. After World formation of acetone and butanol, along with acetic
,',a' ll, acetone and butanol became readily acid and butyric acid is depicted in Fig. 23.3.
316 B IOTE C H N OLO CY

Clucose, on degradationforms pyruvate (glycolysis)

which then gets converted to acetyl CoA. A part of
this acetyl CoA can form acetic acid. Two
molecules of acetyl CoA condense to produce Solvent
acetoacetyl CoA. The latter undergoes a sequence
of reactionsto form B-hydroxybutyrylCoA, crotonyl
CoA, butyryl CoA and butyric acid. This butyric
acid is reduced to butanol. For the synthesis of Titratable
acidity
acetone, acetoacetyl CoA combines with acetyl
CoA to form B-hydroxy B-methylglutaryl CoA,
acetoacetic acid, and finally acetone.
91827 36
process Fermentationtime (hours)
Production of acetone and
butanol
Fig. 23.4 : Acetone-butanol fermentation with
Ac et one and but anol pr oduc in g b a c t e r i u m
reference to solvent production and titrutable aciditjl.
Clostridium acetobutylicum can be stored in the
form of spores for 25-30 years. The inoculum is
bu ild up in s ev er al s t ages .Suc h ino c u l u m , m o r e
butanol.Contamination due to bacteriophages
and
efficiently ferments sugars to give a high yield.
lactobacilli(particularlyLactobacillus
leichmannii)
These organismsare also resistantto contamination.
is a major problemthat hindersthe yield.
The f er m ent at ion is us ually c ar r ie d o u t i n c o r n
Product yield : Approximately,3Ooh of the
or molas s esbas ed m edium . The m ola s s e sm e d i u m getsconvertedto neutralsolventsin
carbohydrate
co nta ins m olas s es , am m onium s ulf a t e , c a l c i u m
the fermentation. In molasses medium,the ratio of
carbonate and sometimes corn steep liquor.
butanoland acetonei s7' .3, w hi l e i n cornmed ium
The fermentersafter sterilization are gased with it is 6 : 3. The actual productionof butanol is
COr. After the addition of the medium and influencedby its toxicityto the organi'sm. Butanol
inoculum (approximately 2-4% by volume), the i s toxi cat a concentrati on
hi eherthan 13.5%i n the
fermenter contents are again stirred with COr. The medi um.
fermentation is carried out with a starting pH
Product recovery : Acetone and butanol are
5.8-6.0, and temperature 34oC. The actual process
recovered through continuous distillation and
of fermentation, spread over 36 hours, occurs rn
fractionation.
The left over residueafterdistillation
three phases.
can be dried and usedas animal feed.
Phase I is characterized by rapid growth of the
organism and production acetic acid and butyric
acid There is an increase in titratable acidity. The
pH o f t he m edium dr ops t o ar ound 5 . 2 . I
In phase 2, an increased production of acetone Gl ycerol i s w i del y used i n i ndustry and
and butanol (solvents)coupled with a decrease in commerce.lt is particularlyimportantas a starting
titratable acidity (the latter phenomenon is referred
material for the manufacture of explosives.
to as acid break). Clycerol is commercially produced by the
saponification of fats and oils. lt can also [re
During the third phase, there is a decrease rn
chemicallysynthesized from propyleneor propane.
the solvent production with a slight or no increase
As such, glycerol is not usually produced by
in oH .
fermentation.
ln Fig. 23.4, Ihe fermentation processes with
Duringthe FirstWorld War,the Cermanscould
reference to solvent production and titratabre
not importvegetableoils to produceglycerol,due
acidity are depicted.
to British naval blockade.And for this reason,
Contamination : Absolute sterile conditions are Cermans were forced to produce glycerol by
required for good production of acetone and microbial fermentation. At present, glycerol
 OF OR C A N ICS OLV E N TS
Chapt er23 : MIC R OB IALP R OD U C T IO N 317

Glucose
+
1,G-bisphosphate
Fructose

3-phosphate
Glyceraldehyde phosphate
Dihydroxyacetone
NAD+-! I
Dihydroxy- I,-NADH + H+
NADH+ H* +4 acetone (
+ . phosphate l\runO*
1,3-Bisphosphoglycerate qenyorogenase
I
+
Y Glycerol
3-phosphate
Pyruvate
I
+
cH2-oh
ncetailenyde
I
NADH+ H*-11 Sodium
t-
CH-OH
i bisulfite
NAD+</l blocks I
cH2-oh
+
Ethanol GlYcerol

Fig. 23.5 : Biosynthesis of glycerol.

fermentation is of no significanceexcept theoretical Further, the recovery of glycerol from the

an d histor ic al im por t anc e. I n addit io n , t h r s fermentati on
broth al so i s i ncompl ete.
fermentation process also demonstrates how a
modification in the fermentation condition leads to Gl ycerol producti on by al ga
a modification in the product formed.
A novel fermentation process for glycerol
production by the alga Dunaliellasaiinahas been
Production process of glycerol
devel oped i n l sraelThi
. sorgani sm, commonl yfound
Basically,glycerol is produced by yeast during i n sal ty (hypersal i ne) l akes synthesi zes gl ycerol
the course of alcohol fermentation.However, in the w i thi nthe cel l sto bal ance the hi ghosmoti cpressure
normal process,the quantity of glycerol formed is in the surroundings (dueto salts).Thus,the higher
very low. Addition of sodium sulfite blocks alcohol is the salt concentrationin the surroundings, the
production and diverts the pathway for the large more i s the i ntracel l ul argl ycerol producti on.
scale production of glycerol (Fig. 23.5). Sodium However,when the surrounding salt concentration
sulfite reacts with CO, in the medium and gets is suddenlyreduced,glycerolis excretedinto the
converted to sodium bisulfite. The latter combines medi um.Thi spri nci pl ei s successfulexpl l y oi tedfor
with acetaldehyde (an intermediate in ethanol the productionglycerolby D. salina.
formation) to form acetaldehyde-sulfitecomplex
an d th us bloc k s alc ohol s y nt hes is . N A D H , Gfycerol production by Bacillus subtr-lis
generatedin one of the early reactionsof glycolysis Bacillus subtilis is normally an aerobic
is utilized for glycerol production (instead of mi croorgani sm.l t i s capabl eof converti nggl ucose
ethanol formation due to inhibition). to glycerol,ethanol,lacticacid, butanedioletc. But
The fermentation is run for 2-3 days. The yield the producti on of gl ycerolunderaerobi ccondi ti ons
of glycerol is in the range of 2O-3O"/"of the sugar is very low. However,under anaerobicconditions,
used. This yield is about half of the theoretical the productionof glyceroll:y B. subtilisis quite
value (- 50%) due to the fact that ethanol high. And the yield of glycerolcomparableto that
fermentation cannot be completely inhibited. obtainedbv veastfermentation.
 h" m aior or ganic ac id s o f c o m m e r c i a l
T
I importance produced by fermentation are citric
acid, gluconic acid, Iactic acid, acetic acid,
ascorbic acid and itaconic acid. These comoounds
m ay be pr oduc ed dir ec t ly f r o m g l u c o s e ( e . g . ,
gluc onic ac id) or f or m ed as e n d p r o d u c t s f r o m
pyruvate or ethanol (e.g., lactic acid and acetic
acid). The production of most of the organic acids
is dir ec t ly or indir ec t ly link ed w i t h K r e b s c y c l e .
Besides microbiological production by
fermentation,some organic acids are also produced
by chenrical methods e.g. acetic acid and lactic
acid. Some details on the production of important
organic acids by fermentation are given in the
following pages.
Citric acici was first discovered as a constituent
of lemon. Today, we know citric acid as an
intermediate of ubiquitous Krebs cycle (citric acid
cycle), and therefore, it is present in every living
or ganis m . I n t he ear ly day s , c it r i c a c i d w a s i s o l a t e d
from lemons (that contain 7-9"h citric acid), anct
today about 99"/' of the world's citric acid comes
from microbial fermentarron.
Applications of citric acid
1 . Citric acid, due to its pleasant taste and A. clavatus, A. wentii, Penicillium luteum, Candida
palatability, is used as a flavouring agenf in foods
and bev er agese. g. , jam s , jellie s , c a n d i e s , d e s s e r t s , s p .
318
f r o z e n f r u i t s , s o f t d r i n k s , w i n e . B e s i d e sb r i g h te n i n g
the colour, citric acid acts as an antioxidant ano
preservesthe flavors of foods.
2. lt is used in Ihe chemical industry as an
antifoarn agent, and for the treatment of textiles. In
metal industry, pure metals are complexed with
citrate and produced as metal citrates.
3. ln pharmaceutical industry, as trisodium
citrate, it is used as a blood preservative.Citric acid
is also used for preservation of ointments ano
cosmotic preparations.As iron citrate, it serve as a
good source of iron
4. Citric acid can be utilized as an agent for
stabilization of fats, oils or ascorbic acid lt fornrs
a complex with metal ions (iron, copper) and
prevents metal catalysed reactions. Citric acid is
a l s o u s e d a s a s t a b i l i s e r o f e m u l s i o ns i n th e
preparation of cheese.
5. ln detergent/cleaning industry, citric acid has
slowly replaced polyphosphates.
Microbial strains for
citric acid production
M a n y m i c r o o r g a n i s m sc a n p r o d u c e c i tr i c a ci d .
The fungus Aspergillus niger is most commonly
u s e d I o r i n d u s t r i a l p r o d u c t i o n o f c i t r i c aci d . Th e
o t h e r o r g a r r i s m s( a l t h o u g h l e s s i m p o r t a n t) i n cl u d e
catenula, C. guilliermondii and Corynebacterium
Chapt er24 : M IC R O B IALPR O D U C T IO N
OF OR C A N ICA C ID S 319

Glucose
MEDIUM

Pyruvate CYTOPLASM
pyruvare
Ror---.]
I Jcarboxylase

MITOCHONDRION

Citricacid

MEDIUM
Citricacid

Fig. 24.1 : An outline of metabolic pathway for the biosynthesis of citric acid
(TCA cycle-Tricarboxylic acid cycle).

Forimprovedindgstrialproductionof citricacid, the biosynthesisof citric acid are depicted rn

mutant strainsof A. niger have been developed. Fig. 24.1.
The strains that can tolerate high sugar
Enzymaticregulationof citric acid production :
concentration and low pH with reduced
D uri ng the synthesi sof ci tri c aci d, there i s
synthesisof undersirablebyproducts(oxalic acid,
a tenfold increase in the activity of the
is oc it r icac i d a n d g l u c o n i ca c i d ) a re i n d u stri al l y
enzyme citrate synfhase while the activities
rmportant.
of other enzymes (aconitase, isocitrate
dehydrogenase)that degrade citric acid are
Microbial biosynthesis of citric acid
reduced. However, recent evidence does not
Citric acid is a primary metabolic product (of supportthe theorythat reductionin the operation
primary metabolism)formed in the tricarboxylic of tricarboxylicacid (i.e. degradationof citric
acid (Krebs)cycle. Clucose is the predominant aci d) contri butes to accumul ati on of ci tnc
carbon source for citric acid oroduction. The aci d. Increasedci tri c aci d i s more l i kel y due
biosyntheticpathway for citric acid production to enhanced biosynthesisrather than inhibited
involvesglycalysiswhereinglucoseis convertedfo degradation. Further, there are anaplerotic
two molecules of pyruvate. Pyruvate in turn reactions that replenish the TCA cycle
forms acetyl CoA and oxaloacetate which intermediates to keep the cycle continuouslyin
condenseto finally give citrate.The major stepsin ooeration.
32() B IOTE C H N OLO CY

Pyruvate carboxylase that converts pyruvate to

oxaloacetate is also a kev enzvme in citric acid
production. xd
YX
=<'
Yield of citric acid : Theoretically,the yield of 66
ov
citric acid for the most commonly used substrate
sucrose has been calculated. It is worked out thar :-\
1.1O
fro m 100 I s uc r os e,112 g of anhy d r o u s c i t r i c a c i d
or 123 g of citric acid- t hydrate can be formed. o->
However, due to oxidation of sugar to CO, during
tro phophas e,t he y ield of c it r ic ac id i s l o w e r t h a n
5 10 t3
th e c alc ulat ed.
Sucroseconcentration(7")
FACTORS IN THE REGULATION OF
CITRIC ACID PRODUCTION Fig. 24.2 : Effect of sugar concentration on cittic
acid production.
Strict maintenance of controlled nutrienr
con dit ions is v er y c r uc ial f or m ax im a l p r o d u c t i o n
o f c it r ic ac id. The opt im al c ondit i o n s t h a t h a v e The concentration of carbohydrate significantly
been worked out for A. niger for the production of influencescitric acid production.ldeally,the sugar
citric acid are briefly described (Table 24.1). concentration should be 12-25%. At a
concentrationless than 57o sucrose,citric acid
Garbohydrate source formati on i s negl i gi bl e,and i ncreasesas t he
A wide range of raw materials can be used for concentrationis raisedto 1O"hand then stablises
th e s upply of c ar bohy dr at es . T h e s e i n c l u d e Gig. 2 .2). lt is believed that a high sugar
molasses(sugar cane or sugar beet), sfarch (from concentration induces increased glucose uptake
potatoes), date syrup, cotton wastes, banana and consequentlyenhancedcitric acid production.
extract, sweet potato pulp, brewery waste and
pin eapple was t e wat er . A high y ield o f c i t r i c a c i d Trace metals
production occurs if the sugars that are rapidly Certaintraceelements(Fe,Cu, Zn, Mn, Mg, Co)
metabolised are used e.B. sucrose, glucose, are essentialfor the growth of A. niger.Someof the
maltose. At present, cane molasses and beet trace metals particularlyMtf*, Fe3* and Znz+
mo las s esar e c om m only us ed. The va r i a t i o n si n t h e increase the yield of citric acid.
composition of molasses(seasonaland production
level), have to be carefully considered for The effect of manganese ions has been
o pti m is ing c it r ic ac id pr oduc t ion. investigatedto some extent.These ions promote
glycolysis and reduce respiration,both these
processespromotecitric acid production.
As regardsiron, it is a cofactorfor the enzyme
citric acid production aconitase(of TCA cycle).It is estimatedthat an Fe
concentration of 0.05-0.5ppm is idealfor optimal
Condition/parameter Optimum citric acid production.At higherFe concentration,
Sugarconcentration 10-25% the yield is lower which can be reversedto some
Tracemetalconcentration extentby addingcopper.
Manganese <10-8M
pH
Zinc <10-7M
tr0n <10-4M The pH of the medi umi nfl uences the yi e ld of
pH 1.y25 citric acid, and it is maximal when pH is below
DissolvedO, tension 150mbr 2.5. Al this pH, the productionof oxalic acid and
gl uconi caci d i s suppressed.Further,at l ow pH,
Ammonium saltsconcentration >0.2%
transportof citric acid is much higher.lf the pH is
Time 150-250hours
above4, gluconicacid accumulates at the expense
 Chapt er24 : M IC R O B IALPR O D U C T IO N
O F OR GA N ICA C ID S 321

of citric acid. And when the pH goes beyond 6,

ox alic ac id ac c u m u l a te s .
Anotheradvantage with low pH is that the risk
of c ont am in a ti o ni s v e ry mi n i ma l , s i n c e m any
or ganis mcsa n n o tg ro w a t th i s p H .

Dissolved O, Solidmedium
Liquidmedium
The yield of citric acid production substantially
increaseswhen the dissolvedO, tension is higher.
Fig. 24.3 : The industrid processes for the
This can be achievedby strong aerationor by production of citric acid.
spargingwith pure Or. lt has been observedthat
s uddenint er ru p ti o ni ns O , s u p p l y(a so c c u rsduri ng
powerbreakdovvns) causedrasticreductionin citric wheat bran or pulp from sweetpotato starchare
acid productionwithout harmingthe growthof the used,as cul turemedi a.The pH of the medi umi s
orSanrsm. adlusted to 4-5, and then sterilized.Now the
inoculumin the form of sporesol A. nigeris spread
Nitrogen source as l ayers(3-6cm thi ckness) and i ncubated at 28" C .
The growthof the organisms can be accelerated by
Ammonium salts, nitrates and urea are the
the additionof cr-amylase. Solid-state fermentation
nitrogensourcesused in the media for citric acid
takes about B0 to 100 hours for maxirrtal
pr oduc t ion.Al l th e th re ec o mp o u n d sa re e qual l y
productionof citric acid.At the end of the process,
good sources,as long as they do not adversely
citric acid can be extractedinto hot water and
effectthe pH of the medium.lf molasses are used
i sol ated.
for nutrientsupply,additionof extranitrogensource
is not required. However, some workers have
Liquid surface fermentation
shown that exogenousaddition of ammonium ions
stimulates citric acid production. Surfacefermentationusing liquid as nutrient
medium is the oldest method for citric acid
PRODUCTION PROCESSES producti on.l t i s sti l l i n use due to a si mpl e
F O R CI T RI C A C ID technology, low energy costs and h igher
reproducibility.Further,the interferenceof trace
There are two processes by which citric acid
metal sand di ssol ved O, tensi onare mi ni mal .The
can be industriallyproduced-the surfaceprocess
labour costs are however, higher since the
and submergedprocess(Fig. 24.3).
manpowerrequirements are more for cleaningthe
The surfaceprocess: This is characterized by systems. About 20% of the citric acid in the world
growing rhe microorganismsas a layeror a film on is producedby surfaceprocesses.
a surface in contact with the nutrient medium,
The nutrient supply for surfacefermentation
which may be solid or liquid in nature.Thus,the
normally comes from beet molasses. The
surfaceprocesshas supported-growth systems.
fermentati on i s usual l ycarri edout i n al umi ni um
T he s ubme rg e dp ro c e s s : In th i s c a s e , the trays fi l l ed w i th steri l e nutri ent medi um. The
organismsare immersedin or dispersedthroughout inoculumin the form of sporesis sprayedover the
the nutrient medium. There are two types of medi um.A steri l eai r i s passedfor suppl yi ngO, as
submerged fermenters (bioreactors) stirred well as cooling. The temperatureis maintained
bioreactors and airlift bioreactors. around 30"C during fermentation.As the spores
germinate (that occurs within 24 hours of
SURFACE PBOCESSES i nocul ati on),a l ayerof mycel i umi s formedover
the medi um.The pH of the nutri entmedi umfal l sto
Solid surface fermentation
less than 2, as the mycelium grows in size and
Surface processesusing solid substratesare forms a thick layer on the surfaceof the nutrient
particularlycarriedout in lessdevelopedareasof solution.The fermentationis stopped after 7-15
rcme Asiancountries.The solid substrates such as days.

)cr=:,hnology[21]
322 B IOTE C HNO LO CY

T h e mv c e l i u m a n d n u tri ent sol uti on are

6
separated. The mycelium is mechanicallypressed o
+F
and thoroughlywashedto obtainmaximumamount l6
of citric acid. The nutrientsolutionis subjectedto t-
processing for the recoveryof citric acid. The final lo
IR
yield of citric acid is in the rangeof 0.7-0.9of per _o
6d
fram of sugar.
Ep
SUBMERGED PROGESSES
Around80% of the world'ssupplyof citric acid
is producedby submergedprocesses. This is the
65
6 100 2o0 300
most preferredmethod due to its high efficiency -----*
time(hours)
Cultivation
and easy automation. The disadvantagesof
submerged fermentation are- adverseinfluenceof Fig. 24.4 : A diagrcmmatic representation of citric
tracemetalsand other impurities,variationsin O, acid (-) production along"with sucrose (-) and
tension, and advanced control technology that bionass (- - -) concentration in rclation to time.
requireshighly trainedpersonnel.
Two types of bioreactorsare in use- stirred
hydrocarbons). The citric acid yield is betterfrom
tanks and aerated towers. The vessels of the
hydrocarbonscomparedto sugarsi.e. 145"h of
bioreactorsare made up of high-qualitystainless
citric acid from paraffin.The mostcommonlyused
steel.The spargingof air occursfrom the baseof
organismis Candidalipolytica.The fermentation
the fermenter.
can be carried out in batch, semi-continuous or
The successand yield of citric acid production conti nuous modes.The pH shoul dbe kep tabove5.
ma i n l yd e p e n do n th e s tru c tu re of mycel i um.The
The maj or l i mi tati onsof ci tri c aci d pr oduct ion
m y c e l i u mw i th fo rk e d a n d b ul boushyphaeand
from alkanesare-very low solubilityof alkanes
brancheswhich aggregateinto pelletsis ideal for
and increasedproductionof unwanted isocitrrc
citric acid formation.On the other hand, no citric
aci d.
acid productionoccurs if the mycelium is loose
a n d fi l a m e n to u s w i th Ii mi ted branches. A n RECOVERY OF CITRIC ACID
adequatesupplyof O2 QO-25"/' of saturationvalue)
The stepsfor the recoveryof citric acid either
is requiredfor good productionof citric acid. The
from surface process or submergedprocess are
ideal aerationrate is in the rangeof 0.2-1 vvm
comparable(Fig.2a.fl. The recoverystartswith the
(volume/volume/minute).
filtration of the culture broth and washing of
The submerged fermentershavethe prclblemof mycel i urn(w hi chmay contai nabout10% of cit r ic
foam formationwhich may occupy about 1/3rd of acid produced). Oxalic acid is an unwanted
the bioreactor.Antifoamagents(e.g.lard oil) and byproductand it can be removedby precipitation
mechanicalantifoamdevicesare used to prevent by addi ngl i me at pH < 3.The cul turebrot his t hen
fo a mi n g . subjectedto pH 7 2 and temperature70-90"C for
Nutrientconcentrationis very importantin the preci pi tati ng ci tri c aci d. For further pu r if icat ion,
i n d u s tri apl ro d u c ti o no f c i tri ca ci d.A di agrammati c ci tri c aci d i s di ssol vedi n sul furi caci d ( calcium
representation of sucrose,citric acid and biomass sulfateprecipitateseparates). The final steps for
concentrationwith respectto cultivationtime rs citric acid recovery are- treatment with activated
shown in Fig. 24.4. lt is estimatedthat under charcoal, cation and anion-exchangers and
o p ti m a lc o n d i ti o n si,n a b o u t2 50-2B Ohours,100- crystallization. Citric acid monohydrate formed
11Odl of citric acid is obtainedfrom 140 gl of below 36"C is the maincommercialproduct.Above
sucrosewith a biomass(dry weight)o{ B-12 g/1. 40" C ,ci tri caci d crystal l i zes i n an anhyd r ous f or m .
The degree of purity of citric acid produced
PRODUCTION OF CITRIC ACID FROM dependson the purposefor w hi ch i t i s r equir ed.
ALKANES For instance,pure forms of citric acid are needed
Both yeastsand bacteriacan be usedfor citric for usein food preparations, while for industrialuse
acid production from n-alkanes (Cs-Cz: it can be crude form.
 o
f-

-o
5

Air tn F
-
L
Nutrient
medium
(raw material)

Surfacefermentation
Air in

C)
c
(g
-c
c)
I
c
o
6
()

g)
or submerged processes N
Fig. 24.5 : Flow chart for industrial praduction of citric acid by surface GI
 324 BIOTECHNOLOCY

(A)
POQ , Glucbse',, ,
rdehydrsgenase Heo
(bacteria)
D-Glucose D-Gluconolactone
S Gluconicacid
Lactonase
(fungi)
(B)

Fig. 24.6 : Biosynthesisof gluconic acid (A) rn Gluconobactersuboxidans(B) rn Aspergillusniger.

(PQQ-PyrroIoquinoline quin one\

Principle of production : The enzymatic

reactions for the formation of gluconic acid
in Cluconobacter suboxidans (bacteria) and
Cluc onic ac id c an be pr od u c e d b y s e v e r a l Aspergillusniger (fungus)are depicted in Fig. 24.6.
b ac t er ia and f ungi. Cluc os e, on a s i m p l e d i r e c t
dehydrogenation,forms D-gluconolactonewhich is In bacteria,intracellular glucoseis convertedto
I then converted to gluconic acid. gl uconi c aci d. A membranebound
extracel l ul ar
I
enzyme, glucose dehydrogenase utilizes
Applications of gluconic
pyrroloquinoline quinone (PQQ) as coenzyme ano
acid
I
.l converts glucose to 6-D-gluconolactonewhich
I . Cluconic acid is used in the manufactureof
undergoes (spontaneous
hydrolysis or enzymatic;to
metals, stainlesssteel and leather,as it can remove
form gl uconi caci d.
the calcareous and rust deoosits.
As regards fungal production, glucose is
2. lt is usedas an additivetofoods and beverages.
oxidized by the extracellularenzyme glucose
3. Cluc onic ac id has p h a r m a c e u t i c a r oxidase to form 6-D-gluconolactone,which
a pplic at ions- c alc ium and ir on t h e r a p y . subsequentlygets convertedto gluconic acid bv
4. Sodium gluc onat e is us ed a s a s e q u e s t e r i n g Iactonase. Clucoseoxidaseis an inducibleenzvme
agent in many detergents. that can be induced by high concentrations of
glucose,and at pH above 4. lt is believedthat
5. Cluconate is used for desizing polyester or
HrO, produced by glucoseoxidase acts as an
polyamide fabrics.
antagonist against other microorganisms
6. lt is ut iliz ed in t he m anu f a c t u r e o f h i g h l y (anti mi crobi al
acti vi ty)i n the surroundi ngs.
resistant(to frost and cracking) concrete.
Production process for gluconic acid
Microbial production of gluconic acid
Submergedprocesses, by employingeither A.
Cluconic acid can be produced by a wide variery niger or G. suboxidans,are used for producing
of prokaryotic and eukaryotic microorganisms. gl uconi c aci d. The cul ture medi um c ont ains
Bacterial
glucose at a concentrationof 12-15% (usually
species of the genem-
C I u co n oba cte r, Acetob acte r, Pseu d om o n as, Vib r i o. obtainedfrom corn). The fermentationis carried
out at pH 4.5-6.5,and at temperature 2B-30"Cfor
Fungal species of the genera-Aspergillus, a periodof about24 hours.Increasing the supplyof
Penici II i um, G liocladium. O, enhances gl uconi caci d yi el d.
 O F O R GA N ICA C ID S
C hapt er24 : M IC R OB IALP R OD U C T IO N 325

Biote ch no log is t s ex ploit t he f er m ent at i o n Glucose

pfocess of gluconic acid for the production of the
en zyme g lucose ox idas e, bes ides pr oduc i n g
ca lciu m glu co na t e and s odium gluc onat e.

Chemical synthesis of gluconic acid : By Glyceraldehyde

3-phosphate Labticacid
emp loying the im m obiliz ed enz y m e gluc o s e
oxida se , g lucon ic ac id c an als o be pr oduc ed.

1, 3-Bisphosphoglycerate
Lactic acid occurs in two isomeric forms i.e.
L(+) and D(-) isomers, and as a racernic mixture Fig. 24.7 : An outline of the pathway (glycolysis) for
(DL-la ctic acid). The is olat ion of lac t ic ac id f r o m the biosvnthesis of lactic acid.
milk wa s d on e in 1798. lt was t he f ir s t or ganic ac i d
produced by microorganisrnsin 1880. Today, lactic
acid is competitively produced both by Lactobacillus sa are used for lactic acid
micro bio log ica l and c hem ic al m et hods procjuction. However, there are variations in the
substratesutilised as indicated below.
Applications of lactic acid
L. delbrueckli l
The re are d iff er ent gr ades of lac t ic ac id m ain l y . I Clucose
L- letcnamannt )
based on the percentageof lactic acid. The grades
and their applicatiorrsare giverr in Table 24.2. L. bulearicus l
I Whey (lactose)
L. netveilt )
Microorganisms for production of
L. lactis - Maltose
lactic acid
L. amylophilus - Starch
Lactic acid producing bacteria are broadly
L. pentosus - Sulfitewaste liquor
categorized into two types.
Biosynthesis of lactic acid : The synthesis of
Heterofermentative bacteria-produce other
lactic acid occurs through glucose oxidation by
byproducts, besides lactic acid, and therefore are
glycolysisto produce pyruvate which on reduction
not useful for industrial production of lactic acid.
gives laciic acid. The reducing equivalents
These bacteria are employed in food or feed
( N A D H ++ H +) p r o c i u c e d d u r i n g t h e o x i d a t i o n o f
oreservation.
glyceraldelyde 3-phosphate are utilised by the
Homofermentative bacteria-specialised lor enzyme lactate dehydrogenase to form lactate
exclusive oroduction of lactic acid and therefore Gig. za,V. Most of the lactic acid producing
a re su itab le fo r indus t r ial pur pos e. m i c r o o r g a n i s n r sn o r m a l l y p r o c i u c eo n l y o n e i s o m e r
of lactic acid L(+) or D(-). However, some bacieria
which usually occLrras infection can form racenric
Tastl 24.2 Commerclalgrades of lactic acid nrixture.
along with their applications
Production process for lactic acid
Grade (% lactic acid) Application(s)
T h e f e r m e n t a t i o n m e d i u m c o n t a i n s 1 2 - - 1 5 'h o f
grade
Technical Estermanulacture, glucose, nitrogen and phosphate containing salts
(20-50%) industry
textiie anci nticronutrients.The orocess is carried out at
Foodgrade (sour
Foodadditive oH 5.5-6.5 and temoerature 45--50"C for about 75
(>80%) flouranddough) h o u r s . C e n e r a l l y , t h e s t r a i n s o p e i - a t i n ga t h i g h e r
temperature (45-60'C) aie preferred, since it
grade
Pharmaceutical lntestinal
treatment
r e d u c e st h e n e e d f c r m e d i u m s t e r i l i z a t i o r rA . s tne
(>90%) (metalionlactates) lactic acid is oroduced, it has to be removed since
326 B IOTE C H N O LO CY

Microorganisms used for production

Fermentationbroth
of acetic acid
J The commercialproductionof acetic acid is
Heatedto dissolvecalciumlactate
carried out by a special group of acetic acid
+
I bacteria,which are divided into two genera.
Additionof H2SOa Gluconobacterthat oxidizesethanolexclusively
(removescalciumsulfate)
to aceticacid.
+
I Acetobacterthat oxidizesethanol first to acetic
Filterandconcentrate
acid, and then to CO, and HrO. These over-
j oxidizersare Cram-negativeand acid tolerante.g.
Additionof hexacyanof
errant A. aceti, A. peroxidans,A. pasteurianus.
(to removeheavy metals)
Biosvnthesis of acetic acid : Acetic acid is a
+
I productof incompleteoxidationof ethanol.Ethanol
Purification is first oxidized by alcohol dehydrogenaseto
(ionexchange) acetaldehydewhich then gets hydratedto fornr
+
I acetaldehyde hydrate.The latteris then actedupon
by acetaldehyde dehydrogenase to form aceticacid
Concentration
Gig" 2a.e).
+
I
LACTIC
ACID Production process for acetic acid
For every moleculeof ethanol oxidised,one
Fig. 24.8 : Flow chart for recovery of lactic acid moleculeof acetic acid is produced.Thus, high-
from termentation broth. yielding strainscan produce 11-12% acetic acid
trom12o/o alcohol.Foroptimalproduction, adequate
supplvof oxygenis very essential. InsufficientOr,
it is toxic to the organisms. Thiscan achievedeither coupled with high concentrationof alcohol and
by a c o n ti n u o ucsu l tu rete c h n i q u eo r by removalof
aceticacid resultin the deathof microorganisms.
lactic acid by electrodialysis. Theoretically, every
moleculeof glucoseformstwo moleculesof lactic Surfacefermentation or submerged fermentation
acid. About 9O"/,oi theoreticalyield is possibleIn processes can be carried out to produce acetic
fermentation industry. L(+) Lactic acid is acid. Trickling generation process, a type of surface
predominantlyproduced.The outline of the steps fermentation, is very commonlyused.
involvedin the recoverylactic acid is depictedin Recovery: The aceticacid producedis clarified
Fig. 24.8. by filtrationand then subjectedto decolourization
by Ko(FeCN)u.

Production of vinegar
Vinegaris an aqueoussolutioncontainingabouf
4% by volume aceticacid and small quantitiesof
The productionof acetic acid, in the form o{ alcohol,salts,sugarsand esters.lt is widely usedas
vinegar(usedas a refreshing
drink),from alcoholic a flavouringag,entfot processedliquid foods such
liquidshas been known for centuries. as saucesand ketchuos-

NADH+ H+ NADP+ NADP + H+

t'oa
cH3cH2oH cHscHo , cH3cH(oH)2 cH3cooH
Ethanol Acetaldehyde Acetaldehyde Aceticacid
hydrate

Fig. 24.9 : Biosynthesis of acetic acid from ethanol.

Chapter24 : MICROBIALPRODUCTIONOF ORGANICACIDS 327

Ervviniasp
2, s-Diketo- Acetobactersp
D-Glucose Glucuronic
acid
gluconic
acid Gluconobactersp
I c"tutyti"
reduction
J

Glucuronolactone
2-KetoL-
gluconicacid

:
L-Gulonolactone
2-Keto-Lgulonicacid I

ACI D
L -ASCOBBI C I
+
I
l.^
Enolformof 2-ketoL-gulonic
acid i L-uuronoraclone
dehydrogenase
I
l, e " id ,
Jtreatment
J
L.ASCORBICACID ACID
L-ASCORBIC
Reichstei n-Grussner sy nthesis l I

Fig. 24.10 : Pathways for the commercial production of ascorbic acid. (A) Two-step fermentation process
(B) Reichstein.Grussner synthesis (C) Production via L-gulonolactone.

The startingmaterialsfor vinegarproductionare in the humanbody.VitaminC alsoprotects the body

wine, whey, malt (with low alcohol content).
Vinegarproductioncan be carried out either by
surface process (trickling generator) or by
submerged process.
Surfaceprocess: The fermentationmaterialis
sprhyedover the surfacewhich tricklesthroughthe
shavingsthat contain the acetic acid producing
bacteria.The temperatureis around 30oCon the
upper par t w h i l e i t i s a ro u n d3 5 " C o n th e l ow er
part. Vinegaris producedin about 3 days.
Submerged process : The fermentation
bioreactors are made up of stainless steel.Aeration
is done by a suction pump from the top. The
productionrate in the submergedprocessis about
10 t im eshi g h e rth a n th e s u rfa c ep ro c e s s .
L-Ascorbic acid is the commonlyusedchemical
namefor the watersolublevitaminC. This vitamin
forms a redox systemand participatesin several
biologicalprocesses. lt is intimatelyinvolvedin the
biosynthesisof collagen,the mostabundantprotein
againstcarcinogenicnitrosaminesand free radicals.
The deficiencyof ascorbicacid causesscurvy.
Applications of ascorbic acid
Becauseof the wide rangeof physiologicaland
beneficial functions of ascorbic acid, its
commerci al producti on assumes si gni fi cance.
Vitamin C is mainly used in food and pharma-
ceutical industries.
Industrial production of ascorbic acid
Ascorbicacid is commerciallyproducedby a
combinationof severalchemical steps,and one
reaction of biotransformation brought out by
microorganisms. This process is referred to as
Reichstein-Grussner synthesis (Fig. 24.10A.
D-Glucose is first converted to D-sorbitol.
Oxidationof D-sorbitolto L-sorboseis carriedout
by Acetobacterxvlinum or A. suboxvdans(The
enzyme being sorbitol dehydrogenase).A
submerged bioreactorfermentation processis ideal
for thi s reacti on. l t takes about' 24 hours at
temperature30-35"C. Sorboseby a couple of
chemical reactionscan be finally convertedto
 324 B IOTE C HNO LO CY

Glucose Itaconicacid
MEDIUM
CYTOPLASM Calcium
Glucose Itaconicacid
^^l
v v 2\ | Itaconic
) Acotinicacid acidoxidase
decarboxylase
YY I
Pyruvate Pyruvate Cls-Aconiticacrd
Itatarta-ric
acid

Fig. 24.11 : Biosynthesis of itaconic acid-an outline of metabolic pathway-

L- as c or bicac id. Nor m ally , about 1 0 0 g o f a s c o r b i c process(Fig. 24.10A) has been reduced to one. The
a c id is pr oduc ed f r om 200 g o f g l u c o s e i n genetically engineered Erwinia cells were able to
Reicl'rstein-Crussner synthesis. convert D-glucose directly to 2-keto-L-gluconic
Two-step fermentation process : In th is, a c i d . T h i s i s c e r t a i n l y a d v a n t a g e o u s s i nce th e
D- gluc os e is c onv er t ed t o 2, 5 - d i k e t o g l u c o n i c metabolic caoabilities of two different micro-
acid by Erwinia, Acetobacter or Gluconobacter sp. o r g a n i s m sc o u l d b e c o m b i n e d i n t o o n e o rg a n i sm .
ln the second step, Corynebacterium sp converts 2, However, the yield of ascorbic acid by the hybrid
5 - dik et ogluc onic ac id t o 2- k et o - L - g i u c o n i c acid, strain rvas very low. Scientistsare now trying to
(Fig. 24.10A). lt is also possible to invclve Bacillus a l t e r c e r t a i n a m i n o a c i d s i n 2 - 5 d i k e t o g l u c on i ca ci d
nnegateriunl for converting L-sorbose to 2-keto-L- reductaseand increasethe catalvtic activitv of this
g luc onic ac id. The lat t er , by c h e m i c a l r e a c t i o n s , e n z y m e .
be converted to ascorbic acid.
^" nfiltrt$:an
/ilfrf'" F _ * -,F
\ Production via L-gulonolactone
Ascorbic acid can also be synthesized via-
gulonolactone which can be directly converted to Itaconic acid is used in plastic industry, paper
L-ascorbic acid by the enzyme L-gulonolactone industry and in the manufacture of adhesives.
dehydrogenase(Fig. 24.1 OCt.
I t a c o n i c a c i d c a n b e c o m m e r c i a l l y p r o d uce d b y
Direct production of ascolbic acid by Aspergillus itoconicus and A. terreus. The
fermentation biosynthesis of itaconic acid occurs by way of
Krebs cycle. The metabolite cis-aconitic acid
Several workers are trying to produce ascorbic
(formed from citric acid) undergoesdecarboxylation
acid directly from glucose. Microalgae o{ Chlorella
catalysedby the enzyme cls-aconiticdecarboxylase
have shown some promising results, although the
(Fig. 24.11). ltaconic acid is oxidised to itatartaric
yield is very low.
acid by itacorric acid oxidase. Tl-risenzyme has to
Genetic engineering for ascorbic acid b e i n h i b i t e d f o r a m a x i m u m y i e l d o f i t a c o ni c a ci d .
production This can be achieved by adding calcium.

Biotechnologistshave been successfulin cloning Batch submerged fermentation process rs

and expressing the gene for 2, S-diketogluconic commonly used for itaconic acid production. The
acid reductase of Corynebacterium sp into Erwinia yield is around 75"h of the theoretical calculation
herbicola. By cloing this, the two step fermentation when the medium contains 157o sucrose.
 A ntibioticsare the chemical substances that can biochemicalbasiswas not known. Ihe penicillin
A *ru microorganismsor inhibit their growth, discovery of Fleming has revolutionised antibiotic
and arethereforeusedto fight infectionsin humans research.
or animals.Mostof the antibioticsare producedby
m ic r oor ga n i s m(is.e .p ro d u c to f o n e o rg a ni smthat W i de range ol anti bi oti cs
can kill other organism).Certain semi-synthetic
Antibiotics are the most important class of
ant ibiot ic sa re th e c h e mi c a l l ym o d i fi e d natural
pharmaceuti cal s produced by mi crobi al
antibiotics.
biotechnological processes.
They are the products
Antibioticshaveundoubtedly changedthe world of secondary metabolism.
we live in, and have certainlycontributedto the Around 10,000differentantibioticsare known,
e th e h u ma nl i fe -s p a nT.h i si s ma i nl ydue and 200-300 new
inc r eas in
onesare beingaddedeachyear.
to the fact that severallife-threatening infectious Most of
these antibioticsare not of commercial
diseases could be conveniently cured by importance due to various reasons-toxicity,
adm inis t r a ti oonf a n ti b i o ti c s . ineffectiveness or high cost of production.There
are around 50 antibioticswhich are mosi widely
useo.
ln Table 25.1, a selected list of important
antibiotics,their propertiesand the producing
A brief history of antibioticsalong with the organi smsi s gi ven.
m ic r oor ga n i s m sp ro d u c i n g th e m , a n d thei r
applicationsare given hereunder. Broad spectrum antibiotics : They can control
the growth of several unrelatedorganismse.g.
History of antibiotic discovery tetracyclines,
chloramphenicol.
Narrow spectrumantibiotics: They are effective
I t was i n 1 9 2 8 , A l e x a n d e rF l e m i n gmade an
agai nstsel ectedspeci esof bacteri ae.g. peni ci l l i n,
accidental discovery that the fungus Penicillium
streptomycin.
notatum produced a compound (penicillin)that
selectivelykilled a wide rangeof bacteriawithout
Mi croorgani sms produci ng anti bi oti cs
adversely affecting the host cells. There are
recordsthat in some pafts of Europe(in 1908) A great majority of antibioticsare produced
extractsof moldy breadwere appliedto woundsor by actinomycetes particularly of the genus
abrasionsto prevent infections, although the Streptomycese.g. tetracyclines,actinomycin D.

329
 330 B IOTE C H N OL O G Y

Besides serving as antimicrobial agents, there are

several other applications of antibiotics.
antibiotics along with pyoducingorganisns
Antitumor antibiotics : There are a selectedfew
Antibiotic activity Producing antibiotics that are in use for control of cancer
specturm and antibiotic microorganism g r o w t h , a l t h o u g h w i t h a l i m i t e d s u c c e s s e.g .
actinomycin D, mitomycin C.
Antibacterial
Penicillin
G Penicillium
sp Food preservative antibiotics : Certain
(l onhalnennr in
Acremonium sp a n t i b i o t i c s a r e u s e d i n c a n n i n g i n d u s t r y ( e .g .
Streptomycin Streptonycessp chlortetracycline),and for preservationof fish, meat
Tetracycline Streptonycessp and pclultry (e.g. pimaricin, nisin). The use of
antibiotics in food preservationis usually under the
Chloramphenicol Cephalosporiunsp
control of the Covernments.
Bacitracin Bacillus
sp
Antibiotics used in animal feed and veterinary
Antitumor
medicine: T i l l s o m e t i m e a g o , a n t i b i oti cs
Actinomycin
D Streptomyces
sp (penicillins, tetracyclines,erythromycins)were very
Mitomycin
C Streptomyces
sp w i d e l y u s e d i n p r o c e s s i n go f a n i m a l f e e d s . S u c h a n
Bleomycin Streptonyces
sp i n d i s c r i m i n a t e u s e r e s u l t e d i n t h e d e v e l o p m e nt o f
Adriamycin Streptonyces
sp a n t i b i o t i c r e s i s t a n c ei n a n i m a l s a n d h u m a n s .A n e w
Daunomycin Streptonyces
sp classof antibiotics have been developed for specific
u s e i n a n i m a l f e e d e . g . e n d u r a c i d i n , t y l o si n .
Antifungal
Likewise, specific antibiotics have been developed
r Gi'iseofulvin Penicillium
sp f o r e x c l u s i v e u s e i n v e t e r i n a r y m e d i c i n e e.g .
Food preservative h y g r o m y c i n B , t h e o s t r e p t o n ,s a l i n o m y c i n .
Natamycin Streptomyces
sp
'It
Antibiotics for control of plant diseases : In
Nisin Streptomyces
sp recent years, several antibiotics have been
I
I t developed for exclusive use to control plant
I
Antiprotozoal
d i s e a s e se . g . b l a s t i c i d i n , t e t r a n a c t i n ,p o l y o x i n .
Daunorubicin Steptonyces
sp
Antituberculosis Antibiotics as tools in molecular biology : Some
o f t h e a n t i b i o t i c s c a n s e l e c t i v e l y i n h i b i t c e r ta i n
Refamycin Nocardia
sp
b i o l o g i c a l r e a c t i o n s a t t h e m o l e c u l a r l e v e l . T he se
Antiamoebic antibiotics do in fact serve as tools for exploring the
Tetracycline Streptonyces
sp knorvledgeof life sciences.Thus, certain antibiotics
Fumagillin Aspergillus
sp have been used to obtain some important
information on DNA replication, transcription and
translation.
The bacteria other than actinomyces also produce
ce rtain ant ibiot ic s e. g. bac it r ac in.

Amorrg the fungi, the two groups Aspergillaceae

and Monilialesare inrpodant for antibiotic prodLrction
e.g , penic illin,c ephalos por in,gr is eof u l v i n

APPLICATIONS OF ANTIBIOTICS
Ant ibiot ic s ar e par t ic ular ly im p o r t a n t a s
antimicrobial agents for chemotherapy. A large
n umb er of bac t er ial dis eas es hav e b e e n b r o u g h t
u nd er c ont r ol bv us e of ant ibiot ic s .T h e s e i n c l u o e
pneumonia, cholera, tuberculosisand leprosy. The
antifungal antibiotic griseofulvin has controlled the
de bil it at ingf ungal s k in dis eas ess uc h a s r i n g w o r m .
The commercialoroductionof antibioticsis a
hi ghl y profi tabl ei ndustryw orl dover.A nnualsales
w i l l run i nto severalbi l l i onsof dollar s
of anti bi oti cs
with an annualgrowth potentialof about 10%.
Antibiotics may be produced by microbial
fermentation, or chemical synthesis, or a
combination of both. For certain antibiotics,the
Chaot er25 : MIC R OB IALP R OD U C T IO N
O F A N TIB IOTIC S 331

basic molecule is produced by fermentation and its

therapeutic value can be increased by chemical
mo dificatio ns .The c os t inv olv ed in pr oduc t i o n a n d
chemical modifications, besidesthe efficacy of the
a ntib iotic is v er y im por t ant in it s m anuf ac tu r e .

Biotechnologists continue their efforts to

increase the fermentation yield and recovery
processesto produce pure antibiotics. : Lactamring
i 6-Aminopenicillanlc
acid
The industrial production of selectedantibiotics
is b riefly d es c r ibed in t he f ollowing pages . R-group Name of the
penicillin
Biosynthetic penicillins

/-\
Penicillin
G
(benzylpenicillin)
P enic illi n sa re a g ro u p o f p -l a c ta mc o ntai ni ng \_/
bac t er ic idaal n ti b i o ti c sBe
. i n gth e fi rs t a m o ngthe
ant ibiot ic s to b e d i s c o v e re d ,p e n i c i l l i ns are
historicallyimportant.The structuresof important
ll-"-cH,-co Penicillin
V

s y nt het ic a n d s e m i -s y n th e ti cp e n i c .i l l i nsare \_/

depic t edin Fi g .2 5 .1. T h e b a s i cs tru c tu re o f al l the Semi-synthetic pen icillins
penic illins c o n s i s ts o f a l a c ta m ri n g and
/-\
a thizolidine ring fused together to form Ampicillin
6- am inooe n i c i l l a nai c i d . \ )FcH-co-
\-/ NHZ

Action of penicillins
HO cH-co- Amoxicillin
Natural penicillins(penicillinsV and C) are I
NHe
effective against several Gram-positive bacteria.
T hey inhib i t th e b a c te ri a l c e l l w a l l (i .e.
peptoglycan) synthesis and causecell death.Some Oxacillin
persons(approximately 0.5-2% of population)are
aller gict o p e n i c i l l i n .
Natural penicillins are ineffective against
microorganisms that produce pJactamase, since
t his enz y m e c a n h y d ro l y s e p e n i c i l l i ns e.B . Cloxacillin
Staphylococcus aureus. Several semi-synthetic
penic illinst h a t a re re s i s ta nto t B -l a c ta m a se have cHs
been developedand successfullyused againsta
large number of Cram-negative bacteria.
Clox ac illina, m p i c i l l i nfl, o x a c i l l i na n d a z l o ci l l i nare
s om eex am p l e o s f s e mi -s y n th e tipce n i c i l l i n s. These
co- Floxaciflin
are quite comparablein actionto cephalosporins.
F r omt he h u g eq u a n ti ti eosf p e n i c i l l i n ps ro duced cHs
by ferrnentation,about 40"/" are used for human
healthcare, 15o/o for animalhealthcareand45o/o for
Ticarcillin
t he pr epar a ti oonf s e mi -s y n th e tipce n l c i l l i ns.

Organisms for penicillin production

ln the early days,Penicilliumnotatum was used Fig. 25.1 : Structures of important penicillins.
f or t he la rg e -s c a l ep ro d u c ti o n o f p e n i ci l l i ns.
 332 BIOTECHNOLOCY

Currently, Penicillium chrysogenum and its L-c-Aminoadipicacid

improvedmutant strainsare preferred.Previously,
lr.
the penicillin productionused to be less than 2 L-cvsteine '='-,r
units/ml,and with the new strains,the production nl L-Lysine
runs into severalthousandsof units/ml.One of the J
high yielding strainsr,r,isQ176 is preferredby L-a-Aminoadipylcysteine
severalpenicillinmanufacturers.
L-Val,ne
Genetic engineering for improved penicillin \/ 'l
pr odu c ti o n : S o m e o f th e g e n e s i nvol ved i n J
penicillin biosynthesisby P. chrysogenumhave cr-L-Am
inoadipylcysteinylvali
ne
been identified. Cenetic manipulations were (tripeptide)
carried out so as to substantiallyincreasethe
penic i l l i n p ro d u c ti o n .F o r i n s ta n c e ,extra genes I cv"t*t
codingfor the enzymescyclaseand acyltransferase t
have been insertedinto C. chrysogenum. J
lsopenicillinN
Biosynthesis of penicillin
Phenylacetyl-,I
L- a -A m i n o a d i p i c a c i d c o m b i nes w i th coA ) nqnrrarnrurwa
L-cysteine,and then with L-viline to form a s-Ar{A+CoAYI
t r ipep ti d en a m e l y c x -L -a mi n o a d i p yl cystei nyl val i ne. J
This compound undergoescyclization to form PENICILLIN G
isopenicillinwhich reactswith phenylacetylCoA
(catalysed by the enzyme acyltransferase) to Fig. 25.2 : Biosynthesis of penicillin by Penicillium
pr odu c ep e n i c i l l i nC (b e n z y lp e n i ci l l i n).In thi s chrysogenum (a-AAA - a-Amino adipic acid;
reaction,aminoadipicacid gets exchangedwith CoA-Coenzyme A.
phenylaceticacid (Fig. 25.2).
Regulation of biosynthesis: Some of the sugar like lactose. The concentrations of
bioc he m i c are l a c ti o nfo
s r th e s y n th e siof s peni ci l l i n phosphateand ammoni aal so i nfl uencepeni ci llin
and lysine are common. Thus, L-o,-aminoadipicsynthesis.
acid is a common intermediate for the synthesis of
penic i l l i n a n d l y s i n e . T h e a v a i l abi l i ty of PRODUCTION PROCESS OF PENICILLII.I
am ino a d i p i c a c i d p l a y s a s i g n i fi cantrol e i n
An outline of the flow chart for the industrial
r egula ti n thg e s y n th e s iosf p e n i c i l l i n .
productionof penicillin is depicted in Fig. 25.3.
P en i c i l l i nb i o s y n th e s iiss i n h i b i te dby gl ucose The lyophilizedcultureof sporesis cultivatedfor
through catabolite repressiorr.For this reason, inoculum developmentwhich is transferredto
penic i l l i n w a s p ro d u c e db y a s l o w l y degraded prefermenter,and then to fermenter.

Nutrients

Lyophilized Inoculum Prefermenter Ferm

culture

Fig. 25.3 : An outline of the flow chaft for penicillin fermentation.

Chapt er2 5 : MIC R OB IALPR O D U C T IO N
OF A N TIB IOTIC S 333

40 hours with a doubling time of 6-8 hours. After

the grornth phase is stabilized, the penicillin
production exponentially increases with
appropriate culture conditions. The penicillin

iI
il:11.,'""
phase can be extendedto 150-180

o Recovery of penicillin
(U
E
c
q) As the fermentationis complete, the broth
c contai ni ngabout 1% peni ci l l i n i s processedfor
O extraction.The myceliumis removedby filtration.
Penicillinis recoveredby solvent(n-butylacetate or
methylketone) extraction at low temperature
(< 10' C ) and aci di c pH (< 3.0).B y thi s w ay, the
chemi caland enzymati c(bacteri alpeni ci l l i nase)
degradati ons of peni ci l l i ncan be mi ni mi zed.
The peni ci l l i ncontai ni ngsol venti s treatedw i th
Fig. 25.4 : Penicillin production in relation to activated carbon to rernove imourities and
substrates utilization and biomass formation.
pi gments.P eni ci l l i ncan be recoveredby addi ng
potassiumor sodium acetate.The potassiumor
sodi umsal tsof peni ci l l i ncan be furtherprocessed
Penicillin production is an aerobic process ancl (i n dry sol ventssuch as n-butanolor i sopropano l)
therefore,a continuous supply of O, to the growing
to remove i mpuri ti es.The yi el d of peni ci l l i n i s
culture is very essential.The required aeration rate
around907o.
is 0 .5-1.0 v v m . The pH is m aint ained ar o u n d 6 . 5 ,
and the optimal temperature is in the range of As the water is totally removed,penicillinsalts
2 5-2 7"C. Penc illin pr oduc t ion is us uall y c a r r i e d can be crystallizedand dried under required
out by submerged processes. pressure.This can be then processedto finally
producethe pharmaceutical dosageforms.
The medium used for fermentation consists of
corn steep liquor (4-5% dry weight) and carbon
sou rce (us ually lac t os e) . An addit ion o f y e a s t
extract, soy meal or whey is done for a good supply
o f nitro ge n.Som et im es ,am m onium s ulf at ei s a d d e d
for the supply of nitrogen. Phenylacetic acid (or
phenoxyaceticacid) which servesas a precursorfor
pe nicillin bios y nt hes isis c ont inuous ly f ed . F u r t h e r ,
continuous feeding of sugar is advantageousfor a
g oo d yie ld of penic illin. The penic illin pr o d u c t i o n
profiles are depicted in Figs. 25.4 and Fig. 25.5.

It is estimated that approximately 10% of the E

C
metabolised carbon contributes to penicillin o
o
c
p rod uction, while 65% is uiilis ed t owar d s e n e r g y
supply and 25"h for growth of the organisms.The
e fficie ncyof penic illin pr oduc t ion c an be o p t i m i z e d
by adequate supply of carbon source. Thus, by
a dd ing g luc os e and ac et ic ac id, t he y ield c a n b e
increa se dby about 25% .

For efficient synthesisof penicillin, the grolvth

Fiq.25.5:Penicillin production in rclation to continuous
of the organism from spores must be in a loose
feeding of sugar, O, utilizatlon, and CO, formatian.
form and not as pellets.The growth phase is around
334 B IOTE C H N OLOCY

Penicillins C and H are the fermented products Several mutants of C. acremonium have been
obtained from the fungus Penicillium chrysogenum. developed for improved production of
cephalosporin. Mutants with defective sulfur
PRgDUCTTON OF 6.AM!NO metabolism or those with resistance to sulfur
PENIC I LLANI C ACI D analogs have high yielding capacity. Certain
Th e penic illins C and H ar e nr os t ly u s e d a s t h e regulatory genes of cephalosporin biosynthesis
starting materials for the production of several ( e . g . , i s o p e n i c i l l i n N s y n t h e t a s e )h a v e b e e n c l o n e d
synthetic penicillins containing the hasic nucleus a n d g e n e t i c m a n i p u l a t i o n sc a r r i e d o u t f o r i n c r e a s e d
namely 6-amino penicillanic acid (6-APA). About production of cephalosporins.
10 yearsago, only chemical methods were available
Biosynthesis of cephalosPorin
for hydrolysisof penicillins to produce 6-APA. Now
a days, enzymatic methods are preferred. The early stages of the biosynthetic pathway
for cephalosporin are the same as that for
lmm obiliz ed penic illin am idas esen z y m e s h a v e
penicillin synthesis(SeeFig. 25.2). As the tripeptide
been developed for specific hyclrolysisof penicillin (aminoadipylcysteinylvaline) is formed, it
C an d penic illin V. Penic illin s alt of ei t h e r C o r V p r o d u c e i s o p e n i c i l l i n N.
undergoes c y c l i z a t i o n t o
can be us ed f or hy dr oly s isby im m obili z e d e n z y m e
B y t h e a c t i o n o f e p i m e r a s e ,p e n i c i l l i n N i s f o r m e ci
system. The pH during hydrolysis is kept around
f r o m i s o p e n i c i l l i n N . T h e n , p e n i c i l l i n N g e ts
7-8, and the product 6-APA can be recovered by
converted to cephalosporin C by a three stage
b ring ing down t he pH t o 4. At pH 4 , 6 - a m i n o
reaction catalysed by three distinct enzymes
penicillanic acid gets precipitatedalmost completely
n a m e l y e x p a n d a s e , h y d r o x y l a s e a n d a c e tyl
in the oresenceof a water immiscible solvent.
transferase (Fig. 25.71.
In general, the enzymatic hydrolysis is more Regulationof biosynthesis: A low concentration
e fficie nt f or penic illin V t han f or p e n i c i l l i n C . of lysine promotes cephal<lsporin synthesis. The
Howe v er , penic illin C is a m or e v e r s a t i l e inhibitory effect of lysine at a higher concentration
compound, as it is required for ring expansions. can be overcome by adding L-aininoadipic acid.
The carbon sources that get rapidly degraded (e.g.
glucose, glycerol) reduce c e p h a l o s p or i n
production. Methionine promotes cephalosporin
T he p h a rm a c e u ti c aul s e s o f p e ni ci l l i ns are synthesis in C. acremonium, but has no influence
associated with allergic reactions in some on Streptomycetes.
indiv id u a l sT. o o v e rc o m eth e s ea l l e rg i cprobl ems,
PRODUCTION PROCESS
cephalosporins were developed. They have
OF CEPHALOSPORIN
improved stability againstB-lactamases, and are
more active against Cram-negative bacteria. The fermentation process concerned with the
Cephalosporins production of cephalosporin is similar to that of
are broadspectrumantibioticswith
low toxicity. p e n i c i l l i n . T h e c u l t u r e m e d i a c o n s i s t so f c o r n s t ee p
liquor and soy flour-based media in a continuous
The structures of different cephalosporins
feeding system. The other ingradients of the
are shown in Fig. 25.6. Basically cephalosporins
m e d i u m i n c l u d e s u c r o s e /g l u c o s e a n d a m m o n i um
have a p-lactam ring fused with a dihydrothiazine
s a l t s . M e t h i o n i n e i s a d d e d a s a s o u r c e o f s u l f u r.
ring
The fermentation is carried out at temperature
Organisms for cephalosporin 25-28"C and pH 6-7. fhe growth of micro-
production o r g a n i s m s s u b s t a n t i a l l y i n c r e a s e s w i t h g o o d Ot
a l t h o u g h d u r i n g p r o d u c t i o n p h a s e , O,
CephalosporinC was first discoveredin the s u p p l y ,
c o n s u m o tion declines.
cultures of fungus Cephalosporiurnacremonium
(later renamedas Acremoniumchreysogenum)and Cephalosporin C from the culture broth can be
this organismcontinuousto be used even today. recovered by ion-exchange resins, and by using
The other organismsemployedfor cephalosporin column chromatography.Cephalosporin C can be
production are Emericellopsissp, Paecilomycessp precipitated as zinc, sodium or potassiumsalt, and
and Streptomycessp. isolated.
 r 25 : M IC R O B IALPR O D U C T IO NO F A N TIB IOTIC S

p-Lactam
nng Dihydrothiazine
flng

R1 R2
Cephalosporin

7-Aminocephalo- -NHz -o-co-cH3

sporanicacid (7-ACA)*
Hztl-1--t-,A -o-co-cH3
co-
CephalosporinC
cooH

co- -cHg
Cephalexin NHz

:
/ \ .co-
Cefadroxil Ho-(\ -cHs
,r/{lrH,
\___-,

-cl
Cefaclor

N-:--N
Cefazolin
,l=- ,1,.--."o- -tyt\.,-
N_N

Fig. 25,6 : A selected list of cephalcsporins (*-Active nucleus of cephalosporins)'

Chemical synthesis of cephalosporin Recently, enzymatichydrolysisof cephalosporin

C to 7-ACA has been developed.This is mainly
I n r ec en ty e a rs ,b y u s i n g p e n i c i l l i nV a s the
carried out by two enzymes-D-aminoacid oxidase
starting material, chemical synthesis of (isolated from Trigonopsis variabilis) and
cephalosporin has becomepossible.This is being (source-Pseudomonas sp). Bio-
glutaryl amidase
done due t o l o w c o s to f p ro d u c ti o no f p e n i ci l l i n' havebeensuccessful in immobilizing
technologists
these two enzymesfor efficient and large scale
PRODUCTION OF of 7-ACA.
manufacture
T.AMINOCEPHALOSPOBANIC ACID
CephalosporinC
7-Aminocephalosporonic acid (7-ACA) is the
nucleusstructurepresentin all the cephalosporins. I o-o.,no acidoxidase
Cephalosporin C, produced by fermentation, can +
to form 7-ACA' I
be subjectedto chemicalhydrolysis amidase
I Glutaryl
This is tedious, and is associatedwith several + acid
7-Aminocephalosporanic
drawbacks.
336 B IOTE C H N OLOCY

L-Aminoadipicacid
I
_. l, L-Valine
L-Cysteine
Amirroglycosides are oligosaccharide (carbohy-
Y drate) antibiofics. They contain an aminocyclo-
J h e x a n o l m o i e t y w h i c h i s b o u n d t o o t h e r a m i no
Tripeptide
(aminoadipylcysteinylvaline) sugars by glycosidic linkages. More than 100
a m i n o g l y c o s i d e s a r e k n o w n e . g . ' s t r e p t o m y c in ,
lCyclase neomycin, kanamycin, gentamicin, hygromycin,
+ stsomtctn.
lsopenicillin
N
I A m i n o g l y c o s i d e sa r e v e r y p o t e n t a n t i b i o t i c sa n d
lEpimera$e act atainst Crarn-positive and Cram-negatrve
+ b a c t e r i a . b e s i d e s m v c o b a c t e r i a . A t t h e m o l e c u l ar
Penicillin
N l e v e l , a m i n o g l y c o s i d e sb i n d t o 3 0 S r i b o s o m e a n d
t_
I EXpanoase
block protein biosynthesis. Prolonged use of
J a m i n o g l y c o s i d e sc a u s e s d a m a g e t o k i d n e y s , a n d
Deacetoxycephalosporin
C hearing impairment.
I For the treatment of severe and chronic
I Hydroxylase
+ i n f e c t i o n s , a m i n o g l y c o s i d e sa r e t h e a n t i b i o t i c s o f
c h o i c e . S t r e p t o m y c i nw a s t h e f i r s t a m i n o g l y c o s i de
Deacetylcephalosporin
C
t h a t w a s s u c c e s s f u l l yu s e d t o t r e a i t u b e r c u l o s i s( i . e .
IAcetyltransferase against Mycobacterium tuberculosis). Usually,
I
J aminoglycosidesare regardedas reserveantibiotics,
CEPHALO SPO RIC
N s i n c e r e s i s t a n c em a y d e v e l o p e a s i l y .

Organisms for aminoglycoside

Fig. 25,7 : Biosynthesis of cephaiosporin C by
production
A- chrysogenum.
Aminoglycoside antibiotics are produced by
Actinomyces sp. Some examples are given rn
Table 25.2. Recombinant DNA techniques have
NEIIT' B.LACTAM TECHNOLOGY
FOR PRODUCTION OF 7-ACA
Scientists have been successful in producing Adipicacid
7-aminocephalosporanicacid by P. chrysogenum +
fermentation. This is possible through genetic Y

rna nip ulat ions . As alr eady desc r i b e d In Adipyl6-aminopeniciilanic

acid
cephalosporin biosynthesis(Fig. 25.7), penicillin N I expandaseand hydroxylase
is the substratefor the enzyme expandase.Adipyl- + (genecefEF)
6-a minopenic illanic ac id ( pr oduc e d b y P. I Acetvltransferase
chrysogenum on adding adipic acid) which | .'
J {genaoeffr)
re se mblesin s t r uc t ur e r v it h penic illin N , c a n a l s o
Adipyl7-aminccephalosporanic
acid
serve as a substrate for exprandase.By inserting
expandaseand hydroxylasegene (cefED,and acetyl o,n*ino,ao,oioxidas'
transferase gene (cefO from 5. clavuligerus into P.
+I
I
chrysogenum,the production of adipyl-7-ACA has I Glutarylamidase
become possible (Fig. 25.8\. Further, the genes +
responsiblefor the enzymes D-amino acid oxidase 7-Aminocephalosporanic
acid
(from Pseudomonas diminuta) have also been
inserted into P. chrysogenurn. Both these enzyrnes Fig. 25.8 : Biosynthesis of 7-aminocephalosporanic
act on adipyl-7-ACA to produce 7-amino- acid by P. chrysogenum transformants
(after suitable gene insertions)
cep ha los por anicac id.
 Chapt er25 : M IC R O B IALPR O D U C T IO N
O F A N TIB IOTIC S 337

PBODUCTION PROCESS
OF STREPTOMYCIN

for thelr production The medium used for streptomycinusually

consistsof soy meal or soy flour or corn syrupthat
Aminoglycoside Organism can supplyglucoseat a slow rate(amylaseactivity
is poor in Streptomyces sp). The initial supply of
Streptomycin griseus
Streptomyces nitrogen(NHr) and phosphateis alsoobtainedfrom
Neomycin
B andC S. fradiae soy meal.This is requiredsinceglucose,ammonia
Kanamycin
A, B andC S. kanamyceticus and phosphate i n hi gh quanti ti es i nhi bi t
streptomycin synthesis.
Hygromycin
B S. hygroscopicus
The fermentatiorr conditions for ootimal
Gentamicin Microm a.purpurea
onospor
production of streptomycin are- temperature
Sisomicin M.inyoensis 27-3O' C ,pH 6.5-7.5,aerati onrate 0.5-1.0vvm.
The durationof fermentationprocessdependson
the strainused,and is between6 to 8 days.
beenusedto producehybridaminoglycosides,
and
the fermentationyield.
for increasing
Recovery of streptomycin
Biosynthesis of aminoglycosides Streptomycinor other aminoglycosidesare
basic in nature. They can De recovered
All the ring structuresin the molecules of
aminoglycosidesare ultimately derived from
glucose. Most of the biosynthetic pathways D-Glucose
concerned with the formation of at least some
aminoglycosides have been elucidated. +
I
D-Glucose
Biosynthesis of streptomycin 6-phosphate

The outline of the pathwayfor the synthesisof

streptomycinis depictedin Fig. 25.9, More than 30 myolnositol D-Glucose D-Glucosamine
enzymatic steps have been identified. Glucose 1-phosphate 1-phosphate 6-phosphate
O-phosphateobtained from glucose takes three I
independent routes to respectively produce +
myolnositol
streptidine6-phosphate,L-dehydrostreptose and N-
i
methylglucosamine. The former two compounds
condenseto form an intermediatewhich later Streptidine L-Dehydro- UDP-Nmethyl-
combines with methylglucosamine to produce 6-phosphate streptose-1
-dTDP D-glucosamine
dihydrostreptomyci n-6-phosphate. This compound, 6-phosphate
in the next of couple of reactions,getsconvertedto
streptomycin. 4-(O-Dihydrostreptosyl)- N Methyl
streptidine6-phosphate L-glucosamine
Regulationof biosynthesis: Very little is known
about the regulationof streptomycinsynthesis. A
compound named as factor A (chemically Dihydrostreptomycin
isocapryloyl-hydroxymethyl-y-butyrate) has been 6-phosphate
isolated from streptomycin-producing strains of
S. griseus. Factor A promotes streptomycin Streptomycin
production.In fact, factorA- mutantsthat cannot 6-phosphate
synthesizestreptomycinhave been isolated.They I
+
can synthesizestreptomycinon adding factor A.
The nutrient sources-carbohydrates (glucose), STREPTOMYCIN
ammoniaand phosphate also regulate(by feedback
Fig. 25.9 : An outline of streptomycin biosynthesis.
mechanism)streptomycinproduction.

Biotechnology[22]
 338 B IOTE C HNO LO CY

Ra g(cHe)z

H2

OH o OH o
Tetracycline R1 R2 Ft^
''J R, Examples of producing
organrsms

Tetracycline cHe OH Streptomycesaureus,

S. flavus, S. antibioticus

Chlortetracycline CI cHg OH S. aureofaciciens, S. viridifaciens,

S. flavus

Oxytetracycline H cHs OH OH S. antibioticus,S. cellulosae

S. pan'us, S. rimosus

Minocycline N( CH3 ) 2 H Semisynthetic

Doxycycline H cHs OH Semisvnthetic

Fig. 25.10 : Structures of some imporTanttetracyclines along with the examples

t of organisms for their production.
l1

I
I'
ir b y weak c at ionic ex c hange r e s i n s i n a n i o n - treatment of human and veterinary deseases,
Ir
I I exchange column. Treatment with activated of fish,meatand poultry
besidesin the preservation
I (in some countries)
carbon is often necessary to remove impurities.
Streptomycin can be precipitated in the form of
sulfate salt. Organisms for tetracycline production

The firsttetracyclineantibioticthat was isolated

was chlortetracyclinefrom the cultures of
Streptomyces aureofaciens (in 1945).Thereare at
least20 streptomycetes identifiednow that usually
Tetracyclinesare broad spectrum antibiotics produce
a mixture of tetracyclines. In the
with widespreadmedical use. They are effective
Fig. 25.10, a selectedlist of these organismsfor
againstCram-positive and Cram-negative bacteria, produci ngtetracycl i nes i s al sogi ven.
besidesotherorganisms (mycoplasmas, chlamydias
rickettsias). Tetracyclines are used to High-yieldingstrains of S. aureofaciensand
combat stomach ulcers (against Helicobacter 5. rimosushavebeendevelopedby usingultraviolet
pylori). They are the most commonly used radiationand/orothermutagens(nitrosoguan idi ne).
a n ti b i o ti c sn, e x tto c e p h a l o s p o ri ns
and peni ci l l i ns. Such strains
are very efficient for the production of
Tetracyclines inhibit protein biosynthesis by chl ortetracycl i ne. Further,geneti cal l yen gineer ed
b l o c k i n g th e b i n d i n g o f a mi noacyl IR N A to strains of 5. rimosus have been developed for
ribosomes(A site). increasedsynthesis of oxytetracycline.
The basicstructureof tetracyclines
is composed
Biosynthesis of tetracyclines
of a naphthacenering (a four ring structure).The
substituentBroupsof the commontetracyclines are of tetracyclines
The pathwayfor the biosynthesis
given in Fig.25.10.Amongthese,chlortetracycline is very complex. An outline of the synthesisof
are mostcommonlyusedin the chlortetracycline
and oxytetracycline by S. aureofaciensis given in
Chapter25 : MICROBIALPRODUCTIONOF ANTIBIOTICS 339

Glucose tetracyclinesis a good exarnple of polyketiCe

anti bi oti csynthesi s.
i As glucosegets oxidised,it forms acetyl CoA
Pyruvate and then malonyl CoA. On transamination, the
Phosphoenol-
pyruvate(PEP) II
l ater gi ves mal onomoyl C oA . The enzyme
I
I
+ anthracene synthase
complexbindsto malonomoyl
I PEP carboxylase C oA and bri ngs out the condensati onof B
+ AcetylCoA
Oxaloacetate moleculesof malonyl CoA to form a polyketide
I AcetylCoA
ca#oxylase intermediates (tbur ring structures). These
Oxidative
+I intermediates undergoa seriesof reactions
to finally
MalonylCoA producechlortetracycline.
decarboxylalion
I T.ransamination Regulation of biosyntlresis: Carbohydrate
+
metabol i sm (parti cul arl y gl ycol ysi s) control s
Malonomoyl CoA- E
I chlortetracyclinesynthesis.For more efficient
B molecules | synthesis of the antibiotic, glycolysishas to be
- \
-+
ol malonvlCoA
substantiallylow. The addition of phosphate
lntermediates reduceschlortetracycline production.
t,
I Cyclization
+ PRODUCTION PROCESS
Polyketide intermediates OF CHLORTETRAGYCLINE
(fourringstructures)
The fernrentationmediumconsistsof corn steep
Y liquor,soy flour or peanutmeal for the supplyof
6-Methylpretetramide nitrogenand carbonsources. Continuous feeciing of
carbohydrateis desirablefor good growth of the
.', organi smand producti on Thi scan
of the anti bi oti c.
Anhydrochlorotetracyclitre
be done eitherbv additionof crudecarbonsources
; (asgiven above)or by supplyingglucoseor starch.
rl.
For more efficientproductioncf chlortetracycline,
Dehydrochlorotetracycline
the supplyof arnmoniumand phosphatehasto be
maintainedat a low concentration.
+
CHLORTETRACYCLINE An outline of the production process for
chlortetracyclineis depicted in Fig. 25J2. fhe
ideal fermentationconditions are- temperature
Fig. 25.11 : An outline of the biosynthesis of
27-30' C , pH -6.5-7.5,aerati on0.8-1.0vvm. The
chloftetracycline by S. aureofaciens
durationof fermentationis around4 days.
( E -Anthracene synthase enzyme camplex)

Recgvery of chlortetracycline
Fig. 25.11. There are at least 72 intermediates At the end of the fermentation, the cuiturebroth
formed during the course of chlortetracycline is filteredto removethe mycelium.The filtrateis
biosynthesis,some of them have not been fully treatedwith n-butanolor methvlisobutvlketone in
characterized. aci di c or al kal i ne condi ti on for extracti ngthe
antibiotic.lt is then absorbedto activatedcharcoar
Polyketide antibiotic synthesis : The term rs
to remove other i mpuri ti es.C hi ortetraycl i ne
polyketiderefersto a group of antibiotics that are
elutedancicrystallized.
synthesized by successivecondensation of small
carboxylic acids such as acetate, butyrate,
PRODUCTION OF TETRACYCLINE
propionate and malonate. The synthesis of
-DIFFERENT PROCESSES
polyketideantibioticsis comparableto that of long
chainfattyacids.That is the carbonchaingrowsby The productionof tetracyclinecan be achieved
cvclic condensation process. The synthesisof by one or more of the following ways.
340 B IOTE C H N O LO CY

Recovery
and
purification

Fig. 25.12 : An outline of production chart for chloftetracycline.

a By chemical treatment of chlortetracycline. butyratewhile the sugar units are derived from
a By carrying out fermentation in a chloride-free gl ucose.Macrol i de bi osynthesi si s a com plex
cult ur e m edium . processand a good exampleof polyketide synthesis
By em ploy ing m ut ant s in whic h c h l o r i n a t i o n
which is analogousto fatty acid biosynthesis. The
reaction does not occur.
enzymelactonesynthase is a multienzyme complex
w hi ch i s comparabl ei n i ts structureand funct ion
By bloc k ing c hlor inat ion r eac t ion b y t h e a d d i t i o n
to fatty acid synthasecomplex. An outline of
o f inhibit or s e. g. t hiour ea, 2{ hiou r a c i l .
the bi osynthesi sof erythromyci ni s gi ven I n
Fig. 25.13.
Regulation of biosynthesis: End product
i nhi bi ti on of erythromyci n synthesi si s well
Macrolides are a group of antibiotics with large documented. E rythronol i de
B i nhi bi tsthe enzym e
Iactone rings (i.e. macrocylic lactone rings). They lactonesynthase.The final product erythromycrn
co ns is t of 12- , 14- , or 16- m em ber e d l a c t o n e r i r r g s hasal sobeenshow nto i nhi bi tcertai nenzymesof
with 1- 3 s ugar s link ed by gly c os i d i c b o n d s . l - h e the pathway (e.g. transmethylase). Addition of
sugars may be 6-deoxyhexoses or aminosugars. propanol to the cul ture medi um i nduces t he
Erythromycin and oleandomycin are 14-membered synthesiso{ acetyl CoA carboxylase,and almost
(lac t one r ing c ont aining) m ac r o l i d e s w h i l e doubl esthe producti onof erythromyci n.
le uc om y c in and t y los in ar e ex am pl e s t , o r 1 6 - m e m -
be r ed m ic r olides . PRODUCTION PROCESS
OF ERYTHROMYCIN
E r y t hr om y c in and it s der iv at iv e c l a r i t h r o m y c i n
are the most commonly prescribedmicrolides.They producti onof erythromyci ins ca r r ied
Industri al
are effective against Gram-positive bacteria, and out by aerobic submerged fermentation. fhe
are f r equent ly us ed t o k ill pen i c i l l i n - r e s i s t a n t of soy mealor cor n
cul turemedi ummai nl yconsi sts
org anis m s . Clar it hr om y c in is c ur r e n t l y u s e d t o steepliquor,glucose(or starch),yeastextractand
combat stomach ulcers caused by H. pylori. fhe ammoni umsul fate.Fermentati on i s carri edout at
macr olides inhibit t he pr ot ein b i o s y n t h e s i s b y 30-34'C for about3-7 dat1s.Conventional methods
bin ding t o 50S r ibos om e. are used for the recoverv and purification of
erythromycin.
Polyene macrolides is the term applied for very
large ring macrolides that many contain lactone
ring s in t he r ange of 26- 28. e . g . n y s t a t i n ,
amp hot er ic in. Thes e poly ene m a c r o l i d e s a r e Tmrr 25.3 lmportant macrolide antibiotics
antifungal.
with the organlsmsproducingthem

Macrolid antibiotic Producing organism

Production of_ macrolides
Macrolides are produced by actinomycetes. The Erythromycin Streptomyces erythreus
major macrolide antibiotics and the corresponding Oleandomycin S. antibiotics
organisms synthesizing them are given in lable Pikromycin S. felleus
25.3. Megalomicin Micrononospora inositola
Tylosin S. fradiae
Biosynthesis of erythromycin
A
Carbomycin ' S. halstedii
In the biosynthesisof erythromycin, the lactone
n n8 s are contributed by acetate, propionate or
Leucomycins ium kitasatoensi
Streptoverticill s
Chapt e r2 5 : MIC R OB IALPR O D L J C TION
OF A N TIB IOTIC S 341

Propionate chlorampherricolis associatedwith side effects,the

I
+ most significant being damage to bone-marrow. As
Propionyl------------* Methylmalonyl such, chloramphenicol is treated as a reserve
CoA CoA a n t i b i o t i c a n d s e l e c t i v e l yu s e d .

C h l o ? a m p h e n i c o lb i n d s t o 5 0 S r i b o s m a l s u b u n i t
1 molecule 6 molecules and blocks (peptidyltransferasereaction) protein
dTDP-Glucose b i o s y n t h e si s .
Lactonesynthase
J
dTDp_4_ Polyketideintermediate Production of chloramphenicol
fet-o-O--deoxy- (enzYmebound)
D glucose- Chloramphenicol can be produced by
dTDp_ + Streptomyces venezuelae and 5. omiyanesis.
I L-Mycarose J
I However, chemical synthesisis mostly preferredfor
| \ ErvthronolideB
the commercial production of chloramphenicol.
L+orop-o---
Desosamine
\ 3-L-Mycarosyl- GRISEOFULVIN
\ erythronolideB
--------t-l C r i s e o f u l v i ni s a n a n t i b i o t i c t h a t a c t ss p e c i f i c a l l y
on fungi with chitinous cell walls. lt is used in the
J treatment of various fungal skin infections. Further,
Erythromycin
D
griseofulvin is also employed in the treatment of
Methylation
-&:in;' ,/\ plant diseases caused by Biotrytis and Alternaria
/ \Hvdroxvration solani.
t\
Erythromycin
B Erythromycin
C Although the exact mechanism of action of
tr"n"- griseofulvin is not known, it is believed that chitin
I
(SAM) biosvnthesisis adverselv affected.
Imethylation
:RYTHROMYCINA

Fig. 25.13 : An outlineof the biosynthesisof Dichloroacetylamino

erythromycin by Streptomyces erythreus moiety
(SAM- S-adenosylmethionine) o
HN-C-CHCt2
.._..
..,..1,.__..............__..:..
cH-cH-cH20H
t-
OH
p-Nitrophenyl Propanodiol
Th e a nt ibiot ic s wit h ar om at ic r ings i n t h e i r moiety moiety
structure are regarded as aromatic antibiotics. ln a Chloramphenicol
strict sin c e, all t he ant ibiot ic s c ont aining a r o m a t i c
n uclei s hould be c ons ider ed in t hi s g r o u p .
However, most authors prefer to treat the three
impo rtan t ant ibiot ic s nam ely c hlor am p h e n i c o t ,
griseofulvin (Fig, 25.141 and novobiocin in the
category of aromatic antibiotics, and the same is
do ne in t his book als o.
n 3L/ \-,

CHLOFAM PHENI CO L

Chloramphenicol is a broad spectrum antibiotic Griseofulvin

tha t can ac t agains t Cr am - pos it iv e an d C r a m -
Fig. 25.14 : Structures of chloramphenicol and
negativebacteria,besidesrickettsias,actinomycetes
oriseofulvin.
an d chl am y dias . Howev er , adm inis tr a t i o n o f
342 B IOTE C H N O LO CY

Production of griseofulvin
o f g ri s e o ful viins carri ed
C o m m e rc i apl ro d u c ti o n
out by employing Penicillium patulum. The
chemicalsynthesisis lessfrequentlyused due to
high cost.
Mycelialfilaments
The fermentationis carriedout by an aerobic
submergedprocesswith a glucoserich medium. l_
I Enzymallc
Nit r o g e ni s s u p p l i e db y s o d i u mn i tra te.Theopti mal
Itreatment
conditions for fermentation are-temperature +
23-26"C,pH 6.8-7.3,aeration0.8-1 vvm, and the \J l) v/-\
^
per i o di s 7 -10 d a v s . Xoo,Po"r-t
vn
,"r\J r-.,-
v^v v
a-!],

Protoplasts
I
DNA
{-lnsert
There are several antibiotics (more than 200 or glycol
Polyethylene
so ) w hic h hav e nuc leos ide lik e s t r u c t u r e s e . g . I
pu rom y c in, blas t ic idin S. Nuc leosi d e a n t i b i o t i c s
have diverse structuresand biological activities.

Puronrycin is used to understand the ribosomal

fun c t ion in pr ot ein bios y nt hes i s . N e p l a n o s i n
possesses antiviral activity. Blasticidin S is a
fungicide antibiotic usecj in plant pathology.

Production of nucleoside antibiotics /oooo\

\nn.O)
.<--.-__=--l
Selectedexamples of nucleoside antibiotics and
lndividualcells
the respective organisms from which they are
produced are given below.

Puromycin Streptomycesalboniger
N e p l a mo s i A
n AmpulIarielIa reguIaris Desiredcell
in a selective
B l a s ti c i d i S
n S. griseochromogenes medium
Polyoxins S. cacaoi
Fig. 25.15 : A diagrammaticrepresentationol
transformation rn Streptomyces sp.

Transformation of stleptomyces
Streptomycesstrains exist as aggregatesof
A greatmajorityof antibioticsare producedby
mycel i alfi l amentsand not as i ndi vi dualcel l s.This
Streptomycessp, a Cram-positivebacteria. Of
is in contrastto E. co!i. lt is thereforeessential
course,thereare someother bacteria(both Cram-
that the cell walls of Streptomycesare broken to
positiveand Gram-negative)and fungithat can also
releaseprotoplastsfor transformation(Fig. 25.15).
produceantibiotics.
B y addi ng the desi redD N A (i n pl asmi ds)and
Cenetic manipulationsin Streptomyceshave polyethyleneglycol, the cells can be transformed.
been extensivelycarried out to enhancethe yield These protoplastsare grown on a solid medium
of antihiotics and reduce cost of production, to regenerate the cell walls. The transformed cells
besides developing newer and more effective rvith desiredpropertiescan be isolatedfor further
ant ib i o ti c s . use.
Chapter2s : tvttcRogtAl pRoDUCTtoN oF ANT|BIOT|CS
Gloning of antibiotic
biosynthesis genes
In general, biosynthesisof antibiotics involves
several reactions and participation of a large
number of enzymes (and of course, several genes).
It is rather difficult to clone so many genes.Mutant
strains of Streptomyces that totally lack the
synthesizing machinery for a particular antibiotic
are very useful. Genes from a clone bank can be
incoroorted into such mutants and screenedfor the
d esired pr oper t ies . This is a lengt hy a n d t e d i o u s
procedure but has been successfullyused for the
improved production of certain antibiotics e p
un de y lpr odigios in.
Direct strategies of gene cloning : lt is often
possibleto identify one or a few important enzymes
in th e s y nt hes isof ant ibiot ic s .Fr om t he s e q u e n c eo f
a mino ac ids in t he enz y m e, ge n e c a n b e
constructed,and cloned. For instance,the gene for
isopenicillin N synthase from P. chrysogenum has
been successfullyconstructed in this fashion and
used f or inc r eas ed pr oduc t ion of pe n i c i l l i n s a n d
cephalosporins.
Genetic engineering for the
production of novel antibiotics
Ce net ic , m anipulat ions c an be d o n e i n t h e
a ntib iot ic s y nt hes iz ing or ganis m s t o u l t i m a t e l y
produce totally new and novel antibiotics-
Wi Id+ype Streptom y ces coe I i co Io r genes encode
the enzymes to produce the antibiotic
actinorhodine. S. violaceoruber produces a related
antibiotic namely granaticin.Cenetic manipulations
can be done between these organisms to produce
novel hybrid antibiotics such as medarrhodine A
an d di hy dr ogr anat ir hodi
ne.
The newly synthesized antibiotics are in fact
structuralvariants of the existing antibiotics. As the
biosyntheticpathways for antibiotic production and
their corresponding genes are better understood, it
becomes possible to design newer antibiotics with
more efficient action.
343
Genetic engineering for improving
antibiotic production
For aer.obic microorganisms (e.g. Streptomyces
sp), there is often the limitation of oxygen supply
that hinders antibiotic oroduction. Some workers
have isolated a lremoglobin-like protein producecl
by the bacterium Vitreoscilla sp. The gene
synthesizingthis protein was isolated and cloned in
a plasmid vector. The hemoglobin gene of
Vitroscilla sp was finally incorporated into
Streptomyces.These newly transformed strains have
better capacity to take O, from the medium even at
a low concentration.
The new strain of S. coelicolor (with hemoglobin
.l
gene) was found to produce 0 times more
a n t i b i o t i c a c t i n o r h o d i n et h a n t h e w i l d s t r a i n , e ve n
at a low concentration of oxygen.
Manufacture of antibiotics is highly
commercialiseddue to a heavy demand worldwide.
It is mandatory that detailed clinical trials are
carried out before considering the manufacture
of any antibiotic. lr4orethan half of the antibiotics
produced are for human use. lf is therefore
absolutely essential that each antibiotic produced
is safe, consistent and poses no health
complications. Covernment authorities play a
p r e d o m i n a n t r o l e i n r e g u l a t i n g t h e p r o d u c t io n
o f a n t i b i o t i c s . T h e s e g u i d e l i n e s e n s u r e t ha t
correct procedures are followed at each stage
of manufacturing the antibiotics. The product
produced should be of consistently high quality.
Quality of the products should be checked at
different stagesof manufacture.
It is exoected that the antibiotic manufacturet.r
should be in the purest form, although 100% purity
is not practicable.lt is mandatorythat the impurities
( i f a n y ) , t h e i r q u a n t i t i e s ,a n d t h e i r i l l e f f e c t ss h o u l d
be made known.
 lUlicrobiql
Production
ofAminoAcids

ht ' jir s t c onr r eT( i. t l pr or lr r c t ion o f t l r o ; r r n r n o F o o r l i n c l u ''t r t ' ( r 5 ') i ,

ar ir l- glut anr ic ar ir ls , r v r s s f . r r t ' r Li ln J . r ; t a ni r r , r :
F - - e r i. i r i r i i l i i c s . l [ ] 'l l ,
t'.rrly. r : , I 90B li n, , r s llic t i. r { of J . r yr a n rt v l r o i i r s l
P i r a r n r aec , L r l r c
,rl
irlcntit iec l t hat glut anr ir ar ir l ( in t h e t o r n r o t - r 'l i
n rorro s oci ul nr glLrt . , inr . lt)cl) os s es s es t as t e - e n c b n a r i I rg i h r . i r c l j r r r l r r . r iI , r n r i r r o a c i c l - ct c , x c c ! i , r : , l vci n ct
p rop c r t ic s lr r t ir e e. r r lv r lal' s , r 'r " r o r r o sior rcnl r , r l o r r , lr ^ i,t h t l r c i r p r o r l l r cl i o n n r e t l r o c l s. t n r l L r s t 's.tr t'
g iut.n r . lt c iM SC) r ' r , . r er
s t r . ic t c r l f r onr t i r e \ c g ( 't J l ) l ( , q l \ '1 , ni n T a h l e 2 6 . 1 T h t ', r p p l i c a t i ( ) n s . r r ( ' l t r r r acl l v
p rotcins ( r v he. it; r nc is o' , , ) I t n' as r - r nl , vi n l 9 5 z , i l r c , i i scrr';sr,.cl lrc,rcti ni lcr
la rg,t s c alc .indus t r ial lr r or lr - rlir r . r nol M S C l r i , L r r i r r i l
lricroc lr ganis nr sc om nr c nt ' c r i Today , c o n r n t c r ri . r l
lrro rlLrl cion of am ir - r oat ic ls is oner o i t h e b i g , l 3 c , s t
ittclr- r s t r ier
r v or
s lt ior , err v r lir . t r - ,rt ni- r ualr r i c r e . r s cr n l l r t ' , \ i r t i n o a c i c l s , r ( ' r - t s e c le i t h c r . r l o t r t ' o i i r r
rlcn r. r r r l bv about I O ' 1, , r o r l i r i r . l r t i c r , . r : I l , r r r - r r rtr'r . l r a n c e r - sM , o n o s o d i u ttt
glttt,rmate is the ntost frequently, ,.tscrl itr loori
Glutamic acld continLrr':to irr. largest producer
i r c l i r : L r v C i v c i i r r ' . r n i l a l i l n i n e a l s o e n h , r r t ( ' t .l 5 tL '
a rr)on g ilr e . t r lr ir r o : r r ir ls , lollt - ir v e d b , v l , v s i n t '. 'l r y 'p f o p h . r n ,
,rrrrJ iilvorrr, i r r J S s u c i J l i , ) r r r i i th
n retlr ionir r e,t hr eonine ar r r l ls par t ic a c i d .
l r l s t i c l i n e , , r ( - 1lss a n . r n t i o x i d . t r r t o p t o s e r v e n r i l l <
lrovvdc-F T o i -[ h c p r c s t 'r r , . r t i o ot tf f r u i t j t r i co s , ( y s l cl r e
\ ( 'r - v L rass a n a t - r t i r . r x i t l . t , r 1
AMINO ACID PRODUCTION
-
GENERALCONSIDERATIONS Aspartame, r r Ii pt'1rticie i aslrart,vi -1therr1'l.il,in i nt:
n r e i l r v e s t e r ; l r r o c l t rir'c l b t a c o n t b i n a t i o n o f
So nr egener aIc or r s ic lc r . r t ioror r s- rt h e c . c - l n t n t ei .rri i aspartic acid and phenylalanine, i-caboLrt20() tinrcs
a lrpli c at ions of anr ino . r rir ls , t ir c i r p r o c l L r c l i o n \ \ \ e {, t i 'T i i r . r r s L r r o s t ' l t i s i r s e d a s . t l o r 'r '-itt l or tt'
n rclh oc ls , anc l t lr c ilr ' r , c lolr nr c ni o i : l r . t i r r r ( ) l . r t i t i r i a l s \ \ '( 'c t ( 'n ( 'ri n s o l l - c l r i n ki n c l L r s t r l . '
n rit ro or - g. t t - t is m sf or ir r lr r ov c c l . r n r i n o , t ( r . i T h e r c a r t ' c . c r l i l i nc s s r - n t i a a l m i l r r - :rr c i t l s l h , r l a r c
p rorltr r t ion ar c lr r ief ly ,r lc s r . r ilr t , r l, i l c l i r i r : n t o r l i n r i t i r r r iSr r p l a n t p r o t e i n s T h e s ei n t l L r r l c:
J r 's i r i e ',i - n e f h i o n i n t ', l l r r e o r r i n e a n c l t r v l r t o p hu n
. \ r l r l t i o r o r l l r r : r l r 'l i r i t 'n t . r n r i n o a c . i r l i s )i n r l r t ove 's
l r r . ' r r r t r i t i r . r l r . rrl l L r . r l i t r o i i r u r r r a n f c l o t l s a s n e l l
, ' \ tllitr t.ra c jci- slr i' .' c a tt itlt' r - ,llr g eo f .r111l l i c.tl i .rr: , r s a r r r n r a li e e r l s i h L r s ,l r r r . a c cl n r i c i r c c ln 'i t i r l ', si r r r - .
I i r r ' p r Olti.r r tiOir a te L r 5 c ( ) l in r ir o .r cicl s i -s gi ri en rn s o r , p r o c l u c t : .s r r ; r p l c 'n r c n l c cr ^l , 'i i hr - n t : t h i o n i n r.tte '
t l r ( ' n (' xt r r ) ltillr n , o 1 l r c t i t : r 'r u t r i t i o n . r l , , '. r l r r c A 1 c 'i i - r i o i r i nacr l r l c r l so v
 OF AMINO ACIDS
Chapter26 : M|CROB|ALPRODUCTTON 345

bean meal is a better feed for pigs and other S o m e a m i n o a c i d s i n t h e f o r m o f N - a c y l

anim als . derivatives are useful for the preparation of
cosmotics.
Pharmaceutical industry
METHODS FOR PRODUCTION OF
T he a m i n o a c i d s c a n b e u s e d a s medi ci nes. AMINO ACIDS
Essentialamino acids are usefulas ingradientsof
infusion fluids, for administrationto patientsrn The i ndustri aloroducti onof ami no aci ds i s
post-operative treatment. carriedout by one or more of the followingthree
processes.
Chemical industry i . E xtracti on: A mi no aci ds are the bui l di ng
blocks in protein structure.The proteinscan be
Amino acids serve as starting materialsfor
producing severalcompounds.Clycine is used subjected to hydrolysis,and the requisiteamino
acids can be isolated e.g. cysteine, tyrosine,
as a precursor for the synthesisol glyphosate
( a her b i c i d e ),w h i l e th re o n i n e i s th e starti ng l euci ne.
material for the oroduction of azthreonam 2. Chemical synthesis: Chemical synthesis
( anot he r h e rb i c i d e ). Po l y -me th y l g l utamate i s resul tsi n a mi xtureof D - and L-ami noaci ds.Most
utilised for manufacturing synthetic leather of the ami no aci ds requi red for commerci a l

Amino acid* Preferred production method Application(s)

Glutamic
acid Fermentation Flavour
enhancer
Lysine Fermentation Feedadditive,infusionsolution
Methionine Chemical
synthesis Feedadditive
Threonine Fermentation Feedadditive
Phenylalanine Fermentation Aspartameproduction
Aspartate Enzymatic
synthesis,
extraction Aspartameproduction
Glycine Chemical
synthesis Sweetener,
foodadditive
Arginine Fermentation,
extraction Infusions,
therapy lorliverdiseases,
cosmotics
Valine Fermentation,
extraction pesticides
Infusions,
Tryptophan immobilized
Extraction, cells Infusions,
antioxidant
lsoleucine fermentation
Extraction, Infusions
Alanine Exlraction,
enzymatic Flavour
enhancer
Leucine Extraction,
enzymatic Infusions
Proline Extraction Infusions
Serine Extraction,
chemicalsynthesis Cosmotics
Histidine iermentation
Extraction, Therapyforulcers
Asparagine Extraction,
chemicalsynthesis Diuretic
Glutamine Fermentation Therapyforulcers
Cysteine Exlraction Infusion
Tyrosine Extraction,
enzymatic Infusion
Ornithine Extraction,
enzymatic TherapyJorliverdiseases
* Theorderof amino acidsis approximately in the decreasingorderol thei annualproduction;Extractionrefersto theextnctionol the
specilicanino acid tron proteinhydrolysates
346
applications are of L-category. However, for the
synthesis of glycine (optically inactive) and some
ot her am ino ac ids whic h c an b e u s e d i n L - o r
D-form (D, L-alanine, D, L-methionine) for certatn
purposes,chemical methods are employed.
3. Microbiological production : For the large-
scale production of amino acids, microbiological
rnethods are employed. There are three different
approaches.
(a) Direct fermentation methods : Amrncr
acids can be produced by microorganisms
by ut ilis ing s ev er al c ar b o n s o u r c e s e . g .
glucose,fructose,alkanes,ethanol,glycerol,
pr opionat e. Cer t ain indu s t r i a l b y p r o d u c t s
like molassesand starch hydrolysate can
als o be us ed. M et hano l , b e i n g a c h e a p
carbon source, is tried for amino acid
pr oduc t ion, but wit h I im i t e d s u c c e s s .
(b) Conversion of metabolic intermediates
into amino acids : In this approach, the
microorganisms are used to carry out
selected reactions for amino acid
pr oduc t ion e. g. c onv er s i o n o f g l y c i n e t o
s enne.
(c) Direct use of microbial enzvmes or
immobilized cells : Sometimes resting
c ells , im m obiliz ed c e l l s , c r u d e c e l l
extracts or enzyme-membrane reactors
can be used for the productiolr of amino
. acids. Some examples are given below.
Amino acid dehvdrogenases from certain
bacteria (e.9. Bacillus megaterium) can be used for
the amination of cr-keto acids to produce L-amino
a c ids e. g. alanine ( f r om py r uv a t e ) , l e u c i n e ( f r o m
rx - k et ois oc apr oicac id) and phe n y l a l a n i n e ( f r o m
p heny lpy r uv at e) . lm m obiliz ed c e l l s o r e n z y m e -
membrane reactors can be used.
Enz y m esor im m obilis ed c ells a r e a l s o e m p l o y e d
for t he pr oduc t ion of s ev er alot he r a m i n o a c i d s e . g .
tr y pt ophan, t y r os ine, ly s ine, v alin e .
STRAIN DEVELOPMENT FOR
AMINO ACID PRODUCTION
The metabolic pathways, for the synthesis of
am ; no ac ids by m ic r oor gani s m s , a r e t i g h t l y
controlled and they operate in an economical way.
Therefore,a natural overproduction of amino acids
is a fare occurrence. Some strains that excrete
B IOTE C HNO LO CY
certain amino acids have been isolated e.g.
gl utami caci d, al ani ne,val i ne.
In order to achievean overproductionof any
amino acid by a microorganism, methodshave to
be devisedfor the eliminationof the metabolic
processes.
regulatory/control In fact, severalamino
acid-producinI microorganisms have been
developed by mutagenesis and screening
proqrammes.
The following are the major ways of strain
developmeirt. In f act, several methods are
combinedto successfullv developa new strainfor
produci ngami noaci ds.
Auxotrophic mutation : These mutants are
characterizedby a lack of the formation of
regulatoryend product(i.e. repressor
or regulatory
effector). The intermediatesof the metabolic
pathwaysaccumulateand get excreted.
Genetic recombination : Mutants can oe
developed by genetic recombinationfor over-
productionof amino acids. Protoplastfusion in
certainbacteriais usedfor developmentof hybrids
e.g. Corynebacteriumglutamicum and Bacillus
flavum.
RecombinantDNA technology: The classical
techniquesof geneticengineering can be usedfor
strain development. Strains with increasing
activities of rate-limiting enzymes have been
developed.In one of the techniques,E. coli and
cloning vector pBR322were usedto increasethe
genes for the production of amino acids e s
gl utami caci d, l ysi ne,phenyl al ani ne,
val i ne.
Functional genomics : a new approach
Analysisof genomesfrom the wild and mutant
strai nsof mi croorgani sms w i l l hel p i n cr eat ing
improvedstrains.Once the entiresequenceof the
chromosomes in the organisms (e.9.C. glutamicum,
E. coli) is established,effortscan be made to carry
out geneti c mani pul ati ons for e f f icient
overproductionof desired amino acids. Chip
technologycan be used to detect new mutations
and consequently the fermentationprocesses.
L-Clutamicacid was the first amino acid to be
produced by microorganisms. The original
Chapt e r2 6 : M IC R O B IALP R OD U C T TON
OF A MTN OA C TD S 347

Glucose
I
I
I
I
I

Phosphoenol
pyruvate Pyruvate
/.\^-

Oxaloacetate

Malate Krebs cycle

lsocitrate

SuccinylCoA 0,-Ketoglutarate

L.GLUTAMIC
ACID

bacterium, Corynebacteriumglutamicum, that was strains to produce and excrete more and more of
first used for largescale manufactureof glutamic glutamic acid. These include the strainsthat can
acid continuesto be successfully usedeven today. tolerate high concentrations of biotin, and
The other importantorganisms(althoughusedto a lysozyme-sensitivemutants with high yield.
lesser extent due to low yield) employed for
glutamic acid production belong to genera Biosynthesis of L.glutamic acid
M i crobacteri um, Brevi bacteri um and A rthrobacter. The pathway for the synthesisof glutamic acid
All these organismshave certain morphological with glucose as the carbon source is depicted in
and physiological characters comparable to Fig. 26.1 . Clucose is broken down to phosphoenol
C. glutamicum. Biochemically, glutamic acid- pyruvate and then to pyruvate. Pyruvate is
producing bacteria have a high activity of convefted to acetyl CoA. Phosphoenolpyruvate (by
glutamate dehydrogenaseand a low activity of the enzyme phosphoenol pyruvate carboxylase)can
a-ketoglutarate dehydrogenase.They also requrre be independently converted to oxaloacetate. Both
t he v it a mi nb i o ti n . these carboxylation reactionsare quite critical, and
require biotin as the cofactor.
lmproved production strains
The next series of reactionsthat follow are the
Several improvements have been made, familiar citric acid (Krebs)cycle reactions wherein
particularlyin C. glutamicum,for improvingthe the k"y metabolite namely o-ketoglutarate is
348
_ N A D P +\
Isocitrate
Fig. 26.2 : Biosynthesis of L. glutamic acid-role
pr oduc ed. I n t he r out ine c it r i c a c i d c y c l e , a -
ketoglutarate is acted upon by the enzyme o-
ketoglutaratedehydrogenaseto form succinyl CoA.
For t he pr oduc t ion of glutamic acid,
a-ketoglutarate is converted to L-glutamic acid by
the enzyme glutamate dehydrogenase (CDH). This
e nz y m e is a m ult im er , eac h s u b u n i t w i t h a
m olec ular weight of 49, 000 . T h e r e d u c i n g
equiv alent s , in t he f or m of NA D P H + H +, a r e
r equir ed by CDH. They ar e g e n e r a t e d i n t h e
preceeding reaction of Krebs cycle (catalysed by
the enzyme isocitrate dehydrogenase) while
converting isocitrateto o-ketoglutarate.The supply
a nd ut iliz at ion of NADPH + H+ o c c u r s i n a c y c l i c
f as hion t hr ough t he par t ic ipa t i o n o f t h e t w o
enzymes, namely isocitrate dehydrogenase and
giutamate dehydrogenase(Fig. 26.2).
Theor et ic ally , one m olec ule o f g l u t a m i c a c i d
c an be f or m ed f r om one m olec u l e o f g l u c o s e . I n
practice, the conversion efficiency of glucose to
g lut am ic ac id was f ound t o be a r o u n d 7 0 7 " .
Regulation of glutamic acid
biosynthesis
The es s ent ial r equir em ent f o r g l u t a m i c a c i d
pr oduc t ion is t he high c apabilit y f o r t h e s u p p l y o f
the c it r ic ac id c y c le m et aboli t e s . T h i s i s m a d e
pos s ibleby an ef f ic ient c onv er s i o no f p h o s p h o e n o l
pyruvate as well as pyruvate to oxaloacetate(Refer
Fig. 26.1) Thus, there are two enzymes
(phosphoenol pyruvate carboxylase and pyruvate
carboxylase) to efficiently produce oxaloacetate,
wh i le there is on ly one enzyme (pyruvate
dehydrogenase)for the formation of acetyl CoA.
Cer t ain m ic r oor ganis m s whi c h have either
phosphoenol pyruvate carboxylase (e.9.,
B IOTE C H NO LO CY
\
\
Glutamate
of NADP*'
E. coli) or pyruvate carboxylase(e.8. B. subtilis) are
n o t c a p a b l e o f p r o d u c i n g g l u t a m i c a c i d to a n y
significant extent. C. glutamicttm has both the
enzynres and therefore can replenish citric acid
cycle intermediates (through oxaloacetate) while
t h e s y n t h e s i so f g l u t a m i c a c i d o c c u r s .
Another key enzyme that can facilitate optimal
production of glutamic acid is a-ketoglutarate
dehydrbgena-se(Refer Fig. 26.1) of citric acid cycle.
Its activity has to be substantially low for good
synthesis of glutamic acid, as is the case in
C. glutamicum. Further, exposing the cells to
a n t i b i o t i c s ( o e n i c i l l i n ) a n d s u r f a c t a n t sr e d uce s th e
activity of ct-ketoglutaratedehydrogenase while
glutamate dehydrogenase activity remains
unaltered. By this way, oxidation of ct-ketoglutarate
v i a c i t r i c a c i d c y c l e c a n b e m i n i m i s e d , w h i l e th e
f o r m a t i o n o f g l u t a m i c a c i d i s m a d e m a xi m u m
oossible.
Release of glutamic acid
Clutamic acid is synthesized intracellularly, and
therefore its release or export is equally important.
lt now appears that there is a carrier-mediated
energy-dependent active process involved for the
export of glutamic acid. There are several ways of
i n c r e a s i n gt h e m e m b r a n e p e r m e a b i l i t yf o r exp o r ti n g
glutamic acid
. Biotin limitation
. Addition of saturatedfatty acids
. Addition of penicillin
. Use of oleic acid auxotrophs
. Use of glycerol auxotrophs
. Addition of local anesthetics
. Addition of surfactants(Tween 40).
 ChA O t Cr OF A MIN O A C ID S
26 : MIC R OB IALP R OD U C T IO N 349

Nutrients
J
lDissolv r nql .f--
- l- - - - \ . lBuf f er l f c.{r-l ai-,b. )
L_t"t__J-[''"1'""', -tjalJ I seoaratorl
\l \:i ,
'l exchanqa-'

Sterileair

I
L-GLUTAMICACID
(as monosodiumglutamate)

Fig. 26.3 : Diagrammatic representation of glutamic acid production plant.

The effect of biotin deficiency in facilitating the i s u t i l i z e d . O t h e r s u b s t r a t e sl i k e a l k a n e s , e t h a n o l

re lea se of int r ac ellular glr - it am ic ac id ha s b e e n and methanol are lessfrequently used.
worked out. Biotin is an essentialcofactor (required Nitrogen sources : The concentration of
by the enzyme acetyl CoA carboxylase) for the ammonia is very crucial for converting carbon
bio synth esisof f at t y ac ids . Due t o a lim it ed s u p p l y source to glutamic acid. However, high
or deficiency of biotin, fatty acid biosynthesisand concentration of ammonia inhibits the growth of
co nseq ue nt ly phos pholipid s y nt hes isis dr a s t i c a l l y the organisms. In the beginning of fermentation,
reduced. As a result, membrane formation (protein- ammonium salts and a low concentration of
phospholipid complex) is defective which alters
ammonia are added. During the course of
p erme ab ilit yf or an inc r eas edex por t of int r a c e l l u l a r
fermentation, ammonia in aqueous solution is
glu tamic acid.
c o n t i n u o u s l yf e d . I n t h i s w a y , p H c a n b e c o n t r o l l e d ,
It is fou nd t hat t her e is an alt er at ion i n t h e besides continuous supply of nitrogen source.
me mbra ne c onr pos it ion of phos pholipids i n o l e i c Sometimes, urea is also used as a nitrogen
acid an d gly c er ol aux ot r oph m ut ant s . T h i s source, since glutamic acid-producing bacteria
fa cilita tesreleas eof int r ac ellularglut am ic a c i d . possess urease that can split urea and release
ammonra.
Th e kn owledge on t he m em br ane per m e a b i l i t y
Growth factors : Biotin is an important growth
o f g luta m ic ac id is s uc c es s f ully ex p l o i t e d
factor and its concentration in the medium is
fo r in cre ased indus t r ial pr oduc t ion of g l u t a m i c
influenced by the carbon source. For instance, a
acid.
supply 5 pg of biotin per liter medium is
recommended if the carbon source is lOok glucose,
Production of glutamic acid-
while for acetate as the carbon soLrrce,the biotrn
requirements and influencing factors
r e q u i r e m e n t i s m u c h l o w e r ( 0 . 1- 1 . O S r g / l )A
. ddition
The ind us t r ial pr oduc t ion of glut am ic a c i d i s of L-cysteine in the medium is recommended for
influenced by carbon sources, nitrogen sources, certain strains.
a,rorvthfactors, pl-l and O, supply. The relevant
Supply ol Or O, supply should be adequately
:lDects are brieflv described
a n d c o n t i n u o u s l y m a i n t a i n e d . l t i s o b s e r v e dt h a t a
Carbon sources : Either refined (glucose, high O, concentration inhibits growth of the
:JCrose,fructose, maltose) or unrefined (sugarbeet o r g a n i s m s w h i l e a l o w O , s u p p l y l e a d s t o t h e
-'.:lasses,sugar cane molasses)carbon sources are p r o d u c t i o n o f l a c t i c a c i d a n d s u c c i n i c a c i d . I n b o t h
( inex p e n s i v e ) instances,glutamic acid formation is low.
-==rj, In cou nt r ies lik e J apan,ac et at e
350
Process of production and recovery
Some important information on the production
of glutamic acid by Brevibacterium divaricatum rs
eiven below.
Carbon source G l u cose(12% )
Nitrogen source A m moni um
acetate(0.5%)
pH 7 .8
Temperature 3 B" C
Period for fermentation 3 0 -35 hours
Yield of glut am ic ac id 1 00 g/l medi um.
A schematic representation of glutamic acid
production plant is shown in Fig. 26.3. As the
fermentation is complete, the cells are separated,
the c ult ur e br ot h is pas s ed t h r o u g h a n i o n
exch anger .The glut am ic ac id bound t o t h e r e s i n si s
elu ted in NaO H, while t he am m onia r e l e a s e dc a n
b e r eus ed. W it h NaO H, glut am ic a c i d f o r m s
mo no s odium glut am at e ( M SC) wh i c h c a n b e
purified by passingthrough anion exchanger.MSC
can be subjectedto evaporation and crystallization.
Lysine is present at a low concentration in most
of th e plant pr ot eins . Being an ess e n t i a l a m i n o
acid, supplementation of plant foods with lysine
in cre as est heir nut r it ional qualit y .
L - Ly s ine is pr edom inant ly p r o d u c e d b y
Corynebacterium glutamicum and to some extent
l:y Brevibacterium flavum or B. lactofermentum.
Biosynthesis of L.lysine
The pathway for the synthesis of L-lysine rs
co mplex , and an out line of it is d e p i c t e d r n
Fig. 26.4. This metabolic pathway is also involved
in the formation of 3 other amino acids, namely
me thionine, t hr eonine and is oleuc in e
As the glucose gets oxidised by glycolysls,
phosphoenol pyruvate and pyruvate are formed. Both
these metabolitescan be converted to oxaloacetate,a
key componentof citric acid cycle.On transamination,
oxaloacetate forms aspartate.The enzyme aspartate
kinase converts aspadateto aspartyl phosphatewhich
later forms aspaftate semialdehyde. Aspartate
semialdehydehas two fates-the biosynthesisof lysine
and formation of 3 other amino acids (methionine,
threonine and isoleucine). When homoserine
dehydrogenaseacts on aspaftate semialdehyde, it is
B IOTE C H N OLO CY
divertedfor the synthesisof 3 amino acids.The enzyme
dihydrodipicolinate synthase converts aspartate
semialdehyde (and pyruvate) to piperideine 2,
6-dicarboxylate.There are two distinct enzymes
succinylasevariant (catalyses4-step reaction) and
dehydrogenase variant(catalysesa singlestepreaction)
that can conveft piperideine2, 6-dicarboxylateto D,
L-diaminopimelate which later forms L-lysine.
Regulation of L.lysine biosynthesis
The following are the regulatoryprocessesin the
production of lysine (Fig. 26.Q.
Aspartate kinase : This enzyme is controlled by
feedback inhibition of the end products. Three
isoenzymes of aspartate kinase have been
i d e n t i f i e d - o n e r e p r e s s e d b y L - m e t h i o n i n e , th e
second one repressed by L-threonine and
L - i s o l e u c i n e ,a n d t h e t h i r d o n e b e i n g i n h i b i t e d a n d
repressedby L-lysine.The amino acid sequenceand
structureof asoartatekinasehave been elucidated.And
by genetic manipulations, it has been possible to
create mutants (of aspartate kinase) that are
i n s e n s i t i v et o f e e d b a c k r e g u l a t i o n b y L - l y s i n e .
Dihydrodipicolinate synthase : This enzyme
competes with homoserine dehydrogenaseto act
on aspartate semialdehyde. Overexpression of
d i h y d r o d i p i c o l i n a t e s y n t h a s e h a s b e e n s h o w n to
i n c r e a s et h e p r o d u c t i o n o f L - l v s i n e .
Succinylase and dehydrogenase variants : The
c o n v e r s i o n o f p i p e r i d e i n e 2 , 6 - d i c a r b o x y l a t et o D ,
L - d i a m i n o p i m e l a t e i s c a r r i e d o u t b y t h e s e tw o
enzymes. At the start of the fermentation,
dehydrogenase variant predominantly acts, and
later succinylasevariant comes into picture for the
b i o s y n t h e s i so f L - l y s i n e .
Role of D, L-diaminopimelate : This amino acid,
an immediate precursor for the synthesis of
L-lysine, is also required for the synthesisof a
t r i p e p t i d e ( L - A l a - y - D - C l u - D ,L - D a p ) w h i c h i s p a r t
o f t h e p e p t i d o g l y c a n o f c e l l w a l l . T h e a c t i v i t i e so f
both the enzymes (succinylaseand dehydrogenase)
that form diaminopimelate (Dap) are important for
the production of L-lysine and for the proper
formation of cell wall structure.
lmproved production strains
Based on the biosynthetic pathway and
the regulatory steps (discussed above), certarn
imorovements have been made in the strains of
 OF A MIN O A C ID S
Chapt e r2 6 : MIC R OB IALPR O D U C T I ON 351

Glucose

Phosphoenolpyruvate
+
Pyruvate

Oxaloacetate
:1
Citricacid
i Krebs
Malate cvcle
t-
0,-Ketoglutarate
Asparticacid
"'
I n"p"n","
Krnase
I
Aspartyl
phosphate

+ Homoserine
+ dehydrogenase
Aspartate -----+ -----+ ----J L-Methionine
semialdehyde L-Threonine
I L-lsoleucine
Pyruvate | Dihydrodipicolinate
- ) synthase
+
J
2, 6-
Piperideine

pimelate Ala-Glu-DaP

t
L-LYSINE
(peptidein cell wall)

Fig. 26.4 : Biosynthesis of Llysine rn C. glutamrcum

(Ala-Glu-Dap - Alanybglutamyl-diaminopimelate, a tripeptide)

C. glutamicum and B. flavum for overproduction of o A strainwith reducedcitratesynthaseactivity (to

\ stn e. lower the occurrenceof citric acid cvcle).
. ,llutant organisms resistant to lysine
antimetabolites(e.9. b-aminoethyl-L-lysine). Release of L.lysine
r \ mutant strain with an altered enzyme The export or releaseof L-lysine from the
aspartokinase,so that it is not regulated by end cel l s i nto the surroundi ng
medi umoccursthroug h
oro du c t inhibit ion. a lysine-export(LysE)carrier protein. lt is a trans-
r \ strain with a decreased homoserine membrane protein (mol. wt-25,400) with six
:ehvdrogenase activity (so that diversion for the segmentsthat participatein lysine transport.The
=i:th esis of m et hionine,t hr eonine and i s o l e u c i n e exporter system is very efficient active processto
s -nin im is ed) . export largequantitiesof intracellularlysine.
 352 B IOTE C H N OLO CY

Both the above grades of iysine are suitable for

s u p p l e m e n t a t i o no f f e e d s .

T
L-Threonine is manufactured industrially by
employing either E coli or C. glutamicum. With
c
the mutant strains of E. coli, the product yield rs
E better
E
0)
c
Biosynthesis of L-threonine
O
The metabolic pathrvay for the synthesis of
L-threonine is depicted in Fig. 26.6 Some of the
reactions of this pathway are common for ihe
Fermentationtime ______+ b i o s y n t h e s i so f L - l y s i n e a n d m e t h i o n i n e , b e s i d e s
isoleucine (Refer Fig. 26.4 also). Starting lvith
Fig. 26.5 : Production of L-lysine n relation to
a s p a r t i ca c i d , i n a s e q u e n c eo f f i v e s t e p s ,t h r e o n i n e
substrate (glucose) and biomass concentration.
is produced

Regulation : The regulatory reactions in E. coli

Production process of L-lysine f o r L - t h r e o n i n eb i o s y n t h e s i sh a v e b e e n e l u c i d ate d .
Three isoenzymes of aspartate kinase, separately
The most commonly used carbon sources for
I lysine rnanufactureis molasses(cane or sugar beet),
i n h i b i t e d b v t h e e n d p r o d u c t s h a v e b e e n i d e n t ifi e d -
I starch hvdrolvsatesor sucrose. The other sources
o n e b y L - t h r e o n i n e ,o n e b y L - m e t h i o n i n e a n d o n e
by L-lysine.Further,two isoenzymesof homoserine
I like acetate,ethanol or alkanes are used to a lesser
d e h y d r o g e n a s e - o n ei n h i b i t e d b y L - t h r e o n i n e a n d
n extent.
I
fhe nitrogen sources are ammonium salts,
gaseousammonia. Protein hydrolysatesare added Asparticacid
to s upply c er t ain am ino ac ids ( L - m e t h i o n i n e , I Aspartate
Klnase
protern I
L-h om os er ine, L- t hr eonine) . The Y
Aspartyl
hydrolysates also supply growth factors such as phosphate
b ioti n.
+
I
A time-course graphic representation for the
formation of lysine is depicted in Fig. 26.5. As ts ^^tP311119,^
semlaloenyoe
L-r-ysrne
e vid ent , a c ont inuous s upply of glu c o s e ( o r o t h e r I Homoserine
sug ar )is r equir edf or s us t ainedpr odu c t i o n o f l y s i n e .
Jdehydrogenase
Un der opt im al f er m ent at ionc ondit io n s ,t h e y i e l d o f Homoserine L-Methionine
Iysin e ( in t he f or m of L- ly s ine HCI ) i s 4 0 - 5 0 g p e r
I Homoserine
1 00 g c ar bon s our c e. I Kmase
+
There are different recovery processesfor lysrne Homoserine
phosphate
d ep ending on it s applic at ion. t-.
I Inreonrne
c ont aining a b o u t 5 0 % syntha8e
" An alk aline s olut ion I
L -ly s ine c an be obt ained a f t e r b i o m a s s
L-THREONINE
separation,evaporation and filtration.

o A c r y s t alline pr epar at ion w i t h 98-99% +

I
L -ly s ine ( as L- ly s ine HCI ) c an be o b t a i n e d b y L-lsoleucine
subjec t ing t he c ult ur e br ot h t o i o n - e x c h a n g e
Fig. 26.6 : Biosynthesis of L-threonine ln E. coli.
chromatography,evaporation and crystallization
 26 : MIC R OB IALP R OD U C T IO N
OF A MIN O A C ID S

other by L-methionineare also known. A gene Erythrose Phosphoenol

thrABC that encodes three polypeptides(one 4-phosphate pyruvare
polypeptidepossesses the activity of kinaseand
homoserine dehydrogenase, the secondhomoserine
kinaseand the third threoninesvnthase)in E. coli
has been identified.
lmproved production strains : The efficiencyof
the producerstrainscan be increasedby creating Deoxyarabinoheptulo-
sonatephosphate
E. coli mutantswith high-levelexpressionof the
gene thrABC. Further, mutants with minimar T
pr oduc t io no f L -i s o l e u c i nael s o re s u l ti s h i g h yi el d +
of L-threonine. +
Y
Production p;ocess of L.threonine
T he c u l tu re me d i u m c o n ta i n i n gg l u ' coseor
J
Shikimate
sucrose/yeast extract and ammonium salts rs
adequatefor L-threonineproduction.The sugar
J
I Shikirnate
feedinghas to be continuedfor good yield (about + kinase
I
60"h of the carbon source). +
Chorismate,-f--f-| L-Tryptophan
The downstreamprocessing for the isolationof
L- t hr eoninceo n s i s ts o f c o a g u l a ti oonf th e c el ! mass IChorisrnate
(by heat),filtration,concentrationby evaporation,
I ll
I mutase
+
and c r y s t a l l i z a ti o n .
..>.->-+ L-Tyrosine
Prephenate
I
{ Prephenate
I dehydratase
+
Both E coli and C. glutamicumcan be usedfor L-PHENYLALANINE
the productionof L-phenylalanine. The biosynthetic
pat hwayis q u i tec o mp l e xa n d a n o u tl i n ei s show n Fig. 26.7 : An outline of the pathway for the
in Fig. 26.7. An interestingfeatureis that the same synthesis of L-phenylalanine, L-tyrosine
pathwayis responsible and L-tryptophan-
for the synthesisof all the
three aromatic amino acids-tyrosine and
t r y pt ophanb, e s i d e sp h e n y l a l a n i n e .
The syntheticpathway commenceswith the
condensation of erythrose 4-phosphate with
phosphoenol pyruvate to form deoxy-
arabinoheptulosonate phosphate(DAHP).DAHP in There are different ways of synthesizing
the next series of reactions is converted to L-tryptophan-chemical, enzymaticand fermentation
chorismate which can form L-tryptopnan. methods At present,large scale manufactureof
Chorismate mutase converts chorismate to tryptophanis carried out by using the enzyme
pr ephenate through tryptophan synthaseof E. coli. Tryptophansynthase
w h i c h fo rms L -p h e n y l a l a n i ne
the participation of prephenatedehydrogenase.combinesindolewith L-serineto form tryptophan.
Prephenatealso serves as a precursorfor the
Indole + L-serine
synthesisof tyrosine.
The genesresponsible for the formationof the
regulatoryenzymesof L-phenylalanine have been
ident if ied.B y e m p l o y i n gg e n e ti c m a n i p ul ati ons,
strainsfor improvedproductionof L-phenylalanine
have been develooec.

Biotechnology
[23]
354 B IOTE C H N OLO CY

encoding anthranilate synthase,a key enzyme in

Chorismate.:: ---y L -P henyl al ani ne (Fig,26.A. Further,genesencoding
- - --*L-Tvrosrne its biosynthesis
|
lAnthranilate other important enzymes (deoxyarabino-
I synthase heptulosonatephosphate synthase,anthranilate
+
phosphoribosyltransferase) were also be modified.
Anthranilate
The resultis that the pathwaybecomesinsensitive
to feedbackinhibitionby end products,leadingto
an overproduction of L-tryptophan.
.t

Thereis a growingdemandfor aspartate,as it is

L-TRYPTOPHAN
Fig. 26.8 : Biosynthesis of L-ttyptophan (For the
Indoleis availablefrom oetrochemical industries formationis favoured.
while L-serinecan be recoveredfrom molasses
during sugarrefinement.Mutant strainsof E. coli
with high activityof tryptophansynthasehave been
developed for large scale manufacture of
tryptophan.
Direct fermentation process : Tryptophancan
also be produced by fermentationemploying
C. glutamicum, or E. coli. For the biosynthetic
pathway, refer Fig. 26.7. Mutant strains of both
theseorganismshavebeen developedfor increased
yield of tryptophan.
Mutant strains for overproduction
L.tryptophan
a componentof aspartame(an artificialsweetener),
besides its use as a food additive, and in
pharmaceutical preparations.
The preferredmethodfor aspartateproductionis
enzymatic in nature. The enzyme aspartase
converts fumarate and ammonia to aspartate.
Although this reaction is reversible,aspartate
umarate+ Ammonie
Aspartate
The aspartaseof E. coli is used. lt is a tetramer
w i th a mol ecul arw ei ght 196,000.Thi s enzymeis
quite unstable. lmmobilization of aspartasein
polyacrylamide or carrageenan that enhancesthe
stability of the enzyme is commonly used.

The productionof tryptophanby C. glutamicum lmmobilizedE. coli cells with good activityof
was increasedby introducing a second gene are also usedfor aspartateproduction.
aspartase
\ /itamins are organic compounds that pefbrm
V specific biological functions for normal
nraintenanceand optimal Browth of an organism
These vitamins cannot be synthesized by the
higher organrisrnt including man, and therefore
they have to be suppliedin small amountsin the diet.
Microorganisms are capable of synthesizing the
vitamins. In fact, the bacteria in the gut of humans
ca n pro du c e s om e of t he v it am ins , whi c h i f
appropriately absorbed can partially meet the
body's requirements.lt is an acceptedfact that after
ad ministrati on of s t r ong ant ibiot ic s t o hu m a n s
(which kill bac t er ia in gut ) , addit ional c ons um p t i o n
of vita mins i s r ec om m ended.
Micro org anis m sc an be s uc c es s f ullyus ed fo r t h e
co mmercial pr oduc t ion of m any of t he v it a m l n s
e.g . thia mine, r ibof lav in, py r idox ine, f olic a c i d ,
pa nto the nic ac id, biot in, v it am in B12, asc o r b i c
acid, p-carotene (provitamin A), ergosterol
(provitamin D). However, from economic point
of view, it is feasible to produce vitamin
Btz,ribo flavin, as c or bic ac id and B- c a r o t e n e
by microorganisms. For the production of
ascorhic acid (vitamin C), the reader must refer
Chapter 24.
Tre disease, pernicious anemia, characterized
o u le ve ls of hem oglobin, dec r eas ednum b e r o f
erythrocytes and neurological manifestations,has
b e e n k n o w n {o r s e v e r a l d e c a d e s . l t w a s i n 1 9 2 6
some workers reoorted the liver extracts could
cure pernicious anemia. The active principle was
later identified as vitamin 8,,, a water solubie
B - c o n r o l e xv i t a m i n .
Occurrence
V i t a m i n 8 . ,, i s p r e s e n ti n a n i m a l t i s s u e a t a v e r y
low concentration (e.9. 1 ppm in the liver). It
occurs mostly in the coenzyme forms-
methylcobalamin and deoxyadenosylcobalamin.
l s o l a t i o n o f v i t a m i n 8 . ,, f r o m a n i m a l t i s s u e si s v e r y
e x o e n s i v ea n d t e d i o u s .
Ghemistry
V i t a m i n 8 1 2 ( c y a n o c o b a l a m i n )i s a w a t e r s o l u b l e
vitamin with complex structure. The ernpirical
formula of cyanocobalaminis C63H90N140t4PCO.
T h e s t r u c t u r e o f v i t a m i n 8 . " c o n s i s t so f a c o r r i n
ring with a central cobalt aiom. The corrin ring is
almost similar to the tetrapyrrole ring structure
found in other porphyrin compounds e.g. heme
(with Fe) and chlorophyll (with Mg).
The corrin ring has four pyrrole units. Cobalt
present at the centre of the corrin ring is bonded to
the four pyrrole nitrogens. Cobalt also binds to
dimethylbenzimidazole and aminoisopropanol.
Thus, cobalt atom present in vitamin Bt, is in a
coordination state of six.
355
356 B IOTE CHNO LO CY

SuccinylCoA present, vitamin Btz is entirely produced by

I
fermentation.lt is estimated that the world'sannual
l7-Gtycine producti onof vi tami n8' ' , i s around15,000kg.
J
6-Aminolevulinic
acid of vitamin8,, are detected
High concentrations
solids. This is produced by
I
J
in sewage-sludge
microorganisms.
sewage-sludge
Recoveryof vitamin 8', Trom
was carried out in some parts of
Porphobilinogen United States.
I
I U nl i ke most other vi tami ns, the chem ical
synthesisof vitamin 8.,, is not practicable,since
IV about 20 complicatedreactionstepsneed to be
ProtoporphyrinlX Uroporphyrinogenlll
carried ouL. Fermentation of vitamin B, is the
only choice.

Microorganisms and yields of

vitamin B*
Severalmicroorganisms can be employedfor
Riboflavin Cobinamide the producti onof vi tami nB 1r,w i th varyingyields.
C l ucose i s the most commonl y used car bon
JI source.S ome exampl esof mi crobesand t heir
corresponding yields are given in Table27.1. The
most commonly used microorganismsare-
Propionibacterium freudenreichii, Pseudomonas
denitrificans, Bacillus megaterium and
Streptomyces o Ii vaceus.
5'-Deoxyadenosyl-
cobalaminphosphate Genetically engineered strains for vitamin B'
production: By employingmoderntechniquesof
+
I geneti cengi neeri ng,vi tami nB 12product ion
can be
5'-Deoxyadenosyl- enhanced.A protoplastfusiontechniquebetween
cobalamin Protaminobacter rubber and Rhodopseudomonas
(vitamin812)
spheroidesresulted in a hybrid strain called
Rhodopseudomonas protamicus. This new strain
can produceas hi gh as 135 mgl l o{ vi tam in 8, ,
uti l i zi necarbonsource.
Biosynthesis
Vitamin Brris exclusivelysynthesizedin nature
by microorganisms.An outline of the pathway is
depictedin Fig. 27.1. The biosynthesis of 8,, is Microorganism Yield (mgfl)
comparable with that of chlorophyll lno
hemoglobin.Many of the reactionsin the synthesis Bacillusmegateriun 0.51
of vitamin 8.,, are not yet fully understood. ny
Strepto cesolivaceus
Butyribacte
rium rettgeri 5.0
COMMERCIAL PRODUCTION
o F v tT AMtN B rz
Micromonospora sp 11.5
Propionibacteriun freudenreichii 19.0
Vi ta m i n 8 ,, i s c o mme rci al l yproduced by
fermentation.lt was first obtainedas a byproductof Propionibacterium shermanii 35.0
Streptomycesfermentation in the production of Pseudomonas denitrificans 60.0
certain antibiotics(streptomyci n, chloramphen icol, Hybridstrain
or neomycin).But the yield was very low. Later,
protamicus
Rhodopseudomonas 135.0
high-yielding strains were developed. And at
 OF V ITA MIN S
Chapt e r2 7 : MIC R OB IALPR O D U C T I ON 357

Production of vitamin B* using Garbon sources for

Propionibacterium sp vitamin B* Froduction
Propionibacterium freudenreichii and P. shermanii, Glucose is the most commonly used carbon
a nd th eir m ut ant s t r ains ar e c om m only u s e d f o r s o u r c e f o r l a r g e s c a l e m a n u f a c t u r eo f v i t a m i n 8 . ,,
vitamin 81, pr oduc t ion. The pr oc es s is c a r r i e d o u t Other carbon sources like alcohols (methanol,
b y ad din g c obalt in t wo phas es . ethanol, isopropanol) and hydrocarbons (alkanes,
d e c a n e , h e x a d e c a n e )w i t h v a r y i n g y i e l d s c a n a l s o
An ae r obic phas e : This is a pr elir nin a r y p h a s e
be used.
that may take 2-4 days In tl.re anaerobic phase
5 '-de oxy adenos y lc obinam ide is pr ed o m i n a n t l y A y i e l d o f 4 2 n l {l o f v i t a m i n 8 , , w a s r e p o r t e d
produced. using methanol as the carbon source by ttre
microorgarrism Methanosarcina barkeri, in fed-
Aero bic phas e : I n t his phas e, 5, 6 - d i m e t h y l -
batch culture system.
benzinidazole is oroduced from riboflavin which
geis incorporated to finally form coenzynre of
vitamin B' ' , nam ely 5' - deox y ader r os y lc o b a l a m i n
In recent years, some ferrnentationtechnolog,ists
h ave su c c es s f ullyc lubbed bot l. ran anae r o b i c a n d
R i b o f l a v i n ( v i t a m i n B ''r ) i s a w a t e r s o l u b l e
a ero bic phas es t o c ar r y out t he o p e r a t i o n
v i t a m i n , e s s e n t i a lf o r g r o w t h a n d r e p r o d u c t i o n i n
con tinu ous ly in t wo r eac t ion t ank s .
man and animals. Deficiency of riboflavin in rats
The bulk production of vitamin 8r, is rnostly causes growth retardation, dermatitis and eye
done by submerged bacterial fermentation with lesions ln humans, vitamin Brdeficiency resultsin
beet molasses medium supplemented with cobalt cheilosis(fissuresat the corner of mouth), glossitis
chloride. The specific details of the process are (purplish tourrge) and dermatitis. Riboflavin exerts
kept as a guarded secret by the companies. its biochemical functions through the coenzymes
(FAD) and
Recovery of vitamin 8.,, : The cobalamins namely flavin adenine dinucleotide
produced by fermentation are mostly bound to the flavin mononucleotide (FMN).
cells. Th ey c an be s olubiliz ed by heat t r e a t m e n t a t
Occurrence
B0 -1 20 'C f or about 30 m inut es at pH 6. 5 - 8 . 5 . T h e
solid s a nd m y c elium ar e f ilt er ed or c entr i f i g e da n d R i b o f l a v i n o c c u r s i n m i l k a n d m i l k p r o d u c t s,
the fe rm ent at ion br ot h c ollec t ed. The co b a l a m r n s m e a t , e g g s , l i v e r a n d k i d n e y . Wh i l e i n m i l k a n d
can be converted to more stable cyanocobalamins eggs, it is present in free form, in other foods it rs
(i.e coenzvmes
This vita m in B, , is ar ound B0ol,pur it y a n d c a n b e f o u n d i n t h e f o r m o f f l a v o p r o t e i n s
o f r i b o f l a v i n b o u n d t o p r o t e i n s ) .
directly used as a feed additive. However, for
me dical us e ( par t ic ular lyf or t r eat m entof p e r n i c i o u s
Chemistry
a ne nria ) , v it am in B, s hould be f ur t he r p u r i f i e d
(95-98% purity). Riboflavin contains 6, 7-dimethyl isoalloxazine
(a heterocyclic 3 ring structure) attached to
Production of vitamin Br. D-ribitol by a nitrogen atom. The isoalloxazinering
using Pseudomonas sp p articipates in the oxidation-reduction reactions
b r o u g h t o u t b y t h e c o e n z y m e s( F A D a n d F M N ) .
Pseudomonas denitrificans is also used for large
sca le pro duc t ion of v it am in B, , in a c os t - e f f e c t i v e Biosynthesis
manner Starting vvith a low yield (0.6 mg/l) two The biosynthetic pathway of r i b o f l a v r n,
decades ago, several improvements have been elucidated for the microorganismsAshbya gossypii
made in the strains of P denitrificans ior a and Eremothecium ashbyii is depicted in Fig. 27.2.
tre men dous im pr ov er nentin t he y ield ( 6 0 m g / l ) . The overproduction of riboflavin in these organisms
Ad dition of c obalt and 5, 6 - d i m e t h y l takes place mainly due to the constitutive nature of
be nzimidaz ole t o t he m edium is es s e n t i a l . T h e t h e r i b o f l a v i n s y n t h e s i z i n g e n z y m e s . l r o n w h i c h
rTieldo f vit am in B1, inc r eas eswl. r ent he m e d i r t m t s i n h i b i t s t h e p r o d u c t i o n o f v i t a m i n 8 , , i n c l o s t r i d i a
sup ple m ent ed wit h bet aine ius ual s ou r c e b e i n g and yeasts, has no effect on A. gossypii ancl E.
suq ar b eet m olas s es ) . ashbyii.
358 B IOTE C HNO LO CY

Guanosinetriphosphate riboflavin. In the acetone-butanolfermentation,

II employing the organisms Clostridium aceto-
J butylicum and Clostridiumbutylicum, riboflavin is
2, S-Diamino6-keto4- formed as a byproduct.
(5'-phosphoribosyl
amino)-pyrimidine C ommerci al producti on of ri bof lavin t s
II predominantlycarried out by direct fermentation
+ using the ascomycetes. The different organisms
S-Amino2,S-dioxy4- usedand the corresponding yieldsof riboflavinare
(5'-phosphoribotyl
amine)-pyrimidine given in Table 27.2. The two plant pathogens
I namelyAshbyagossypiiand Eremotheciumashbyii
+
I are most commonl yempl oyeddue to high yield.
Diaminouracil Amongthesetwo organisms, A. gossypiiis preferred
as it is more stablewith a high producingcapacity
II of riboflavin.
+ Genetically engineered strains for riboflavin
6-Methyl7-(1', 2' -dihydroxyethyl)-
producti on: H i gh yi el di ng strai nsof Ashbya
8-ribityllumazine
gossypii have been developed by genetic
II S uchstrai nscan yi el das highas 15
mani pul ati ons.
+ g/1 riboflavin.
6, 7-Dimethyl8-ribityllumazine
II Production process of riboflavin
+ Industrial production of riboflavin is mostly
Riboflavin carried out with the organism , Ashbyagossypiiby
usi ngsi mpl esugarssuchas gl ucoseand cor n st eep
liquor. Clucose can be replacedby sucroseor
maltosefor the supplyof carbonsource.In recent
years,lipids such as corn oil, when addedto the
medium for energy purpose, have a profound
COMMERCIAL PRODUCTION i nfl uence on ri bofl avi n producti on . Fur t her ,
O F R IBOF L A VIN supplementationof the medium with yeast
Therearethreeprocesses employedfor the large extract, peptones, glycine, inositol, purines
scale production of riboflavin. The worldwide (not pyri mi di nes)al so i ncreasethe yield of
requirement of riboflavinis estimated
to be around ri bofl avi n.
2,500 tonesper year. to careful l ysteri l i zethe m edium
It i s essenti al
1. Biotransformation: About 50% of the for good yield of riboflavin.The initial pH of tne
world's requirementof riboflavinis producedby cul turemedi um i s adi ustedto around6- 7. 5.f he
biotransformation, followedby chemicalsynthesis.
For this purpose,glucose is first converted to
D-riboseby mutantstrainsof Bacilluspumilus.fhe
D-riboseso producedis convertedto riboflavinby
chemicalreactions.
2. Chemicalsynthesis: Approximately20'/' oI Microorganism Yield (mp/l)
the world's riboflavin is produced bv direcr Clostridi un acetobutylicum 0.097
c hem ic al s y nt hes is . Clostridiunbutylicum 0.120
3. Fermentation : At leastone third of world's Mycobacterium smegnatis 0.060
riboflavin requirements are met by direct
Mycocandida riboflavi
na 0.200
fermentation orocesses.
Candidaflareri
Microorganisms and yields of riboflavin 2.500
ashbyii
Erenothecium
Several microorganisms (bacteria, yeasts and
Ashbyagossypii 7.500
fungi) can be employed for the production of
 O F V ITA MIN S
Chapt er27 : MIC R OB IALP R OD U C T IO N 359

fermentation 26-28'C riboflavin productionare aliphatic hydrocarbons

is conductedat temperature
with an aeration rate 0.3 vvm. The processis (organismPichiaguilliermoudii)and n-hexadecane
carried out for about 5-7 days by submerged (organisms - Pichia miso).
aeratedfermentation.
Riboflavin fermentation by Eremothecium
ashbyiiis comparableto that describedabovefor
Ashbya gossypii. Candida sp can also produce
riboflavin, but this fermentation process is
p-Caroteneis the provitamin A. When ingested,
extremely sensitive to the presence of iron.
Consequently,,iron or steel equipmentcannot be it gets convertedto vitamin A in the intestine.
used.Suchequipmenthaveto be linedwith plastic Vitamin A is a fat soluble vitamin requiredfor
m at er ial. vision, proper growth and reproduction.The
deficiency of vitamin A causes night blindness,
Fermentation through phases changes i n the ski n and mucosalmembranes.
Some studies have been carried out to Occurrence and chemistry
understand the orocessof fermentation of riboflavrn
p-C arotene i s found i n many ani maland pl ant
particularlyby ascomycetes. lt is now acceptedthat
tissues. However, it originatesexclusivelyfrom
the fermentation occursthroughthree phases.
pl antsor mi croorgani sms. Y el l owand dark green
Phase| : This phaseis characterized by rapid vegetablesand fruits are rich in
B-carotenee.g.
gr owt hof t h e o rg a n i s m . pyruvi c carrots,spi nach,amaranthus,
u ti l i z i n gg l u c o s eAs mango,papaya.
acid accumulates, pH becomesacidic.The growth
of the organismstopsas glucosegetsexhausted. In Carotenoids are isoprene derivatives.
phasel, there is no productionof riboflavin. C hemi cal l they
y, are tetraterpenoi w i th ei ght
ds
isopreneresidues.Thereare around400 naturally
Phasell : Sporulationoccursin this phase,and
occurring carotenoids. The most important
pyruvateconcentrationdecreases. Simultaneously,
carotenoids are $-carotene, G,-carotene, 6-carotene,
t her e is a n a c c u m u l a ti o no f a mmo n i a (due to
lycopeneand zeaxanthin.
enhanceddeaminaseactivity) which makes the
m edium al k a l i n e .P h a s el l i s c h a ra c te rized by a Carotenoids are mainlyusedas colouringagents
m ax im alpro d u c ti o no f ri b o fl a v i nBu . tth i s i s mostl y e-9., p-carotene,lycopene,xanthophylls.Several
in the form of FAD and a small ooftion of it as foods (cheese,meat, egg products)can be made
F M N. attractiveby coloration.lt may be noted that the
phase, get demand for B-caroteneas the provitaminA is
Phaselll : In this last cells disrupted
comparativelyless.
by a processof autolysis.This allows releaseof
FAD, FMN and free riboflavininto the medium. Biosynthesis
Recovery: Riboflavinis founcjin fermentation
The pathwayfor the biosynthesis of B-carotene
broth and in a bound form to the cells.The latter
and some other important carotenoids, elucidated
can be releasedby heat treatmenti.e. 120'C for
in plantsand fungi, is shown in Fig. 27.3.
about t hour. The cells can be discardedafter
filtration or centrifugation.The filtrate can be
COMMERCIAL PRODUCTION
furtherpurifiedand dried,as per the requirements.
OF B.CAROTENE
Other carbon sources for riboflavin B-Carotenecan be produced bry microbial
production fermentation.However, for economic reasons,
Besidessugars,other carbonsourceshave also direct chemicalsynthesis of vitaminA is preferred
production.A pure grade ratherthan using its provitamin (F-carotene).
been usedfor riboflavin
of riboflavin can be prepared by using
Microorganisms
Saccharomyces sp, utilizing acetateas sole carbon
source. Methanol-utilizingorganism Hanbenula The organismsBlakesleatrispora, Phycomyces
polymorpha was found to produce riboflavin. The blakesleeanusand Choanephoracucurbitarum are
-'th,e.carbon sources used with limited success for most frequently used for the production of
360 B IOTE CHNO LO CY

2 AcetylCoA

+
I carotenoids
Acetoacetyl
a:i Carotenoid Organism Production
^ .
CoA yield (g/l)
lr-Acetyl
+ p-Carotene Blakeslea
irispora 3.0
B-HydroxyB-methyl (mixed of
cultures
glutarylCoA
+ and- sexualforms)

+
I Lycopene Blakeshlea trispora 0.4
(mixedculture)
Mevalonicacid5-PP
I Streptomyceschrestomyceticus 0.5

Dimethylallyl-PPH
J s-PP
lsopentenyl
Zeaxanthin Flavobacterium
sp 0.4

Production process of B-carotene

A s a l r e a d y s t a t e d , t h e i n d u s t r i a l p r o d u cti o n o f
B-carotene is mostly carried out by Blakeslea
trispora. The fermentation medium contains corn
s t a r c h , s o y b e a n m e a l , B - i o n o n e , a n t i o x i d a n ts e tc.
Farnesyl-PP
(C15)
Addition of antioxidants imoroves the stabilitv of
B-carotene with in the cells. fhe fermentation is
carried out by submerged process.
Geranyl-geranyl-PP
(C20)
l 'h e f e r r n e n t a t i o ni s u s u a l l y s t a r r e db y m i xi n g th e

+
I cultures of both sexual forms, (+) and (-) strainsof
B. trispora.The vield of B-carotene is significantly
phytoene (Cas) h i g h e r w i t h m i x e d c u l t u r e s , c o m p a r e d to + o r -

J
I strains (Frg. 27.4). fhis is due to the fact that

Phytofluene

+
I
Neurosporene
c

E
c
Lycopene Zea carotene 0)

+
I +
I c
o
o
c)
p-Carolene 6-Carotene c
0)

t
I

+
I 6
Zeaxanthin o-Carotene o
I

Fig. 27"3 : Biosynthesis of carotenes

B-carotene Among these, B/akeslea trispora s Glucoseconcentration

preferreddue to high yield. In the Table 27.3, some
Fiq.27.4: Yield of B-carotene by *, - and mixed
im por t ant c ar ot enoids , t he o r g a n i s r n s a n d t h e
(+ -) cultures o/ Blakesbatrispora.
pr oduc t ion y ields ar e giv en.
Chapter27 : MICROBIALPRODUCTIONOF VITAMINS 361

B-carotene productionpredominantly occursduring

the process of zygospore formation. lt may be
statedhere that the use of mixed strainsdoes not
improve the yield for other microorganisms (as
Cibberellinsare plant hormonesthat stimulate
observedin case o{ Blakesleatrispora). plant growth. They promote growth by cell
Factors affecting production : Trisporic acid enl argementand cel l di vi si on. The observabl e
which can act as a microbial sexual hormone effects of gibberellinsinclude stimulus to seed
improves production yield of B-carotene. germination,floweringand lengtheningof stems.
p-lonones enhance p-carotene synthesis by
increasingthe activityof enzymes,and not by their MICROBIAL PRODUCTION OF
direct incorporationinto B-carotene.When the GIB B E R E LLIN S
fermentationmediumis supplemented with purified So far only one microorganism,the fungus
kerosene, p-carotene productionis almostdoubled. namely Gibberella fujikuroi has been found to
Keroseneincreasesthe solubilityof lrydrophobic produce gibherellins.This is actually a pathogenic
substrates. fungusof ri ce seedl i ngs.
Recovery: The myceliumrich in B-carotene can C i bberel l i nproducti oncan be carri edout by
be directlyusedas a feedadditive.For purification, usi ng a gl ucose-salmedit um at pH 7.5 and
myceliumis removed,subjectedto dehydration(by temperature25oC for 2-3 days. The fermentation
methanol)and extractedin methylenechloride. processis conductedin aeratedsubmerged process.
This product is of 7O-85'h purity which can be After the growth of the fungus is maximum,the
furtherpurifiedas per the iequirements. productionof gibberellinscommences.
 II t is estinrateci that about 20-30% of rne people A selected list of fermented foods along
I h oLr s eholdbudget is s pent t owar d s f o o d s i n t h e w i t h r a w m a t e r i a l sa n d f e r m e n t i n g m i c r o o r g a ni sm s
de ve loped c ount r ies .This m ay be a l i t t l e l e s s i n t h e is given in Table 28.1 .
developing nations. Therefore, food and beverage
bio tec hnology oc c upies a pr om inen t p l a c e w o r l d - Advantages of fermented foods
over. . E n h a n c e dn u t r i t i v e v a r u e .
Ther e ar e r ec or ds t hat m an was m a k i n g b r e a o , . I n c r e a s e dd i g e s t i b i l i t y
win e, c ur d et c , as ear ly as 400 0 B C . T h e s e . Imoroved flavour and texture.
processes,collectively referred to as traditional or .
S e r v ea s s u p p l e m e n t si n p r e p a r i n gs e v e r a ld i sh e s.
old biotechnology, mostly employed for the
preparationof foods and beverages,were based on Besides the Iist given in Table 28.1, there are
the natural capabilities of the microorganisms m a ny other compounds that are directly or
(a lthough t heir ex is t enc e was unk n o w n a t t h a t i n d i r e c t l y u s e d a s f o o d s u p p l e m e n t s w h i c h mu st
u mel also be referred by the reader. These include
vitamins (Chapter 27), amino acids (Chapter 26),
Wit h t he adv anc es m ade in m icr o b i o l o g y a n d
organic acids (Chapter 23), enzymes (Chapter 21),
recently biotechnology, food and beverage
b i o p o l y m e r s ( C h a p t e r3 0 ) , s i n g l e - c e l lp r o t e i n s a n d
p rod uc t ion is a m ajor indus t r y .Food b i o t e c h n o l o g y
m u s h r o o m s ( C h a p t e r2 9 ) .
is a ls o c onc er ned wit h t he im pr o v e d q u a l i t y ,
nutrition, consistency, colour, safety and A selected few of the fermented foods with
preservation of foods, besides making them special referenceto their production are described
a va ilable r ound t he y ear ( Not e : M o s t f o o d s a r e b r i e f l y i n t h i s c h a p t e r .
seasonal in nature and therefore as such are nor
a va ilablet hr oughout t he y ear ) I n add i t i o n , m o d e r n CHEESE
b iote c hnologic al pr oc es s esals o t ak e i n t o a c c o u n t Cheese production is the largest dairy industry
.l
the health aspects of the people in the world. There are around ,000 types of
different cheeses l-hey are broadly of two types -
unripened cheeses (cottage cheese with low fat,
cream cheese with high fat) and ripened cheeses
(hard cheese e.g chedder, blue cheese;soft cheese
The pr oduc t ion of f er m ent ed f ood s i s v a r i a b l e . e.g. limburger,camembert). lrrespectiveof the type
Th is depends on geogr aphic al r egio n , a v a i l a b i l i t y of the cheese,all of thern are invariably made from
of raw materials,traCitions and food habits of tne the casein qf milk, that is produced after separating

362
 OF FOOD SA N D B E V E R A C E S
Chapt er28 : M IC R O B IALP R OD U C T IO N 363

Fermented food/food product Raw material Fermenting organism(s)

(country) (substrate)

Dairy products
(worldwide)
Cheese Milk Streptococcus
sp
Penicilliunroquefortii,
P. camembertii
Yogurl(worldwide) Mitk Streptococcus thermophiIus
Lactobacillus
bulgaricus
Kefir(Russia) Mitk Lactobacillus
sp, Candidasp

Vegetarianproducts
Cocoabeans(worldwide) Cocoafruit Candidakrusei,
Geotrichun sp
Coffeebeans(worldwide) Coffeecherries Edwiniadissolvens,
Saccharonycessp
(lndonesia)
Tempeh Soybeans Rhizopusoryzae,Lactobacillus
delbrueckiia
Soysauce(worldwide) Soybeans Aspergillus
orzyae,
A. soyae
(Europe)
Sauekraut Cabbage Leuconostocnesenteroides,
L.plantarum
Breads(worldwide) Whealflour Saccharomycescerevisiae
Rolls,cakes(worldwide) Wheatflour Saccharomycescerevisiae
ldli(lndia) Riceandblackgram Leuconotocmesenteroides

Non-vegetarian products
(worldwide)
Drysausages Beef,pork Pedicoccuscerevisiae
(worldwide)
Fishsauces Small
fish Halophilic
sp,Eacil/us
sp
Country-cured
ha?ns(worldwide) Pork,hams Aspergillus
sp,Penicillium
sp

t he whey (l i q u i d p o rti o n o f mi l k ). M il k from Production process

differentanimalscan be usede.g.sheep,cow, goat, As alreadystated,cheeseis producedfrom milk.
buffalo. This is carried out by a processof dehydration
Historical perspective w herei n casei n (mi l k protei n) and fats are
T he useo f a n i m a ls to m a c hfo s r c a rry i n gl i qui ds concentrated 5-15 fold. Cheeseproductionis very
is c ent ur i eos l d . Wh e n mi l k w a s tra n s p o r ted i n thi s complicated, and broadly involves four stages-
fashion,the formationof solids (that were tasty) aci di fi cati on of mi l k, coagul um formati on,
was observed.The solidswere concentrated atter separation of curd from whey and ripening of
dr aining l i q u i d s . T h e s e s o l i d s w e re s a l ted and cneese.
consumed later. A good example of food 1. Acidificationof milk : By employinglactic
preservation,long longago!We now know thatthis acid bacteria (StreptococcusIactis, Lactobacillus
solid portion is the cheese.lt is producedby the /actrdthe sugarof milk (lactose)can be converted
combined action of enzymes (rennet) of the to lactic acid. This lowersthe pH to around 4.6,
stomachlivine and the bacterialcontamination. and thus aci di fi esmi l k.
 364 B IOTE C H N O LO CY

Coagulum
formation
r-Casein
Caseinin degraded
solublestate

2. Coagulum formation : When the acidified Sources of chymosin for cheese

milk is t r eat ed wit h r ennet ( i. e. t h e e n z y m e production
ch ym os in of anim al or f ungal or igin ) , c a s e i n g e t s
There are severalsourcesof rennet(chvnrosin
co ag ulat ed. Cas ein m ainly c onsi s t s o f t h r e e
enzyme) for cheese production. These include
co mponent s - ins oluble q and B ca s e i n s a n d a
calves, adult cows, pigs and fungal sources.
r-casein that keeps them in soluble state. By the
Il the fungal(e.g.Mucor meihei)sources
Fortunately,
action of c hy m os in, r - c as ein i s d e g r a d e d .
of chymosi nare al mostcomparabl e to the anim al
ii Consequently,a and B caseins and the degraded
sourcesand are widely used in some countries.
prodtrcts of r casein combine to form a coagulum
somepeoplealwayspreferanimalrennet
Hor,r.,ever,
(curd) (Fig. 28.1). This process of coagulation is
usedcheesedue to i ts sl i ghtsuperi orfl avour.
de oe nc ienton c alc ium ions .
Cenetically engineered microorganismsfor
3. Separation of curd from whey : When the chymosin: Someworkerscould successfully clone
temperature of the coagulum is raised to the genesof animalchymosinand transferthe same
aro und 40' C; t he c oagulum ( c ur d ) a n d w h e y into microorganisms.The chymosinso produced
(fluid portion) get separated. The separated curd by geneticmanipulationis very widely usedthese
is cut into blocks, drained and pressedinto different
shaoes.
Mitk
4. Ripening of cheese : The flavour of raw
I
cheese (with rubber texture) such as cheddar is ACIDIFICATIONI r-scqq,acid
hqstbriE
bla nd. Ripening im par t s f lav our s , be s i d e s m a k i n g J
changes in its texture. The procedures adopted for Acidifiedmilk
ripe ning ( or m at ur at ion) ar e hig h l y v a r i a b l e
depending on the type of cheese to be prepared.
OOAGULUM I n"nn*,
FORMATION J (chVmosin)
The blocks of curd separatedare subjected to the +
action of proteases and/or lipases. Alternativelv, Curd(coagulum)
they may be inoculated with certain fungi (e.g I
SEPARATION
Penicillium roquefortii). The hydrolysis of proteins l_rwn"v
and fats (either by enzymes or microorganisms) J
Curd subjectedto salting
result sin c er t ain c om pounds whic h im p a r t sf l a v o u r
to the cheese. Mild hydrolysis of fats (or cheese), I Proteases,lipases
RIPENING
usually carried out by lipasesor Aspergillusniger or | (or microorganisms)
Mucor maihai resultsin butyric acid formation with J
Cheese
characteristicflavour.

A diagrammatic representation of cheese

production is depicted in Fig. 28.2.
 28 : MIC R OB IALP R OD U C T IO N
O F FOOD SA N D B E V E R A GE S

days in some countries. The taste of cheese t h a t o c c u r s d u r i n g b r e a d f o r m a t i o n i s t h e

ma nu factr-rr ed by m ic r obial c hy m os in ( i . e . fermentation of hexosesto CO, and ethanol.
ge ne tica lly engineer ed)anc i t he anim al c hy m o s i n
CbHt206 -------) 2C2HsOH + 2CO,
are ide ntical Public as well as t he v ege t a r i a n
so cie ties in s om e c ount r ies hav e ac c ept e d t h e The ethanol produced either gets evaporated or
che ese pro duc ed by t he genet ic m anipulat i o n o f forms esters.The CO, gets entrapped in the dougn
th e en zyme, c hy m os in. resulting in its expansion. The expansion and
s t r e t c h i n go f t h e d o u g h , p a r t i c u l a r l y w i t h w , h e a t r s
YOGI{URT due to the unique elastic protein namely gluten.
Yoghurtis producedby fermentingwholemilk C l u t e n i s m a i n l y r e s p o n s i b l ef o r r e t a i n i n gt h e s h a p e
of bread.
by employing a mixed cr-rltureof Lactobacillus
bulgaricus and Streptococcus thermophilus. While Besides yeast enzymes, the enzyrnes (e.g.
L. bulgaricus produces acetaldehydetl'ratimparts a a m y l a s e s ) o f o t h e r m i c r o o r g a n i s m s a l s o h e l p r n
characteristictaste, .9. thermophilus results in the fernrentation and bal<ingof bread. The texture of
fo rmatio n of lac t ic ac id t o giv e ac id f lav o u r . I n bread is influenced by fats and emr-rlsifiers added to
ad ditio n, bo th t hes e bac t er ia pr oduc e ex t r ac e l l u l a r the dough The bread making is carried oui r,r,ith
po lyme rs that inc r eas e t he v is c os it y of t h e three oblectives :
ferme nte d rn ilk . Yoghur t is v er y delic ious a n d i n
. Cood leaveningdue to CO, formation.
fact frozen yoghurt is becoming popular as an
alternative to ice creanr. . Flavour development

SAUEKRAUT
Sauekrautis prepared in most western countries
It is a fermented and preserved form of cabhage.
The sh red ded c abbage is m ix ed wit h s a l t
(approximatelyat 2.5'/o concentration)and packed
anaerobically. High salt concentration promotes
leakageof sugarsfrom the cabbage while reducing
the water activity. As the growth of the lactic acid
b acteriao ccur s , t he pH is lower ed. At t his low p H ,
the putrifying bacteria cannot grow In this way,
sauekraut can be preserved for long. Sauekraut is
n utritio us a s w ell as delic ious .
The traditional pkkles (mango, Iemon, etc.) in
lndia are also good examples of fermentation and
preservation, based on the principles of
biotechnology.
BREAD
Brea d making f r om t he dough by em pl o y i n g
micro org an is m sis one of t he oldes t ex am pl e s o f
fermentationp!'ocessesknown to mankind. There is
evidence that bread was prepared in Egypt in 3000
BC.
Primarily, bread is a fermented product of
cereal flours srrch as wheat and rye. The cerear
flour mixed with water, salt, sugar, fat and other
ingradientstas desired for enrichment of bread) is
subjected to fermentation by yeast, Saccharomyces
cerevisiae(top fermenting strain).The main reaction
. Cood texture.
The yeast fermented bread has the above
c h a r a c t e r i s t i c s .T h i s i s i n c o n t r a s t t o t h e b r e a d
p r o d u c e d w i t h b a k i n g p o w d e r w h i c l - ra l s o p r o d u c e s
COr. Bui this does not have the same flavour and
texture as that produced by yeast. Thus the yeasr,
which is appropriately referred to as baker,s yeast
is a package of enzymes to give a desired product.
In recent years, some workers have reported the
development of genetically engineered strains of
Saccharomyces cerevisiae with improved
ferrnentation properties Such organisms, when
used in baking industry, are believed to furtner
enhance the quality of bread with regardto flavour,
texture etc.
Sour-dough breads : In some parts of the world,
sour breads are prepared by using the yeast,
Candida milleri and bacterium, Lactobacillus
sanfrancisco.
BAKER'S YEAST
The living cells of aerobically grown
Saccharomyces cerevisiae are collectively referrecl
to as baker's yeast. Baker's yeast is commercially
available eiiher as a dried powder i.e. dry yeast
with about 95"k dry u,eight or in the form of cakes
(about 25-30% dry weight). These commercially
available yeast preparationscan be used in breao
making.
366 B IOTE C H N O LO C\

Nutrientmedium

n
I Ll*ll
T
Frozen
culture

Freshyeast

Fig. 28.3 : Flow chart for the production of baker's yeast (1 is the inoculation reactor,
2-5 are production reactors).

Production of baker's yeast : The medium for This block can be cut into small pieces,wraped
baker's yeast production contains molasses, and stored (at -4'C) until used. The samplesof
am m o n i u m s a l ts (o r a mmo ni a), vi tami ns, yeastare usuallytestedfor bakingproperties
before
phosphatesand antifoam agents.Sugar cane or they are put in use.
sugarbeet molassescan be used.A commercially
availablemolasseswith a sugarconcentrationof OTHER VEGETARIAN PRODUCTS
45-50% is usually preferred. Baker's yeast
production is carried out by an aerobic fed-batch Besides the bread which is consumed
worldwide, there are several other vegetarian
process.
fermented foods. These include rolls and cakes
The flow chart for the productionof baker's (worldwide)soy sauce(worldwide)idli (lndia) and
yeastis depictedin Fig.28.3.The actualproduction tempeh(lndonesia).
processand the strainof Saccharomyces cerevisiae
useddependson the company.The desiredstrainis Goffee, tea and cocoa
maintainedin a frozen state. The inoculum is
preparedin stagesso that largevolumesare finally Coffee,tea and cocoa are very popular non-
obtained.Fromthe inoculation (fermenter),
reactor alcoholic fermented beverages,and extensively
the culture is transferredto productionreactors. consumedthroughoutthe world.
The orocessis carried out in air-lift or bubble
When the tea leavesare crushed,the chemical
column type reactors at pH 4-5, temperature
componentsof tea are releasedby enzymatic
28-30"Cfor about 12-1B hours.The yeastcellsare
activity. For coffee and cocoa, a natural
washed, centrifugedand then dewateredon a
fermentationprocess(by bacteria,yeastand fungi)
rotatingdrum. The fresh yeast obtained can be
on the pulp surroundingbeans results in the
eitherdirectlyusedor dried and stored.
developmentof flavour and aroma. The exact
The yeastcells may be mixed with plasticizer natureof the fermentationprocessesinvolved in
(e.g.vegetableoil) and preparedin a block form. tea, coffee and cocoa are not fully known.
Chapt er28 : MIC R OB IALP R OD U C T IO N
OF FOOD SA N D B E V E R A C E S 367

The dried products namely tea leaves, and thaumatin production was very low. Attempts were
coffee and cocoa beans are commercially available also made to express thaumatin genes in yeasts
for beverage preparation. such as S. cerevisiae and Kluyveromyces lactis.
Some successhas been reported in this direction.
SWEETENERS
MONELL'N-A CANDIDA|E FON
Sweetenersoccupy a prominent place in food
SUGAR SUBST'TUTE
con su mptio n, by all t he people. The m o s t
predominantly used natural sweetener is sucrose Monellin is a protein found in the fruit of an
(obtained from sugar cane or beet). Sweetenersare African plant Discorephyllum cumminsii.lt is about
invariably required for the preparation of soft 100,000 times sweeter than sucrose (on molar
d rinks, ice c r eam s , jam s , jellies , s auc es , b r e a d , b a s i s ) .M o n e l l i n i s d i m e r c o m p o s e d o f a n A - c h a i n
confectionery, pickles etc. Hence there is a (45 amino acids) and a B-chain (50 amino acids).
continuous search for low calorie sweeteners. This proiein is optimally sweet when both tne
chains are held together and not when they are
Saccharin, a chemically synthesizedsweetener separated.
(300 times sweeter than sucrose) was used for
several years, and its use is now being restricted I t h a s b e e n p o s s i b l et o c h e m i c a l l y s y n t h e s i z ea
due to certain adverse health effects. High fructose g e n e t h a t e n c o d e s b o t h A a n d B c h a i n s a s a s i n g l e
corn syrup (HFC\, produced from sucrose rs polypeptide. This gene was in fact introduced and
sweeter (1.5 times) than sucrose. Aspartame expressed in tomatoes for the production of
(a sp artylp heny lalanine)is a pr oduc t of inn o v a t i v e monellin. However, the success has been very
biotechnology. lt is about 200 times sweeter than limited.
sucrose and is truely a low calorie sweetener.
Aspartame is approved for human consumption FLAVOUR ENHANCERS
a nd is widely us ed in s of t dr ink and o t h e r Taste and flavour are very important for the
i nd ustries. acceptability of foods. Monosodium glutamate is
the most commonly used flavour enhancer. lts
THAUMAT'N-A SWEET PROTE'N production and other aspects are described
Thaumatin is a orotein extractedfrom the berries elsewhere (Chapter 26). The degraded products of
ol the Thaumatococcus daniellii, a native plant in proteins and RNAs also impart flavour. Thaumatin
Africa. Thaumatin is aboul 3000 times sweeter and monellin (described above) are also flavour
than sucrose. Thaumatin certainly appears to be a e n h a n c i n g a g e n t s , b e s i d e ss w e e t e n i n g .
good sugar substituteand a low-calorie sweetener. The other flavouring agents are citric acid
It is in fact being used as a sweetener and flavour (produced by Aspergillus niger), acetone and
enhancer of various foods. At least five differenr butanol (produced by Clostridium acetobutylicum).
forms of thaum at ins wit h a m olec ular wei g h t s r n The lactones formed by some microbial
the range of 20,OOO-22,000have been isolatedand fermentations also enhance flavour e.g.
characterized. y-butyrolactone produced in yeast fermentation of
wine and beer. Mention must be made abour
Genetic engineering alcohol also which can increase flavour and taste
to produce thaumatin when added at appropriate concentrations e.g.
Natural production of thaumatin is very limited certain medicinal svruos.
and therefore can never meet the demands of
consumption. Biotechnological production of this
:\.,eeteneris the method of choice for its laree scale
-ianufacture.

Tha uma t in- enc odingm RNA s pec ies hav e b e e n
:clated, identified and converted to cDNA and
'-allv to do uble- s t r andedDNA. Thes e DNA s w e r e
: and transferredto E. coli. But the yield of
'rned
Alcoholic beveragesare producedthroughout
the world. The type of the beverageand the
usedfor its productionmostlydependson
substrate
the crops grown in the region. For instance,rn
368 B IOTE C H N O LO CY

Beverage Alcohol (%) SubstrateG)

Beer(ale,lager) 4-8 Barleyandothercereals
Wine(red,white) 10-16 Grapes
Sake 10-14 Rice
Champagne tu-tt Grapes
Cidar 1 14
I-ta Applejuice
Brandy 35-45 Grapes or otherfruits
Whisky 40-55 Cereals(barley,
rye,corn)
Rum 50-60 Molasses
Gin,vodka 40-50 Potato,wheat,rye
Chinesebrandy 30-40 Rice
Toddy 5-15 Palmyrajuice
Arrack 60-75 Rice,jaggery,wastecarbohydrates

Po land, Rus s ia and Sc and i n a v i a , b e e r s There are two types oi S. cerevisiaestrains-

manuf ac t ur edf r om bar ely ar e c or l s u m e d w h i l e r n bottom yeast and top yeast The bottorn yeast
Franc e, it aly and Cr eec e, wines o b t a i n e d f r o m settlesto the bottomwhereasthe top yeastraisesto
pa lm y r a juic e ar e m os t ly c onsu m e d b y r u r a l the top lvhile carryingout fermentation-ropyeast
p eo pr e. arefrequentiyusedfor the productionof beerwhile
bottom yeastare employedfor wine production.
The common types of alcoholic beverageswith However,both strainscan be used for beverage
their alcoholic contents and substrates used for producti on.
their production are given in Table 28.2.
Geneticallyengineeredstrainsof S. cerevisiae;
perspective B y empl oyi ngrecombi nantD N A technol ogy,new
Historical
strainsof Saccharomyces cerevisiaeare constantly
Ar c haelogic al ev idenc e indic a t e s t h a t t h e r e bei ng devel oped.S ome of these strai n s wit h
existed fermentationof grains as early as 4000 B.C. improved production yields of alcoholic beverages,
Production of alcoholic beveragesis considered to are in fact approvedfor use in beverageindustries.
b e a par t of hum an c iv iliz at ion. lhe b i o l o g i c a l a n d
ch er . ' r ic alpr inc iples under ly ing alco h o l i c b e v e r a g e ALCOHOLIC BEVERAGE PRODUCTION
p roduc t ion wer e eluc idat ed by Lou i s P a s t e u ri n t h e -GENERAL ASPECTS
second half of nineteenth century.
The startingmaterialsfor beverageproductron
are sugaryor starchymaterials.
Saccharomyces cerevisia+the key
organism for the production of Sugarymaterials: Fruit juice (or fruits),plant
alcoholic beverages sap and honeythat can be di rectl yused.
Starchymaterials-variousgrains and roots :
The most commonly used organism for the
They have to be subjectedto hydrolysisto yield
production of berrerages is the yeasr,
simplefermentable sugars.This process,referredto
Saccharomycescerevisiae.This organism is capable
as saccharification,is carried out either by planL
of utilizing simple sugars (glucose, fructose) and
materi al s(barl eymal t, rye mal t or mi l l et malt )or
converting them to ethanol. lt is fortunate that
microorganisms(Aspergillus sp, Rhizopus sp.
S. cerevisiaeis a unique organism that can tolerate
Mucor sp) rich in starchhydrolyticenzymes.
and grow even at a high concentration of alcohol.
This is advantageousfor good production yield of Fermentationprocess : When subjectedto
alcoholic beveraees. fermentati onunder i deal condi ti ons, alcohol
 O F FOOD SA N D B E V E R A C E S
Chaot er28 : M IC R O B IALPR O D U C T IO N 369

production ranging from 2 to 16"/" is usually Barley

observed. These alcoholic beveragesmay be
appropriatelyprocessedand drunk. e.g. beers. MALTING I ct"un"o,
soaked,
germinated
and dried
However,for the productionof beverageswith J
higher concentrationof alcohol (brandy,whisky, Malt
rum, gin etc.),distillationhas to be carriedout to MASHING I eo*0"r, add adjunct
increasethe alcohol content. and mix with water
J
Brewing is the technical term used for the Mash
production of malt beverages. Besides5. cerevisiae I rittration
(described already),S. carisbergensrs is alsousedin J
br ewingind u s tryT. h efi n i s h e dp ro d u c tsi n brew i ng Worl
namely the alcoholic beveragesdiffer from the
otherindustrialfermentedproducts.This is because I Inoculated with S. cerevisiae
FERMENTATIONI Fermented for 5-10 days
besidesthe alcohol content,severalother factors +
are importantin finishedbeverages. Theseinclude BEER
flavour,aroma,colour,clarity,foam productionand
I fept at 0'C for severalweeks
stability, and satiety.These factors decide the MATURATIoN I :::;.:
; Pastuerized at 60'Cfor20 min.
consumeracceptance and the comnrercial valueoi +
alcoholic beverages. All these beveragescan be
BEER FOR CONSUMPTION
consumedrn,henfreshlyproduced.However;they
are normally kept for storing or aging before
. g i n g c e rta i n l y i m p ro v e sf l avour,
c ons um pt i o nA
aromaetc.
The commercialproductionprocessesemployed
for the manufactureof beer and wine are briefly 2. Mashing : The powdered nralt is n.rixecl
describedhereunder. w i t h h o t w a t e r ( 5 5 - 6 5 'C ) . D u r i n g t h e c o u r s e o f
mashing, the soluble materials from malt and
malt adjuncts are extracted. Mashing is also
BEER
associated with degradation of starches (by
Beer is an undistilled alcoholic beverage with a m y l a s e s ) t o p r o d u c e d e x t r i n s , m a l t o s e a n d
alc oholc on te n it n th e ra n g eo f 4 -B o h .l its p r oduced g l u c o s e a n d h y d r o l y s i s o f p r o t e i n ( b y p r o t e a s e s )
by fermentationof barley or other cereals,by t o p e p t o n e s , p e p t i d e s a n d a m i n o a c i d s . T h e
employingthe yeasts(mostfrequently) S. cerevisiae t e m p e r a t u r e a n d p H i n f l u e n c e t h e a c t i v i t i e s o f
or (sometimes)S. carlsbergensis. enzymes. At the end of mashing, the medium for
fermentation which is referred Lo as beer wort is
It is often necessaryto add starch- rich
developed. This is rich in sugars, amino acids,
materials(wheat,maize, rice) known as adjuncts,
minerals and vitamins.
to enhancefermentation,besidesreducingthe cost
of raw materials.There are four major steps Hops are the dried female flowers obtained from
involvedin the productionof beer (Fig.28.4). hop plant. Addition of hops to wort is often done to
provide characteristicflavour, aroma and stabilizing
1. M alt i n g : D ri e d b a re l y a re c l e a n ed and
effect to the beer. Besides providing a mild
soaked in water for a period of two days. The
antibacterial activity.
excesswater is drainedand the soakedbarleyare
incubatedlor 4-6 days to germinate.Cermination 3. Fermentation : The beer wort, kept in open
Drocess is associatedwith the formation of or closed bioreactors, is inoculated with pure
enzymes-amylases (starch hydrolysing) and strainsof yeast.5. cerevisiae(usuallytop fermenting
proteases(protein degrading).The germinated strains). The fermentation is carried out at
seedsareslowlysubjected to increased temperature temperature 2O-28"C for 5-1 0 days. In some
(up to BO"C)so that the germinationprocessis countries,the bottom fermenting yeast S. uvarum is
halted,but the enzymesretaintheir activities.Malt used. For this organism, the ideal temperature for
is preparedby powderingthe seeds. ootimal fermentationis 10-] 5"C.

Biotechnology
[24J
37(J B IOTE C HNO LO CY

4. Maturation : The fermented fluid is (mechanically or by treadinBof feet)and the juice

transferred to storage tanks maintained at extracted. Ihis grape juice ready for fermentation
temperature 0-3"C. Duringthe storageperiod(that processis technicallyreferredto as musf. lt is a
may lastfor severalweeks),cold storagematuration oracti ceto add sul furdi oxi deto mustto i nh ibitt ne
occurs. This orocess is associated with growth of non-wine yeast and contaminating
sedimentation of yeast cells and precipitationof bacteri a.S ul fur di oxi de w hi ch can ki ll ot her
nitrogenous substances, resins,phosphates etc.This organismscan be toleratedby wirre-fermenting
p a rti a l l y ma tu re b e e r (u s u al l y w i th turbi d yeasts.Sometimes, the mustmay also be subjected
appearance)is then subjected to chillproofing. to partial or completesterilization.The must in
C h i l l p ro o fi n gb a s i c a l l yi n v o l v esthe removal of suitable bioreactorsis inoculated with desired
re s i d u a l n ro te i n s (b e e r i s tu rb i d due to thei r strains of the yeast Saccharomycescerevisiae.
presence) suspended in the beerby precipitation or Initially, oxygen is bubbled through the
b y e m p l o y i n gp ro te o l y ti ce n z y mes.l t i s advi sabl e fermentationmedium to promotegood growth of
to add antioxidants duringcold-storage maturation yeastcel l sand gradual l yanaerobi ccondi t ionsar e
to preventoxidativedamage. The w i ne producti onnormal lyt akesa
establ i shed.
Carbonation of the beeris usuallycarriedout by .few days (2-5 days).The fermentationconditions
injecting CO, (evolved during the course of (temperature, time etc.)are actuallydependenton
fermentation). Carbonation can also oe the type of wine produced. At the end of
accomplished by addingfermentingyeast,which is fermentation, wines are transferred to storagetanks
l e s sc o mmo n l yd o n e . (or vats)and allowedto age,which may takesome
rnonthsor years.Ageingof wine is very important
The maturedand carbonatedbeer is bottledor
for the development of characteristic flavour and
at 60"C for about20
canned,and then oasteurized
aroma.The alcoholcontentof wines is in the range
m rn u te s .
of 10-16' h.
As alreadystated,there is variationin the raw
materialsusedfor beerproduction.For instance,rn Types of wines
Africa it is sorghum beer that are commonly
produced. This is in contrast Io the barley beer There are hundredsof differenttypesof wines
producedin most Westerncountries. producedin differentpartsof the world.
Red wine : The red colour of this wine is oue
W IN E S the colour extractedfrom the black grape skrns
Wi n e sa re o ri g i n a l l yMi d d l e E astand E uropean (when crushedtotally).Red wines are commonly
drinks, although almost every country now drunk, by many people in the west, along with
producesthem. Largescaleproductionof wines is l unch and di nner.
carriedout by usinggrapesof speciesVitis vinifera. White wine : Blackgrapesafterremovalof skins
Crape juice is a good sourcefor wine production or white grapesare used for the productionof
because of its high concentration of sugar w hi te w i ne.
and other nutrients, natural acidity (that can
Rosewine : Thiscan be producedwith a limited
inhibit unwantedgrowth of microorganisms) and
contactto the skinsof grapesduringfermentation.
the capability to produce pleasantaroma and
flavour. Dry wine : lt contain relativelyhigheralcohol
Louis Pasteuroften used to state 'wine is the contentand producedwhen sugarsare completely
most healthy and most hygienic of beveraget. fermented.
There is some recent scientificevidencealso rn Sweet wine : This is sweet to taste since it
support of this view, since moderate wrne containssome residualsugarsafterfermentation.
consumptionreducesthe risk for coronary heart
Fortifiedwines : The'normal concentrationof
d i s e a s e(C H D ).
al coholi n w i nes i s l essthan 16oh.The w inescan
be fortifiedwith additionof alcohol (usuallydone
Production of wines
by addi ngbrandyor other di sti l l edspi ri ts)t o t he
Quality of the grapesis very importantfor the desiredconcentration. Someexamplesof fortified
production of wines. The grapes are crushed wines are sherry,port and vermouth.
 ChA P I CT O F FOOD SA N D B E V E R A C E S
28 : M IC R OB IALP R OD U C T IO N 371
I

Tlslr 28.3 Enzymesused in various food and beverageindustries

Industry Enzymes
Dairy (animal/microbial),
Chymosins lipase,lactase,
lysozyme
Baking protease,
u-Amylase, phospholipases,
xylanase
Starchandsugar Amylases (o- andB-), glucoamylase,
pollulanase,
invertase,
glucoseisomerase,
xylanase
processing
Fruitandvegetable pectinlyase,hemicellulases,
Pectinesterase, polygalacturanase
Meatprocessing papain
Proleases,
Animalandlishboneprocessing Alkaline
ohosohatase
Eggwhiteprocessing Glucose
oxidase,
catalase
Brewingproduction
of beerand (a- andB-), proteases,
Amylases papain,
cellulases,
alcoholic
beverage aminoglucosidases,
xylanase

p r o t e i n s o f s o y b e a n ; p r o t e i n ( g l u t e n )o f d o u g h i n
preparing bread.

Lactase in dairy industry

There are a large number food products or thetr
ing rad ien ts,pr oduc ed by m ic r oor ganis m s For f u l l
d eta ils on th em , t he r eader m us t r ef er t h e
corre sp on din gChapt er s- s ingle- c ell pr ot ein a n d
mushrooms (Chapter 29), vitamins (ChapIer 27),
amino acids (Chapter 26), or1anic acids (Chapter
24), polysaccharides (Chapter 30), and vinegar
(Chapter 24).
ENZYMES IN FO O D AND
BEVERAGE I NDUSTRI ES
Food and beveragebiotechnology is very closely
lin ke d with th e us e of enz y m es W hile a m aj o r i t y
of enzymes are microbial in nature, some other
sources of enzymes can also be used. In
Table 28.3, a selected list of enzymes employed in
food processing and beverage industries is given.
Some of the aspects of enzymes in dairy, baking
an d b rewin g indus t r ies ar e des c r ibed in t h e
foregoing pages. A few more important ones are
described hereunder.
Proteases in food industry
Proteases (proteolytic enzymes) are used for
tenderisation of meat which flavours the meat,
besides easy digestion Proteasesare employed for
removal of meal attached to bones in the form of a
slurry. This can be used for the preparation of soups
and canned meats. Partial hvdrolvsis of certain
ve ge tab lep rote ins als o inc r eas est heir f lav our e . g .
Milk and whey cohtain about 4-5"/" lactose,
Lactose intolerance, due to a defect in the intestinal
enzyme lactase, is very common in humans. lt is
estimatedthat half of the world's population suffers
f r o m l a c t o s e i n t o l e r a n c e .T h e s e i n d i v i d u a l s w h e n
c o n s u m e m i l k a n d m i l k p r o d u c t sc o n t a i n i n g l a c t o s e
( p a r t i c u l a r l yi n l a r g e q u a n t i t i e s )s u f f e rf r o m d i a r r h e a
and flatulance (abdominal cramps and increased
intestinal motility). Obviously, lactose intolerant
persons require milk products with a low lactose
concentratron.
The enzyme lactase cleaves lactose to glucose
a n d g a l a c t o s ew h i c h c a n b e e a s i l y a s s i m i l a t e db y
the body. The lactase enzyme may be obtained
from microbial sources e.g. Kluyveromyces fragilis,
Aspergillus niger. Sometimes, immobilized lactase
can also be used to degrade lactose.
F o r t h e p r e p a r a t i o no f f l a v o u r e d a n d c o n d e n s e d
m i l k s a n d i c e c r e a m s , l a c t a s ei s w i d e l y u s e d .
ENZYMES IN THE PREPARATION
OF FRUIT JUICES
T h e t u r b i d i t y a n d c l o u d i n e s s ,c o m m o n l y s e e n i n
f r u i t j u i c e s , i s d u e t o t h e p r e s e n c eo f p e c t i n s a n d
cell debris. Enzyme preparation containing pecfin
esterase, polygalacturonase, pectin lyase and
hemicellulase is added to the fruit pulp for the
removal of pectins and other turbid-causing
materials.
372
Removal of glucose and oxygen from
foodstuffs
The presenceof glucose and oxygen in the
foods is often associatedwith certain amount of
damage.Their removalenhancesthe storabilityof
foods.Glucosefrom the foodstuffscan be removeo
by the combined action of glucoseoxidaseand
catalase,as illustratedbelow.
Clu co se o xid a se
^t
UfUCOSe * U2 -
C l u c oni cacfd + H rO,
Catalase
2H2O2 ----------------+2HrO + O,
The egg white used in bakingindustryis made
freefrom glucoseby the enzymatictreatment,given
above. This pair of enzymes(glucoseoxidaser FOOD BIOTECHNOLOGY.PUBLIC
catalase)is also useful for the removal of O, ACCEPTANCE
presentin the head spaceof bottledand canned
ln general, the public have a negative attitude
foods and drinks.
DIAGNOSTICS IN FOOD
BIOTECHNOLOGY
It is essential
to ensurethat the foods oroduced
by biotechnological processesarefreefrom disease-
causingorganisms and toxins.Then only the foods
are suitablefor human consumotion.The most
common pathogenspresentin foods are Salmonella
B IOTE C H NO LO G Y
sp, Campylobacter sp and Listetia sp. The food
toxins are derived from bacterial (endototoxins)ano
fungal (mycotoxins)sources.
Several novel and innovative techniques have
been developed in recent years for testing the
safety of foods. For instance, the presence of
Salmonella in foods can be detected by an
immunoassyon the same day (This is in contrastto
the traditional methods that take 5-7 days).
ln fact, kits are available for the detection of
concentrations of several undesirable compounds
in the foods e.g. endotoxins, mycotoxins,
antibiotics, hormones.
towards the biotechnology based foods, particularly
involving genetic manipulations. For this reason,
t h e a p p l i c a t i o n o f g e n e t i c e n g i n e e r i n gt e c h n i q u e s
for food biotechnology is rather slow. Several
factors influence the public acceptance. These
include the type of biotechnology adopted, tne
regulatory procedures, economics of the people
and consumer acceotance.For more details on this
aspect, refer Chapter 61 .
 r ,::r :t .i r ,. - , :.- i t;,i ,.r ..i - l ,- * r i r ;1.:+tgi ,;tl n;58" r " !$
ing ,le- c ellpr ot ein ( SCP)r c t . er st o t he t l i c r o l l i r l .
'
.-,ce lls or t ot al pr ot eit r c x t r ac t c ' d f r o r l r p L r r c ' ' j-lr
.

m ic-robialcel I c:uItr-r re (t'trotroc.u IlLt re) r'r'lr

ich c..t tr lrt'
T h e ; r r o t c i n - p r o r l uttt 't gc, a l t a l r i l i t i e so f . l 2 5 0 l i g ,
used ns pr ot ein s uplt let ' t r etior r t hum at r sor . l t . l i t r r a l s
corr,' .,rt't(l250 g tll nllcroorganlsllls ATe olt('ll
Th e rvo r c l SCP is c or r s ider c d t o be . r p p r o l r r i a l c .
c o l l r p a r e c i T h c c . o n c a t r i r r o t i t t c c 'a l l r - r t r t2 0 0
sirrcemo s t oi t he m ic r oor ganis nr sSr ow : ls s i n g l e o r other h a r r -c l ,
protein per dav C)n thc
fi lame nti ous indiv ic luals . This is in c o n t r a s t I t r r't,hengror'r'trttnclcr
nricloorganistrrs,tl-reoreticaIIy,
co mple te m ult r c ellLr larplant s ar r d ar r im a l s l f t h e
i c l c . a l c o n d i t i o n s , c r : r r r l c lp r o d t t c c . r b r l t r t 2 0 2 5
SCP is suit able f or hunr an c ons unr llti t - r t r i,t i s oi
tonncs of prot('in Thc-re arc t'tt.-tnyaclvatrtag,es
corrsidercdas food grade SCP is rcgarcleclas feed f o r S C P p i o r l t t c t l o t l
using microorg,anisms
grade, wl-ren it is r-rsecl as atrirral fecd sr-rpplen'rerrt,
b ut n ot s uit able f or hlt m an c ons r - t nr pt io n 1 M i c r o o r g a n i s m s g r o r 'r ,a t a v e r v r i l p l ( l f i r t e
u n d c r o p t i n r a l c - u l l u r ec o t r d i t i o r l s S o t l r c n 't i c t o l l c s
Sin gle - c ell pr r t t ein br o: r dly r ef er s t o t h e d o u b l e t h e i r n r ; r s si n l e s s t h a r r - 1 0 t r l i r r r - r t e s
microbial biomass or protein extract used as food
2 The quality and quantity af protein content
or feed additive Besicles h igh protcirr cotrtcnt
(a bo ut 6 0- 80' f n oi c lr y c ell r ' r , eight ) ,S C P . r l s o in microorg,anisms is better comparat! to higher
leic ac ic l s ,v i t a n ri r r s plants and animals
con tai ns f at s ,c ar bohy dr at esnuc ,
an d min er als Anot her ac lv ant agewit h S C P i s t h a t 3 A wide range of raw materials, whiuh nre
it is rich in c er t ain es s ent ialam ino ac ic l s l l y s i n e , othefwise wasfed, can be fruitfully used for SCP
meth ion ine) whic h ar e us r - lally lim i t i n g i n production
mo st p lant and anir nal f oods Thus , SCP i s o f h i g h
4 T h e c u l t u r e c o n d i t i o n s a r r d t h e i e r t n e t l t a t i o tr
n utritio nalv alue f or hr t m at ror anim al c on s u n r p t i o n
p f o c e s s c sa r e v e r y s i n r p l e
lt is es t r m at edt her t about 25"1, oi t h e w o r l d 's 5 M i c r o o r g a n i s m sc a n b e e a s i l y h a l l c . l l e c al ,r c l
po ou lati on c ur r er r t ly s r - t f f er sf r on' r hu r r g e r a n c l
s r - r b j e c t e tdo g , e n e t i cn r a r r i p t t l a l i o t l s
rlraln utr it ion M os t of t hes e people I i v e i r r
cle ve lop ing c ount r ies Ther ef or e, SCP d e s e r v e s a
i'ef e.iy, a,r:r;exltehil ity and
se riou s cons idr : r at ionf or it s us e as f oo d o r f e e d i..rxl
;oisil.s oi 5i:F'
sr-rp ple m entln addit ion t o it s ut ilit y as a n u t r i t i o n a l
sr-rp ple m entSCP , c an als o be us ed f or t h e r s o l a l i o n T h e r e a r e n r a n y n o r l - t e (h n o i o g i c a i f a c t o r s t h . l t
'l 'h e s e
of several compoulrds e g carbohydrates, f.rts, i n f l u e n c e t h e p r o c l t r c t i o no f S C P i r r c l L r c lteh e
r ita nrirr s ,nr iner als g , e o g , r a p h i c a l
s , o t : i a l , p o l i t i c a l a r r d 1 ; s y c h o l o g i c al

373
 374 B IOTE C H N OLO CY

factors. In many countries, there are social and Substrates

psychological barriers to use microorganisms as
The nature of the iaw materialssupplying
food sources. lt is desirable to first consider tne is very crucial for SCPproduction.The
substrates
safety, acceptability and toxicology of SCP, cost of raw materialsignificantlyinfluencesthe
p ar t ic ular ly when it is c ons ider e d f o r h u m a n
consumption. There are several limitations for the
widespread use of SCP.
1. The nucleic acid content of microbial and substratesused for single-cellprotein
biomass is very high @-6% in algae; 10-1 5% in
b ac t er ia;5- 10% in y eas t ) .This is hig h l y h a z a r d o u s ,
sinc e hum ans hav e a lim it ed c apa c i t y t o d e g r a d e Microorganism Substratek)
nu c leic ac ids .
Eacteria
2. The presence ol carcinogenic and other MethyIophiIus methylotrophus Methane,methanol
toxic substances is often observed in association
Methylononas sp Methanol
with SCP.These include the hydrocarbons, heavy
metals, mycotoxins and some contaminants. The Pseudomonas sp Alkanes
nature and production of these compounds Brevibacterium sp C.,-Cohydrocarbons
depends on the raw materials, and the type of
Yeasts
o rganis m us ed.
I l; lipolytica
Saccharomycopsis Alkanes
3. Ther e is a pos s ibilit y of c o n t a m i n a t i o n o f
(previousnilfla-candida tipotytical
pa t hogenic m ic r oor ganis m sin t he S C P .
Candidautilis liquor
Sulfite
4. The diges t ion of m ic r obial c e l l s i s r a t h e r
Kluyveronyces fragilis Whey
slow. This is frequently associated with indigestion
Saccharomyces cerevisiae Molasses
and allergic reactions in individuals.
yeast)
(baker's
5. Food gr ade pr oduc t ion of S C P i s m o r e
Lactobacillus bulgaricus Whey
expensivethan some other sourcesof proteins e.g.
so y m eal. O f c our s e, t his m ainly d e p e n d s o n t h e Iosulopslssp Methanol
cost of raw materials. In general, SCP for human Fungi
co ns um pt ion is 10 t im es m or e ex pe n s i v et h a n S C P
Chaetomium cellulolyticum wastes
Cellulosic
for anim al f eed.
Paecilomycesvarioti liquor
Sulfite
For the above said reasons,many countries giv'e
niger
Aspergillus Molasses
low priority for the use of SCP for human
consurnption.In fact, mass production of SCP using Trichodermaviride Straw,starch
costly raw materialshas been discontinued in some Algae
countries e.g. Japan, Britain, Italy. However, these
Spirulinamaxima Carbon
dioxide
countries continue their efforts to produce SCP
from cheap raw materials such as organic wastes. Chlorellapyrenoidosa Carbon
dioxide
acutus
Scenedesmus Carbon
dioxide
MICROORGANISMS AND SUBSTRATES
Actinomycetes
USED FOR PRODUCTION OF SCP
Nocardiasp Alkanes
Several microorganisms that include bacteria,
fusca
Thermomonospora Cellulose
yeasts, fungi, algae and actiomycetes utilizing a
wide range of substratesare used for the production Mushrooms (a typeof fungi)
of SCP.A selected list is given in Table 29.1 . Agaricus
biosporus Compost,ricestraw
The selection of microorganisms for SCP Morchella
crassipes Whey, liquor
sulfite
production is based on several criteria. These Auricularia
sp Sawdust,ricebran
in c lu{ p t heir nut r it iv e v alue, n o n - p a t h o g e n i c Sawdust,ricebran
Lentinus
edodes
nature, production cost, raw materials used and
volvaceae
Volvariella Cotton,
straw
growth pattern.
 ChA P I CT
29 : SIN C L E -C E LPLR OT E INAN
, D MU S H R OOMS 375

final cost of SCP.The most commonlv used raw 2. Short chain alkanes with carbon length in
materials may be grouped in the following the range of C',o-C''r, isolated from gas oil bv use
categories. of molecular sieves.
.l
methane,
. High-energysourcese.g. alkanes, Airlift bioreactor system with continuous
m et hanole, th a n o l g
, a so i l . o p e r a t i o n w a s o n c e u s e d ( i n F r a n c e a n d B r i t a i n )t o
wheyisewage, produce SCP from gas oil employing the or5lanism
2. Wasteproductse.g.molasses,
animal manures/straw,begasse. Saccharomycopsis lipolytica. But this is now
d i s c o n t i n u e df o r o o l i t i c a r r e a s o n s .
3. Agricultural and foresty sources e.g.
c ellulos el,i g n i n . Degradation of alkanes : Alkanes harreto be first
brolien down to appropriate metabolites for their
4. Carbondioxide,the simplestcarbonsource.
utilization to form SCP.The most important step in
Some details on the oroductionof SCP from t h i s d i r e c t i o n i s t h e i n t r o d u c t i o n o f o x y g e n i n t o
most importantraw materialsare brieflydescribed. a l k a n e s w h i c h c a n b e b r o u g h t o u t b y t w o
pathways-terminal oxidation and subterminal
PRODUCTION OF SCP FROM HIGH oxidation (Fig. 29.1).
E NE RG Y S O U R C ES
ln terminal oxidation, the terminal carbon gets
Thereare a largenumberof energy-rich carbon oxidized to the corresponding monocarboxylic
comooundsor their derivatives which serveas raw acid. The latter then undergoesB-oridation to form
m at er ialsfo r S C P o ro d u c ti o n . T h e s e i ncl ude a c e t i c a c i d . I n s o m e m i c r o o r g a n i s m st,h e o x i d a t i o n
alkanes, methane, methanol, ethanol and gas oil. may occur at both the terminal carbon atoms (by a
Bacteriaand yeastsare mostly employedfor SCP process referred to as crr-oxidation)to form a
production from high energy sources. Some dicarboxylic acid. This can be further broken down
scientistsquestion the wisdom of using (rather to acetate and succinate bv B-oxidation. Terrninal
m is us ing ) h i g h -e n e rg y c o mp o u n d s for the oxidation is the predominant pathway occurring in
production of food, since they regard it as a majority of yeasts and bacteria
wastefulexercise.
Suhterminal oxidation involves the oxidation of
interminal carbon atoms (any carbon other
Production of SCP from alkanes
than terminal i.e. C2, C3, C4, and so on).
Alkanes can be degraded by many yeasts, The corresponding ketone produced undergoes
certainbacteriaand fungi. The major limitationof a-oxidation, decarboxylation, and finally
alkanesis that they are not easilysoluble,hence p-oxidation to form acetate and propictnate.
they cannot enter the cells rapidly.lt is believed The individual enzymes responsiblefor terminal
thatthe cellsproduceemulsifying substances which oxidation or subterminal oxidation have not been
convert insoluble alkanes into small droplets fully identified.
0.01-0.5pm) that can enter the cells by passrve
dittusion.lt is observedthat when cells are grown Limitations of SCP production from
on a m ediu mo f a l k a n e se n ri c h e dw i th l i p i ds,the alkanes
dirfusionof alkanesinto the cells is enhanced.
The production of SCP from alkanes is a very
Certainyeastshave been successfully used for complex
biotechnological process and has been
producingSCPfrom alkanese.g.Saccharomycopsisextensivelystudied. The malor dral,r,back
of alkanes
Iipolytica, Candida tropicalis, Candida oleophila. as substratesis the formation of carcinogens,along
Petroleum products for SCP production : w i t h S C Pw h i c h a r e h i g h l y h a r m f u l . F o r t h i s r e a s o n ,
Severaloil companieshavedevelopedfermentation many countries have discontinued alkane-based
systems,employingpetroleumproductsfor large production of SCP.
scalemanufactureof SCPby yeasts.Two typesof
petroleumproductsaremainlyusedfor this purpose. Production of SGP from methane

1. Gas oil or dieseloil containing 1O-25"hol lzlethaneis the chief constituentof natural gas in
wit h c a rb o nl e n g thC ,' r-C ro(i .e .l o ngchai n many regions.Although methane can be isolated in
alk anes
aIkanes). p u r e g a s f o r m , i t c a n n o t b e l i q u i f i e d . T h e 'h a n d l i n g
376 B IOTE C H N OLO CY

cH3-(cH2)n-cH2-cH3

cH3-(cH2)2-cH2-cooH fol
acid
Monocarboxylic llll
-(cH2)n-+!ll-
cH3 cH3
i\
i \ r+Oxldation Ketone
iF-oxidation
i\ \ I u-Oxroation
Y

n""I"t" HooC-(CH2)n-CH2COOH
tol
1 ilI
Dicarboxylic
acid cH3-(cH2)n---lel--coo
H
I
Decarboxylation
i P-OxiOatlon +I
CH3-(CH2)n-COOH + COt

Acetate + Succinate I B-orioution

+
Acetate+ Propionate
| :
''" '" " - " r . S I N G L E - C E L L \_
, ri
PROTEIN

Fig. 29.1 : Oxidation of alkanes by yeasts (e.9. Saccharomycopsislipolytica/ to produce single-cell protein.

and transportation of methane (an explosive gas) Hansenula sp, Torulopsis sp) and fungi
are very difficult and expensive. (Tric hoderma Iignorum, GIiocIadi um deIi nquescens)
Certain bacteria that can utilize methane for are capable of producing SCP from methanol.
SC P pr oduc t ion hav e been i d e n t i f i e d e . g . Bacteriaare mostlypreferredbecausethey require
Methylococcus capsulatus, Methylomonas simple fermentationconditions,grow rapidly and
methanica, Methylovibrio soehngenii. So far, yeasts possesshigh contentof protein.
that can utilize methane have not been identifieo. Oxidationof methanol: Methanolgetsoxidized
The bacterial enzyme methane oxygenase to formaldehyde,then to formic acid and finallyto
o xi diz es m et hane t o m et hanol, w h i c h c a n b e carbondioxide,as depictedin Fig. 29.2.
converted to formaldehyde and then to formic acid.
The productsobtainedfrom methanolhave to
Although methane was extensively researched
form C, compounds(such as pyruvate)for final
for its use as a source of SCB it is not widely used
oroductionof SCP.Carbon dioxide formed from
du e t o t ec hnic al dif f ic ult ies . by photosynthetic
methanol can be utilized
organi sms for the formati on of ri bulose
Production of SCP from methanol
diphosphate. Alternately, formaldehyde may
Methanol is a good substratefor producing SCP
condense w i th ri bul ose 5-phosphateto f or m
Methanol as a carbon source for SCP has several
3-keto 6-phosphohexulosewhich then gives
advantagesover alkanes and methane. Methanol is fructose 6-phosphateand finally pyruvate.This
ea s ily s oluble in aqueous p h a s e a t a l l pathway is referredto as ribulose monophosphate
concentrations,and no residue of it remains in the (or
Quayle) cycle.
h ar v es t ed biom as s . Tec hnic allv , m e t h a n o l c a n b e
e as ily handled. The s our c es f or m e t h a n o l a r e Formaldehydecan condensewith glycine to
n atur al gas , c oal, oil and m et hane.M a n y s p e c i e so f form serinewhich in a seriesof reactionsforms
bacteria (Methylobacter, Arthrobacter, Bacillus, phosphoenolpyruvate.This is referredto as serine
Pseudomonas, Vibrio) yeasts (Candida biodinii, pathway.
Chapt er2 9 : S IN C L E-C ELPLR OT E INA, ND MU S H R OOMS 377

cH3oH half of the production cost expenses.In the Middle

Methanol East, due to high availability and low cost of
II Methanol methanol, the production of SCP appeared to be
attractive. In the erstwhile Russia, there were
I dehydrogenase
+ several plants producing SCP from methanol which
HCHO were later closed.
Formaldehyde
I Genetic engineering for improved SGP
NAD*, I Forrnatdehyde production from methanol
GlutathioneI deflydrogenase
+ The efficiency of SCP production has been
HCOOH improved by using genetic engineering. The
Formicacid
assimilation of ammonia by M. methylotrophus is
I a n e s s e n t i a ls t e p f o r c e l l u l a r g r o w t h . T h i s o r g a n i s m
NAD+I Forrnats
I dehydrogenase lacks glutamate dehydrogenase lt possesses
+ glutamine synthase and glutamine ketoglutarate
ww2
transaminaseto utilize ammonia for the forrnation
of glutamate (Fig. 29.4A). This is an energy (ATP)
Fig. 29.2 : Oxidation of methanol.
dependent reaction. By employing recombinant
DNA technology, the gene for the enzyme
glutamate dehydrogenasefrom E. coli was cloned
Production process : lmperial Chemical
and expressed in M. methylotrophus. These
I ndus t r ie s(l c l ), U .K. w a s th e fi rs t c o m pany to
genetically transformed organisms were more
develop a process for continuous methanol
e f f i c i e n t i n a s s i m i l a t i n ga m m o n i a . T h e y c o u l d g r o w
fermentationfor large scale production of SCP.
rapidlv and convert more methanol to SCP.
Later,Hoechst(Cermany)and Mistubishi(Japan)
However, the overall increase in the production of
also developedsimilarfermentationsystems.
SCP did not exceed l0olo.
lCl employed Methylophilus methylotrophus
(formerlycalled Pseudomonas methylotrophus)for Production of SCP from ethanol
producing SCP from methanol. A bioreactor,
referred to as lCl pressure cycle fermenter was Ethanol is a good substratefor the production of
usedfor this purpose(Fig.29.3).Thisfermenterhas SCP for human consumption (feed grade SCP).
three con'rponents-airlift column, down-flow tube
and gas releasespace.The operationwas carried
Waste gas
out at temperature35-37'C and pH 6.5-70. The
cells were subjec,ted to disruptionby heat or acid
treatment.The nutrientsolutioncan be clarifiedbv
dec ant ing .

lCl pruteen
fhe single-cell protein produced by lCl from Downflow
methanol and ammonia using M. methylotrophus tube
was referredto as ICI pruteen. This SCP was
Heat
ex c lus iv e luy s e dfo r a n i ma lfe e d i n g l.C l i nvesteda excnanger
huge am o u n t (a ro u n df4 0 mi l l i o n ) i n 1 979 and
ins t alleda c o n ti n u o u sc u l tu re s y s te mfor S C P
production.Thiswas the world'slargestcontinuous
airliftfermenter.Unfortunately, the plant could not Nutrient
be operatedfor long due to economicreasons. For solution
instance,in 1984the cost of soy meal was around
Fig. 29.3 : A diagrammatic representation of ICI
$125- 200p e r to n w h i l e l C l p ru te e nw a s sol d at
pressure cycle fermenter to produce SCP from
$600 per to n ! T h i s i s m a i n l yb e c a u s eo f the hi gh
methanol.
cost of methanolwhich represents approximately
378
NHf + ATP ADP + Pi
Glutamine
synthase
Glutamate A Glutamlne
Glutamineketo-
glutaratetransaminase
Phase ll : ihe sterilized material is passed
through two bioreactors.The first reactor contains
Glutamate+NADP+ B cr-Ketoqlutarate+NADPH
E. fibuligira which hydrolyses starch. When this
T
Glutamate hydrolysate is passedto the second bioreactor,the
dehydrogenase organism, C. utilis grows to form biomass.
NHi
Fig. 29.4 : Assimilationof ammonia by Methylophilus
methylotrophus (A) Normal pathway, (B) Genetically
engineered pathway.
How ev er , t his pr oc es s ,as s uc h, is no t e c o n o m i c a l l y
feasible. Horvever, several factors-local raw
materials, innovative fermentation technology,
po lit ic al dec is ions and f or eign t r a d e b a l a n c e s
influence production of SCP. lt may not be
su rpr is ing if lar ge s c ale pr oduct i o n o f S C P
commences, on one day, from ethanol for a variety
of reasons.
PRODUCTION OF SCP FROM WASTES
There are several materials that serve no useful
purpose and they are collectively referred to as
wastes e.g. molasses, whey, animal manures,
seulage, straw, date wastes. These waste products,
form ed in v ar ious indus t r ies and o t h e r b i o l o g i c a l
processes, largely contribute to environmental
po llut ion. Ther e ar e s ev er aladv ant a g e so f u t i l i z i n g
wastesfor the production of SCP.These include the
conversion of low-cost organic r,r,astesto useful
pro duc t s ,and r educ t ion in env ir onm e n t a lp o l l u t i o n .
However, there has been very Iimited successfor
the large scale production of SCP from wastes.This
is main ly because of transportation cost and
te ch nic al dif f ic ult ies .The t ec hnolog y a d o p t e d a n d
th e or ganis m em ploy ed f or SC P p r o d u c t i o n
depends on the waste being used as tl.resubstrate.
B IOTE C H N OLO CY
Thus, Saccharomyces cerevisiae is used for
molasses, Kluyveromyces fragilis for cheese whey.
Symba process
Symba processis a novel technology developed
i n S w e d e n t o p r o d u c e S C P b y u t i l i s i n g s t ar ch y
wastes by employing two yeasts, Endomycopsis
fibuligira and Candida utilis. The Symba process is
carried out in three phases.
PhaseI : The waste material containing starch is
sterilized by passing through a heat exchanger.
Phase lll: The microbial biomass can be
separated by centrifugation. The samples of SCP
can be dried, packaged and stored.
Applications of Symba product : The yeast
biomass produced in Symba process is of good
n u t r i t i v e v a l u e . l t i s w i d e l y u s e d a s a n a n i m a l fe e d
for pigs, calves and chicken. The animals grow
ouite well and no adverse effects have been
reoorted.
Pekilo - a fungal protein rich product
A filamentous fungus, Paecilomyces variotii,
with good fibrous structure was used for the
p r o d u c t i o n o f P e k i l o . T h i s p r o t e i n , r i c h i n f un g a l
biomass, was produced by fermentation of wastes
such as molasses, whey, sulfite liquor and
a g r i c u l t u r a l w a s t e s . l t c a n b e p r o d u c e d by a
continuous fermentation process. Pekilo is rich in
proteins (containing essential amino acids),
vitamins and minerals. lt was used as an animal
feed in supplementing the diets of calves, pigs,
chickens and hens without any adverseeffects.lt is
unfortunate that the production of Pekilo has been
discontinuedat most placesdue to economic and
commercial considerations
Quorn-the mycoprotein for humans
The protein Quorn is the mycoprotein produced
by the fungus Fusarium graminearum. Many
companies in the developed countries are engaged
in the production of fungal proteins for human
consumption. Quorn is the trade name for Fusarium
Chapt er2 9 : S IN GL E -C E LPLR OT E INA, ND MU S H R OOMS 379

natural sourcesfor the productionof SCP.lt rs

however,essentialto breakdownthese cellulosic
(in percentage)of mycoprotein in compounds into fermentable sugars. For this
comparisonto beef purpose,extracel l ul arcel l ul asescan be used.
Certain bacteria (Cellulomonassp) and fungi
Nutrient Mycoprotein Beef (Trichodermasp, Penicilliumsp) are good sources
(raw lean beefstreak)
for cel l ul ases.
Protein + 68 Techniques for the productionof cellulases
have
Lipid 15 30 been w el l standardi zedfrom severalorgani sms.
Carbohydrate 10 0 The cost of producti onof cel l ul asesi s a cri ti cal
factor in determiningthe ultimateproductioncost
Fiber 25 Traces of SCP.
Ash(minerals
etc.) 3 1
In some i nstances, the cel l ul osi cmateri al scan
RNA 1 Traces be di rectl y used for bi omassproducti on.The
resultantSCPis usedas animal feed.

mycoprotein produced in Britain by Marlow Foods P B OD U C TTON OF S C P FR OM C O2

(lCl in as s oc iat ionwit h Bank - Hov is - M c D o u g a l l ) .
C ertai nal gaegrow ni n open pondsrequi reonl y
The fungus Fusariumcan be grown continuously CO, as the carbon source. In the presenceof
on simp le c ar bohy dr at e s our c es ( lik e g l u c o s e ) . sunlight, they can effectively carry out
Ammon ium ions s upply nit r ogen.M iner al s a l t sa n d photosynthesis, and produceSCP.The examplesof
vitamins are also added. The fermentation is carried these algae are Chlorella sp, Senedesmus sp and
out at pH 6.0 and temperature 30'C. At the end of Spirulina sp. Chlorella is used as a protein and
fermentation, the culture is heated to 65'C to vi tami n suppl ementfor enri chi ng i ce-creams,
activate RNases.This is necessaryto degrade RNA breadsand yoghurtsin some countries.
and reduce the content from 10"h to around 17o. In some partsof the world, the algae in ponds
The breakdown products of RNA namely the are usedfor the removalof organicpollutants. The
nucleotides diffuse out from the cells and can be resultant algaebiomasscan be harvested, driedand
easily removed. (Reduction in RNA content is powdered.Algae SCP are very useful as animal
desirable to make the product acceptable for suppl ements
h uma n cons um pt ion.This is bec aus ehum a n s h a v e
a very limited capacity to digest nucleic acids). lt is Nutritive value of Spirulina SCP: Traditionally
possible to prod.uce1 kg of fungal biomass with a Spirulina sp have been eaten by people in some
protein content of about 1 35 g from 1 kg glucose parts of Africa and Mexico. SCPof Spirulinais of
utilized i n t he c ult ur e m edium . high nutritive value (protein-65"h,carbohydrate-
20"h, f aI-4o/",fibre-3"h, c hIo rophy||-5'h, ash-3'/.).
The dried Fusarium product is artificially S pi rul i nai s a good sourceof protei nfor human
flavoured and marketed in pieces that resemble consumpti on, parti cul arl iyn devel opi ngcountri es.
beef, pork and chicken. The nutritional composition
of mycoprotein when compared to beef is given in PRODUCTION OF SCP FROM SEWAGE
Table 29.2. Besidesbeing z'ch in essential nutrients,
Domestic sewage is normally used for large
mycoprotein has a good content of dietary fiber.
scal eproducti onof methane,w hi ch i n turn may be
There are several advantagesof fiber consumption-
utilized for the productionof SCP.The sewage
prevents constipation, decreasesintestinal cancers,
obtai ned from i ndustri al w astes i n cel l ul os e
improves glucose tolerance and reduces serum
processing, starchproductionand food processing
cholesterol.
can be utilized for the productionof SCP.The
organismCandidautilis is usedto produceSCPby
PRODUCTION OF SCP FROM WOOD
using effluentformed during the courseof paper
The natural waste wood sources containing manufacture. Other microorganisms namely
cellulose, hemicellulose and lignin are attractive Candida tropicalis, Paecilomyces varioti are
fr
r

380 B IOTE C H N OLO CY

Tnslr 29.3 A selectedlist of the edible mushroomscultivated on comnercial scale

Mushroom species Common name Substrate(s)

Agaricus
bisporus Buttonmushroom Straw,horsemanure,
compost
Leutinule
edodes mushroom
Oakor shiitake Sawdust,woodenlogs,ricebran
Pleurotus
ostreatus Oystermushroom Straw,sawdust,paper
Volvariella
volvacea Chinese mushroom Straw,cotton
or padi-straw
mushroom
Auricularia
sp *:".1mushroom Sawdust,ricebran
Coprinus
sp Straw

e mploy ed t o us e s ulf it e was t e l i q u o r f o r t h e T h e c u l t i v a t i o n o f e d i b l e m u s h r o o m s i s o ne o f

production of SCP the rare examples of a microbial culture wherern
the cultivated macroscopic organism itself is
GE NETI CALLY ENG I NEERED directly used as human food Mushroom growing is
ART I FI CI AL PRO TEI N AS AN I M A L FEED o n e o f t h e f a s t e s t d e v e l o p i n g b i o t e c h n o l o g i ca l
industriesworldover. Further growth of mushroom
R um en bac t er ia c an s y nt hes ize a m i n o a c i d s .
industry is expected for the production of enzymes,
Some workers have developed genetically
and pharmaceutical compounds, including
engineered strains of rumen bacteria that can
antitumor agents and antibiotics.
p roduc e a pr ot ein r ic h in m et hion i n e , t h r e o n i n e ,
lys ine and leuc ine. This ar t if ic ial pr o t e i n h a s a t o t a l Poisonous mushrooms
o f 1 00 am ino ac ids , of whic h 57 a r e e s s e n t i a l .
T h e r e a r e c e r t a i n p o i s o n o u s m u s h r o o m s a l so .
The gene for artificial protein was synthesized They usually possessunpleasant taste and odour.
b y 14 ov er lappingoligonuc leot idesh e l d t o m a l t o s e T h e s e m u s h r o o m s p r o d u c e s o m e p o i s o n o u s
binding protein gene. This gene was expressed rn substances like phallin and muscarine. The
E. coli under the transcriptional control of tac examples of poisonous mushrooms are Amanita
promoter. The production of this artificial protein phalloides, A. muscaria, A. viraosa, Lepiota
a ccount s t o ar ound 12% of t h e i n t r a c e l l u l a r morgani and Boletus satanas.
proteins.

However, the large scale production of artificial
protein by rumen bacteria is yet to be clearly
e si ablis hedand c om m er c ialis ed.
Mushrooms are fungi belonging to the classes
basidiomycetes (Agaricus sp, Auricularia sp,
Tremella sp) and ascomycetes (Morchella sp, Tuber
sp ) . M ajor it y of edible m us hr oom s a r e t h e s p e c i e s
of basidomyces.lt is estimatedthat there are around
4 ,000 s pec ies of bas idiom y c es O f t h e s e , a r o u n d
200 are edible, and a dozen of them are cultivated can be prepared. This actually depends on the
on lar ge s c ale. Som e of t he m os t i m p o r t a n t e d i b l e dietary habits of the people. Some of the common
mus hr oom s , t hbir c om m on na m e s a n d t h e
substratesused are given in Table 29.3.
Nutritive value of edible mushrooms
Some people regard €dible mushrooms as
vegetable meat. Mushrooms contain 80-90%
water, depending on the growth conditions
( t e m p e r a t u r eh
, u m i d i t y ) . E d i b l e m u s h r o o m sa r e r i ch
sources of protein (35-45"/' of dry weight).
However, all these proteins are not easily digestible
b y h u m a n s . M u s h r o o m s a l s o c o n t a i n f a t s a n d fr e e
fatty acids (7-1O%), carbohydrates (5-1 5%) and
m i n e r a l si n g o o d c o n c e n t r a t i o n .C e r t a i n u n d e si r a b l e
substances may also be present in edible
mushroomse.g. cadmium, chromium.
M a n y d e l i c i o u s r e c i p e s o f e d i b l e m u s h ro o m s
recipes are mushroom soup, mushroom paneer,
mushroom puiao, and mushroom omelette.
 PR
Chapt er2 9 : S IN GL E -C E L L O T E INAN
, D MU S H R OOMS 381

Advantages of edible mushroom Formulationand

biotechnology preparation
of compost
1. Mus hr oom s c an be pr oduc ed by u t i l i z i n g I Steritization
cheap and often waste substrates(industrial and J
wood wastes). Compostspread
Stock culture in trays
2 . fh ey ar e of high nut r it iv e v alue bein g r i c h i n
I
o rote ins. v it am ins and m iner als
I II Spawning
3 . Man y delic ious r ec ipesc an be pr epa r e df r o m
mushro om s .
J T
Spawn Spawnrunning
4 . Due to low c ar bohy dr at e c o n t e n t , preparation
ldeal culture
con su motion of m us hr oom s is adv oca t e d t o conditions(temperature,pH,
dia be tic Dat ient s 02, humidity)

PRODUCTI O N O F EDI BLE M USHROO M S Mushrooms

Mu sh room pr oduc t ion is bas ic a l l y a

ferme rrtat ionpr oc es s .This is m os t ly c ar r ie d o u t b y
solid-substrate fermentation A wide range of
substrates(straw,saw dust, compost, wooden logs)
de pe nd ing t he or ganis m c an be us e d ( R e f e r
Table 29.3). Mushroom production is a good
examp le of a low t ec hnology ut iliz at io n i n a n
o the rwise s ophis t ic at edm oder n biot ec hn o l o g y .
The mos t c om m on edible m us hr oom c u l t i v a t e d
worldover (that may constitute about 207o world
mushroom rrroduce) is the whife button mushroom,
Agaricus bisporus. Lentinula edodes is the second
most cul t iv at ed m us hr oom in t he wo r l d . T h e
substratesstraw, compost or horse manure can be
u se d. Th e s ubs t r at es elec t ion depends on t h e l o c a l
factors.
A schem at ic r epr es ent at ion of m u s h r o o m
production is depicted in Fig. 29.5. The compost
with d esir edf or m ulat ion is pr epar edand st e r i l i z e d .
It is spread into the trays which are then transferred
to production room and inoculated with spawn.
Spawn is the term used for the mushroom inoculum
containing spores andlor small pieces of fruiting
b od y. Aft er inoc ulat ion ( s pawning) ,t he cu l t u r e i s
ma inta ined at opt im al gr owt h c ondit ions T h e t r a y s
a re re gu lar ly wat er ed t o m aint ain 7 0 - B O %
h umid ity. The ideal t em per at ur eis about 1 5 'C , a n d
p H ab ou t 7. 0. lt t ak es about 7- 1 0 day s f o r e a c h
crop of m us hr oom pr oduc t ion. lt is pos s ib l et o h a v e
3-4 crop s , bef or e t er m inat ing t he p r o d u c t t o n
process. The mushrooms can be harvested and
marketed
+
I
Harvesting
II
+
Marketing
Fig. 29.5 : An overview of edible mushroom
Droduction.
Mushrooms have a very short life B 12 hours,
unless stored at low temperature (refrigerator
2-5"C). Therefore, they should be immediately
consumed, stored or canned.
Variations in culturing mushrooms : The
production of mushrooms is highly variable and
mostly dependson the organism and the substrate
used, besidesseveral other local factors. There are
distinct differences in the mushroom cultivation
methods between different countries. For instance,
garden and field cultivation methods are used in
Europe, while in USA, cave and house cultivation
techniquesare employed.
Some mushrooms (e.g. Volirariella sp) are
s u i t a b l ef o r c u l t i v a t i o n i n s u m m e r a n d r a i n y r e a s o n
while others grow well in winter (Agaricusbisporus,
Pleurotus sp). lt is however, possible to Srow
these mushrooms any time in a year with
appropriate temperature and humidity control
arrangements.
 [ / ic r oor ganis m s , in t he pr es e n c e o f e x c e s s A p p l i c a t i o n s of microbial
I Y I s upply of c ar bon ( gluc os e) a r e c a p a b l e o f p o l y s a c c h a r i d e s
pro duc ing s t or age c om pounds T h e s e s t o r a g e
com pounds ar e poly s ac c har idesan d p o l y h y d r o x y - M i c r o b i a l o o l v s a c c h a r i d e s h a v e i m me n se
a lka noat es( f r om s om e m ic r oor gani s m s )a, n d l i p i d s c o m m e r c i a l i m p o r t a n c e .T h e y a r e e m p l o y e d i n th e
(from some yeasts and fungi). stabilization of foods, and production of several
i n d u s t r i a l a n d p h a r m a c e u t i c a l c o m p o u n d s . Th e
In gener al, t he s y nt hes isof s t or a g ec o m p o u n d s commercial value of a polysaccharide is based on
by m ic r oor ganis m s is higher on r e s t r i c t i n g t h e i r its ability to modify the flow characteristics of
g rowt h, by lim it ing t he s upply of o n e o r m o r e solutions (techn ically known as rheology)
essential nutrients (other than carbon source). P o l y s a c c h a r i d e sc a n i n c r e a s et h e v i s c o s i t ya n d , a r e
t h e r e f o r e u s e f u l a s t h i c k e n i n g a n d g e l l i n g a g en ts.

Microbial polysaccharides are of great

The m ic r oor ganis m sc an pr oduc e l a r g e a m o u n t s
of poly s ac c har ides in t he pr es e n c e o f s u r p l u s
ca rbon s our c e Som e of t hes e poly s a c c h a r i d e s( e g .
glyc ogen) s er v e as s t or age c om p o u n d s . T h e
polysaccharidesexcreted by the cells, referredto as
exopolysaccharides, are of com merc i al i mportance.
Th e ex opoly s ac c har ides m ay b e f o u n d i n
ass oc iat ionwit h t he c ells or m ay r e m a i n i n t h e
me dium .
importance in oil industry. By conventional
methods, only 50% of the oil can be extracted.And
the rest is either trapped in the rock or too viscous
to be pumped out. lt is now possible to recover
such oils also by a technique called microbial
enhanced oil recovery (MEOR). This can be done
b y i n j e c t i n g s u r f a c t a n t sa n d v i s c o s i t y d e c r ea si n g
b i o l o g i c a l a g e n t s( i . e t h e m i c r o b i a l p o l y s a c c h a r i d e s
e.g. xanthan and emulsan).
Production of microbial
polysaccharides

The m ic r obial poly s ac c har idesm a y b e n e u t r a l The synthesis of polysaccharides favourably

(e.9 . dex t r an, s c ler ogluc an) or ac i d i c ( x a n t h a n ,
ge llan) in nat ur e Ac idic poly s ac c ha r i d e sp o s s e s s i n g
io ni z ed gr oups s uc h as c ar box y l , w h i c h c a n
fu nct ion as poly elec t r oly t esar
, e c om m e r c i a l l y m o r e
rmpor t ant
o c c u r s i n t h e e x c e s ss u p p l y o f c a r b o n s u b s t ra ter n
t h e g r o w t h m e d i u m w h i l e l i m i t i n g n i t r o g e n s u p p l y.
A carbon/nitrogen ratio of around 10 : 1 rs
considered to b e f a v o u r a b l e f o r o o ti m a l
p o l y s a c c h a r i d es y n t h e s i sT
. h e p r o d u c t i o n p r o ce ssr s
Chapter30 : POLYSACCHARIDES,
POLYHYDROXYALKANOATES,
AND LIPIDS 383

mostly carried out by batch culture fermentation. producti on, are gi veni n the
and thei r appl i cati ons
By manipulatingthe nutrient supply, differential Table 30.1. Some of the imoortantfeaturesof
synthesisof polysaccharides can be achieved.By i ndi vi dualmi crobi al pol ysacchari des are bri efl y
lim it ing n i tro g e ns u p p l y i n th e me d i u m , mostl y descri bedhereunder.
neutralpolysaccharides are produced.When metal
ionsar e lim i te d ,a c i d i cp o l y s a c c h a ri daere
s mai nl y XANTIIAN
synthesized. Molecularoxygen supply of around
907" saturationis ideal for good growth and Xanthan or more frequently referred to as
polysaccharide synthesis. xanthangumwasthe firstpolysaccharide available
commerci al l y.
l t i s a w el l studi edand mostw i del y
Biosynthesis of polysaccharides: Micro-
used hexopolysaccharide.
organisms are capableof producinga largenumber
of polysaccharides.The pathways for their Chemistry: Xanthanhas a molecularweight in
biosynthesis are comparableto the processess that the range of 2-15 x 104 dal tons.The basi c
occur for the formationbacterialcell wall. lt is repeati nguni t of xanthan i s a pentasacchari de
estimatedthat there are well over 100 enzymatic contai ni nggl ucose (C l c), mannose(Man) and
reactions,directly or indirectly involved in the glucuronic acid (ClcA) with acetate (Ac) and
synthesisof polysaccharides.
Startingwith glucose, pyruvate(Pyr)as depictedbelow.
appropriate sugars (by transforming glucose to
others) are incorporated in the formation of
polysaccharides.
Recovery of polysaccharides : As the
productionincreases,
polysaccharide there occurs Pyr
a markedincreasein viscosityof the culturebroth.
46
The polysaccharidescan be precipitatedby salts,
acids or organic solvents, and recovered by p-Man-( 1----+4)-p-G lcA-( 1-----;2)-o-Man-6-O-Ac
employingappropriatetechniques.
B a s i c a l l y ,x a n t h a n i s a b r a n c h e d p o l y m e r w i t h
Microbial polysaccharides versus plant (1
0 -++; linked glucan (glucose polymer) backbone
polysaccharides b o u n d t o a t r i s a c c h a r i d e( M a n , C l c A , M a n ) s i d e
c h a i n o n a l t e r n a t eg l u c o s e r e s i d u e s .T h e m a n n o s e
Thereis a lot of comoetitionbetweenmicrobial
and plant polysaccharides for industrial has either acetate or pyruvate groups. The number
applications. Productionof plant polysaccharides is of acetate or pyruvate molecules in xanthan rs
r elat iv elyc h e a p ,a l th o u g hi t i s u n c o n trol l edand variable and is deoendent on the bacterial strain
used. The culture conditions and the recovery
occurs for a short period in a year. In contrast,
processesalso influence the quantities of pyruvate
productionof mic,robialpolysaccharides is well
and acetateresidues.lt is believed that the viscosity
controlled and can be continued throughout the
gum is influenced by the contents of
year. However, fermentation processes for of xanthan
(from plant pyruvate and acetate.
manufacture of cheap sources)
polysaccharides
is not advisable. Applications : Xanthan gum is used as a food
additive for the preparation of saft foods (ice
G E NE RAL F EA T U R ES OF cream, cheese). lt is also used in oil industry for
MICROBIAL POLYSACCHARIDES enhancing oil recovery. Further, xanthan is useful
Of the several microbial polysaccharides,for the preparation of tooth pastes and water based
As already o a i n t s .
around2O areof industrialimportance.
stated,the commercialvalueof a polysaccharideis
' Biosynthesis: For the biosynthesisof xanthan,
mostlydependenton its rheologicalpropertiesi.e.
t h e m o n o m e r sa r e b o u n d t o a c a r r i e r l i p i d m o l e c u l e
its ability to modify the flow characteristics
of
and then transferredto a growing polymer chain.
s olut ions .
The activated monosaccharide nucleotides (e.g.
A selected list of commercially important u r i d i n e d i p h o s p h a t eg l u c o s e , U D P - g l u c o s e )s u p p l y
polysaccharides, usedfor their energy for the formation of glycosidic bonds
the microorganisms
384 B IOTE C H N OLO CY

Polysaccharide Producing organism(s) Application(s)

Xanthan Xanthononas
camDestris Asa foodadditive gelling
forstabilization, andviscosity
control, i.e. for the preparation of soft foods
e.g.rcecream,cneese.
In oil industryfor enhanced oil recovery.
In thepreparation of toothpastes,andwaterbased
oaints.
Dextran Leuconostoc n esenteroides, Bloodplasma expander
Acetobactersp, Usedin theprevenlionn andin wound
of thrombosis,
Streptococcusmutans dressing (asadsorbent).
In the laboratory
lor chromatographicandother
techniques involvedin prification.
As a foodstuff.
Al g i n a te Pseudononas aeruginosa In foodindustry andgelling
as thickening agent.
Azobactervinelandii Alginatebeadsareemployedin immobilization
of cells
anoenzymes.
Usedas ion-exchange agent.
Sc l e ro g l u c a n Sclerotium glucanicun latexpaints,printing
Usedfor stabilizing inks,and
S. rolfsii,S. delphinii muds.
drillino
Gellan Pseudomonaselodea In foodindustry
as a thickner
andsolidifying
agent.
Po l l u a n pollulans
Aureobasidiun polysaccharide,
Beinga biodegradable it is usedin
andpackaging.
foodcoating
Curdlan Alcaligenes
faecalis As a gelling
agentin cookedfoods(formsa stronggel
above55'C)
Usefulfor immobilization
of enzymes.
Emulsan Acinetobacte
r calcoaceticus In oil industry
for enhancedrecoyery.
Arlhrobacter
sp Forcleaning of oilspills.

between adjacent units. The biosynthesisof other Genetic engineering oI Xanthomonas

e xo poly s ac c har ides is c om par able w i t h t h a t o f campestris for xanthan production
xanthan. Dextran synthesis, however is rnuch
'. : The wild type X. campestriscan efficiently
simoler as described later.
utilize glucose, sucroseor starch as a carbon
source.They are however,unableto use lactoseas
P r oduc t ion : Xant han is c om m er c i a l l y p r o d u c e d
by the Cram-negative bacterium, Xanthomonas a carbonsubstrate.
campestris. The culture medium usually consists
Whey is a byproduct obtained in the
of 4-5% carbohydrate (glucose, sucrose,
manufacture of cheese.Disposalof largequantities
cor n s t ar c h hy dr oly s at e) , 0. 05- 0 . 1 % n i t r o g e n
of w hey i s a maj or probl em i n dai ry i ndust r y.
(ammonium nitrate, urea, yeast extract) and salts.
Fortunatel y,w hey i s ri ch ' i n l actose, besides
The pH is m aint ained ar ound 7 . 0 , a n d t h e
contai ni ngsmal l quanti ti esof protei ns,vi tam r ns
fermentation is carried out by batch culture for
and minerals.Atemptsare made to use whey in
2-3 days. Xanthan in the cu lture broth is
fermentati on i ndustri es.
precipitated by isopropanol or methanol. These
a ge nt s als o k ill t he m ic r oor g a n i s m s . T h e Ceneticallyengineered X. campestrishavebeen
precipitated xanthan can be dried and used for developed that can utilize lactose (from whey) for
com m er c ial our oos es . the. producti on of xanthan. For thi s purpose,
 Chapt er30 : P O L Y SA C C H A R ID P
ESO ,L Y HY D R OX Y A LK A N OA A
TENS
D, LIP ID S 385

the E. coli laczy genes (encoding the enzyme
p-galactosidaseand lactose permease respectively)
were cloned under the transcriptional control of
X campestris bacteriophage promoter. This
construct was first introduced into E. coli, and then
transferred to X. campestris. The genetically
engineered strains of X. campestris expressedthe
genes and produced high quantitiesof the enzymes
B-galactosidaseand lactose permease. These new
stra ins u tiliz e lac t os e or whey v er y ef f ic ien t l y f o r
the ind ustrialpr oduc t ion of x ant han. This is a g o o d
example of successfully converting a waste product
(whey) into a commercially important and valuable
product (a biopolymer namely xanthan gum).
DEXTBAN
Chemically, dextrans are glucans (polymers of
glu co se ) co nt aining 1 - + 6 gly c os idic lin k a g e s .
Some dextrans also have cx1 -+2, a.l -+3 and
a1 +4 linkages .The m olec ular weight s of de x t r a n s
a re in th e ra nge of 15, 000 500, 000.
Applications : Dextrans are used as blood
plasma expanders, for the prevention of
thrombosis and in wound dressing. In addition, However, algal alginates lack acetylation
d extran s are us ef ul in t he labor at or y ana l y t i c a l
techn iqu es for pur if ic at ion of biom olec ules .
Production : Dextrans can be produced by a
wid e ra ng e of G r am - pos it iv e and Cr am - ne g a t i v e u n s t a b l e a n d g e t e a s i l y d e g r a d e d . A l g i n a t e s a r e
bacteria e.g. Leuconostoc mesenteroides and useful as thickening agents in food industry, and
Streptococcus mutans. ln contrast to other
e xo po lysacc har ides( whic h ar e s y nt hes iz edw i t h i n
the cells), dextrans are produced by extracellular
en zyme in t he " m edium . The enz y m e t s
dextransucrase(a transglucosidase)which acts on
sucroseand brings about polymerisationof glucose
re sid ue s,a nd s im ult aneous lyliber at esf r ee f r u c t o s e
in to th e med ium .
The commercial production is carried out by
using lactic acid bacterium, L- mesenteroidesby a
batch fermentation process. Besides sucrose, the
cu lture me dium c ont ains or ganic nit r ogen s o u r c e
and inorganic phosphate. The crude dextran
p rod uced is pr ec ipit at ed by alc ohol and t h e n
sub jected to ac id hy dr oly s is . ln r ec ent y ea r s , t h e
alcohol precipitated polymeric dextran is subjected
to enzymatic hydrolysis by using exo- or endo-u
dextranasesto get dextrans of desired molecular
',veight.The resultant dextrans can be fractionated
a nd d ried .
It is also possibleto use a cell free systemfor the
production of dextrans. The extracellular enzyme
dextrasucrasecan transform sucrose into dextran rn
a cell-free nutrient solution. This reaction rs
o p t i m u m a t p H 5 0 - 5 . 5 a n d t e m p e r a t u r e2 5 - 3 0 'C .
ALGINATE
Alginate is a linear polymer composed of
mannuronic acid and glucuronic acid (both of
them being uronic acids) in a proportion ranging
from 4 : I to 20 : 1. Some of the mannuronic acid
residues are acetylated. Alginate is commercially
produced by Gram-negativebacteria, Pseudomonas
aeruginosa and Azobacter vinelandi i.
The type of organism used and the culture
conditions determine the relative orooortion of
m a n n u r o n i c a c i d a n d g l u c u r o n i c a c i d r e s i d u e sa n d
the degree of acetylation in alginate.Alginateswith
high contents of mannuronic acid are elastic in
nature while those with high concentration of
glucuronic acid are strong and brittle.
tl
ll
A l g a l ( s e a w e e d )a l g i n a t e s a r e a l s o p o l y m e r s o f rl
mannuronic acid and glucuronic acid, and
comparable in structure with bacterial alginates.
For commercial purposes,seaweed alginatesare
m o r e c o m m o n l y u s e d t h a n b a c t e r i a la l g i n a t e s .T h i s
i s m a i n l y b e c a u s e b a c t e r i a l a l g i n a t e sa r e r e l a t i v e l y
for immobilization of cells and enzymes.
SCLEROGLUCAN
Scleroglucan is a glucose polymer (glucomer). lt
i s a n e u t r a l p o l y s a c c h a r i d e w i t h p 1 - +3 g l u c a n
backbone and single glucose (Glc) residuebranches
( B 1 - +6 l i n k a g e ) .T h e b r a n c h i n g o c c u r s a t a r e g u l a r
s e q u e n c ea t e v e r y t h i r d g l u c o s e u n i t i n t h e p o l y m e r
backbone chain.
-----+3)-B-Glc ( 1-----+3)-B-Glc (1----+3)-B-G lc (1-----+
I
_r_
1I
=
I
0-Glc
S cl erogl ucani s a fungalheoxpol ysacchari de.lt
is commercially produced by Sclerotium
glucanicum,S. rolfsii and S. delphinii.Scleroglucan
is useful for stabilizing latex paints, printing inks
and dri l l i ngmuds.

Bictechnology [25]
386 B IOTE CHNO LO CY

G E L L AN
' Cellan is a linear heteropolysaccharide. The
re p e a ti n gu n i t o f g e l l a n i s composedof tw o
o-1"-r.rr,-8-]
g l u c o s e ,o n e g l u c u ro n i ca c i d and one rhamnose (A)Generalstructureof a
monomerin PHA
molecules.Cellan is produced by Pseudomonas
elodea f c H" ol
A deacetylatedgellan which forms firm and ll" t t l
brittlegels underthe trade name Gelrite has been {_o-cH-cHz-4n
developedby a reputedcompany in USA (Kalco (B) Polyhydroxybutyrate
l n c ).
CHa
C e l l a n i s u s e d i n fo o d i n d ustry.E venat a l ow t-
c o n c e n tra ti o ni t, i s a th i c k n er. CHr
t-
CH2
POL L U L AN t-
CHr
Po l l u l a ni s a n c ,-g l u c o speol ymer(o-gl ucan)
w i th t-
a 1-+4, and a few s., 1-+6 glycosidic bonds. f CHc ol
Po l l u l a n i s p ro d u c e d b y usi ng the fungus, l t- il 1
Aureobasidiumpollulans.lt is estimatedthat about t-o-cH-cHz-cln
7O"h oI glucose (the substrate)is convertedto (C) PHA with 3-hydroxyoctanoate
p o l l u l a n d u ri n g fe rm e n ta ti on, al thoughthe ti me
taken is ratherlong (5-7 days).
Po l l u l a ni s m a i n l y u s e d i n food coati ngand I CH" Ol I
?'.
CH, Ol
packaging. l r" illl r- tr l
t-o-cH-cHz-cJ t-o-cH-cHz-c+
C U R D L AN (D) 3-Hydroxybutyrate-co-3-hydroxyvalerate
C u rd l a ni s a B-g l u c o speo lymer(F-gl ucan).The
g l u c o s e re s i d u e sa re h e l d t ogetherby p 1-+ 3 Fig. 30.1 : Structures of polyhydroxyalkanoates(PHA).
g l y c o s i d ib
c o n d sT
. h ee x o p o lysaccharicurdl
de ani s
commerciallyproducedby employingAlcaligenes
faecalis. Curdlan-like polysaccharidesare also of yearsand are a major sourceof environmental
produced by other microorganismssuch as pol l uti on,often resul ti ngi n ecol ogi caim
l balance.
Agrobacterium rhizogenes and Rhizobium trifolii. A heavydemandfor biodegradable plasticmaterials
exists. There are some attemptsto chemically
Curdlanformsstronggelswhen heatedto above
synthesize biodegradable polyesters e.g. polylactic
55"C. Therefore,it is used as a gelling agentfor
aci d and pol ygl ycol i caci d. The pro duct ionof
cookedfoods.In addition,curdlanis alsoemployed
polyhydroxyalkanoates by fermentation is preferred
fo r i m m o b i l i z a ti oonf e n z y m es.
for use as biodegradable plastics.

PHA.CHEMISTRY AND PROPERTIES

PHA serveas lipid reservematerials in bacteria.
Polyhydroxyalkanoates (PHA) are intracellular Their functionin bacteriais comoarableto that of
carbon and energy storage compounds, produced fats and oiis in yeastsand fungi. The granulesof
by many microorganisms. They are biodegradable PHA, stored within the cells are clearly visible
polymers,and are elasticin naturedependingon under electron microscope.Some bacteria may
the polymercomposition.PHA are well suitedfor accumulate huge quantities(upto BO"/,of dry
the synthesis of plastics,the biodegradablepacking weight)of PHA.
m a te ri a l s .
are linear polyester
Polyhydroxyalkanoates
The conventionalplastics,made from coal or polymers composed of hydroxyacid monomers
They survivehundreds (Fig.30.1A).The mostcommonlyfound monomers
oil, are not biodegradable.
Chapter30 : POLYSACCHARIDES,
POLYHYDROXYALKANOATES,
AND LIPIDS 387

are 3 hydroxy acids with a carbon length ranging Glucose

from C, to C,'o.

Homopolymer PHA-polyhydroxybutyrate
cn3-do-scon
The most common PHA is polyhydroxybutyrate Acetyl CoA
which is frequently referred to as PHB. lt is a CHq-CO-SCoA----l
-
polyester with 3-hydroxybutyrate as the repeating ) 3-Ketothiolase
CoA{/l
unit (Fig. 30,1B). PHB is the homopolymer PHA.
J
PHB, a s such, is r at her har d and inf lex ible.
o o
Be ing a hig h m olec ular weight c om pound , t h e
a ccumu latio n of PHB in huge quant it iesals o d o e s CH3-C-CH2-C-SCoA
not significantly effect the osmotic pressurewithin AcetoacetylCoA
the cell. The reservecarbon comoound PHB can be NnDeH--11
oxidized to carbon dioxide and water, releasing NAD'*, Acetoacetyl
CoAreductase
large amount of energy. Bacteria require energy, +
although they are not growing, to maintain pH OH
gradient and concentration gradient of several
CH3-CH-CH2-C-SCoA
compounds. This energy, referred to as maintenance
energy essentialfor the survival of the cells, is met CoA
3-Hydroxybulyryl
by the reservematerial PHB.
I eory1e-nyoroxybutyric
CoA<lsynthase
acid) tll
!ll
Heteropolymers of PHA J
Majority of polyhydroxyalkanoates, with the I cu. o- ]
exception of PHB, contain two or more different lr"ill
monomers and are referred to as heteropolymers. t-o-cH-cHzaJ
These heteropolymers are usually composed of a Poly3-hydroxybutyric
acid
random sequence of monomers, and not different
monomers, in different chains. Besides 3-hydroxy Fig. 30.2 : Biosynthesis of polyhydroxybutyric
acids, several other hydroxy acids are found in the acid (PHB).
structure of PHA. e.g. 4-hydroxybutyrate (4HB). In
fact, it has been found that the bacteria are capable
of in co rpo rat ing m or e t han 100 dif fe r e n t eutrophia produces a copolymer of 3-hydroxy-
hydro xylate d m onom er s int o PHA. This dep e n d s butyrate and 3-hydroxyvalerate(abbreviatedPHB/
on the organism and the nature of the carbon V). PHBas suchis hardand brittlebut the presence
sou rce sup pli ed dur ing t he ac c um ulat ion of t h e 3-hydroxyvaleratemonomersmakesPHBIr'flexible
DOtVmer. The properties
and stronger. of PHBIr'are similarto
those of polypropylene, and therefore it is
The properties of PHA mostly depend on the
commerci al l more
y useful .
na ture o f th e m onom er s it c ont ains . I n gen e r a l ,
PHA with longer s ide c hains ( e. g. 3- hy d r o x y -
POLYHYDROXYBUTYRATE (PI{BI
octanoate containing PHA) and heteropolymeric
PHA are more flexible and soft. Biosynthesis of PHB
Among the severalPHA, the pathwayfor the
3.Hydroxybutyrate'cG (PHB)hasbeen
biosynthesisof polyhydroxybutyrate
3.hydroxyvalerate
thoroughlyinvestigated.Startingwith acetyl CoA,
As already stated, by selecting a specific P H B i s synthesi zedi n three reacti on steps
org an ism a nd by m anipulat ing t he c om pos it i o n o f (Fig. 30.21.Acetyl CoA is convertedacetoacetyl
the me diu m, the c hem ic al c ons t r uc t ionof t he P H A CoA bv the enzvme3-ketothiolase which is then
ca n b e alte red. Thus , when t he m edium c on t a i n s reducedto 3-hydroxybutyryl CoA by acetoacetyl
qlu co sea nd p r opionic ac id, t he or ganis m Rals t o n r a CoA r.eductase. The reducing equivalentsare
 388 B IOTE C H NO LO CY

Glucose

i
+
Pyruvate
l/'
I
l>COe
+
Acetyl CoA

OU O A
CoA
NADPH
_--- a.rrr"*
// .dynthase :
p+ Oxaloacetate Cikate

/\
/\

I
/T
I
I

Fig. 30.3 : Regulation of polyhydroxybutyrate (PHB) metabolism.

s u p p l i e db y N AD PH .T h e e n z y meP H A synthase rs C oA )decreases. Thi si s mai nl ydueto the i nhibit ion

responsiblefor the addition of 3-hydroxybutyrateof citratesynthaseby NADH. A decreasein the
r e s i d u etos th e g ro w i n gP H B c h a i n. concentrationof coenzymeA (as citrate is not
formedfrom acetylCoA, CoA is low) relievesthe
Regulation of PHB biosynthesis i nhi bi ti on of 3-ketothi ol ase. Thi s enables t ne
diversionof acetylCoA for the productionof PHB.
The outline pathways concerned with the The suppl y of reduci ng equi val ents(NADPH)
bi o s y n th e s ai sn d d e g ra d a ti oonf P H B i n R al stonta requiredby the enzymeacetoacetyl CoA reductase
eutrophiaaredepictedin Fig.30.3.Theenzymesof also regulates the synthesis of PHB.
PHB biosvnthesr's are constitutivein nature.Thus
the enzymemachineryfor PHBsynthesis PHB,the storageenergyreservecompound/can
is present
al l th e .ti me i n th e c e l l . W h e n th e grow th of the be degradedto acetyl CoA and metabolisedvia
m i c ro o rg a n i s im Krebs cycle. This occurs when the organismis
s re s tri c tebdy l i m i ti ngan essenti al
nu tri e n t,PH B s y n th e s ira deprived of energysupplyingcarbonsources.PHB
s p i d l yo ccurs.
is degraded by PHB depolymeraseto form
Duringthe activegrowthphase,acetylCoA gets 3-hydroxybutyrate. NADH is the inhibitor of the
oxidized through the Krebs cycle. Further,free subsequentreaction, catalysedby the enzyme
coenzymeA inhibits 3-ketothiolase and therefore 3-hydroxybutyrate dehydrogenase i.e. formationof
very little PHB is synthesized. When the growth acetoacetate from 3-hydroxybutyrate. The
ceasesor is restrictedby limiting a nu(rient,the biosynthesis and breakdownof PHB form a cyclic
operationof Krebscycle (i.e. oxidationof acetyl process,as depicted in Fig. 30.3.
 ES , Y H Y DR OX Y A LK A N OA A
Chaot er30 : POL Y SA C C H A R ID POL TENS
D, LIP ID S 389

Biosynthesis of PHBAI
Some strainsof Ralstoniaeutropha(formerly
c
known as Alcaligeneseutrophus)are capable of
o synthesizing poly (hydroxybutyrate-co-hydroxy-
c
0) valerate)(PHB/V).For the formation of PHB/r/,
glucose and propionic acid are required as
substrates Propionic acid as propionyl CoA rs
responsible for the synthesis of 3-hydroxyvalerate
The three enzymesinvolvedin the synthesis of 3-
hydroxybutyrate(3-HB) also participate in the
formation of 3-hydroxyvalerate(3-HV). The
pol ymer P H B /V contai ns 3-H B and 3-H V
monomersi n a randomseouence(no i ndi vi dual
pol ymersof 3-H B or 3-H V exi st).The rel atrve
concentrati ons of gl ucose(precursor for 3-H B )and
propi oni caci d (precursorfor 3-H V ) i n the cul ture
medi um determi nethe chemi calcomoosi ti onof
pftR /\/
Fig. 30.4 : Production of polyhydroxybutyrate (PHB).
The biosynthetic pathway for the formation of
PHBI/ is depicted in Fig. 30.5.
Production of PHB
Polyhydroxybutyrate is mostlymanufactured by
batch culture.PHB oroductionoccurswhen there
> an excesssupplyof carbonsource,and limitation
: i s om eot her e s s e n ti anl u tri e n ts u c h a s n i tro gen,
r hos phor usor s u l fu r s o u rc e . T h e p ro d u c ti on/
:ccumulation of PHB is depicted in Fig. 30.4.
Trere are two distinct phases-a growth phaseand
Acetyl CoA
= polymer accumulation phase. As the growth
: ' as e c eas esd, u e to n u tri e net x h a u s ti o ns,y n t hesi s
: poly m er( P H B)c o mme n c e sl t. i s a l s op o s s i bl e
to
I
+ 3-KetovalerylCoA
g e o x y g e ns u p p l yto AcetoacetylCoA
: ' : ' duc e P HB b y re s tri c ti nth
. = ' obic bac t eri a .
I
+
A pplic at ions o f PH B 3-Hydroxybutyryl
CoA
r HB c an b e i mp l a n te di n th e h u m a n body
'. :^out rejection.This is becausePHB does not
' - , juc e any i mmu n e re s p o n s ea n d th u s i t i s CoA
3-Hydroxyvaleryl
Poly (3-hydroxy-
biocompatible. PHB has several medical butyricacid)
: : : r c at ionse.B. a s d u ra b l e b o n e i mp l a n ts,for
, - . lr d dr es s i n g s .A tte m p ts to u s e PH B as Poly (3-hYdroxy-
butyricacid)
: = : - . : dables u tu re sa n d o th e r i m p l a n tsh a s not synthase
- =: ,r ith successdue to very slow degradationof

Poly (3-hydroxybutyrate-co
'3-hydroxyvalerate)
B I O S Y NT HES IS O F O T H ER PH A
^e bios yn th e s i so f o th e r PH A i s ' q ui te Fig. 30.5 : Biosynthesis of poly (3-hydroxybutyrate-co-
s-hydroxyvalerate) (Note : The same 3 enzynes are
- : : . able t o th a t d e s c ri b e dfo r P H B Some
required for the synthesis of poly
-:, iant featureswith regardto the synthesisof
3-hydroxybutyric acid; For details refer Fig. 30.2)
- - c or t ant P H A a re d e s c ri b e d .
 390 B IOTE CHNO LO CY

Biosynthesis of PHA with conventionalnon-biodegradableplastics.For this

long side chains reason, many companies have stopped the
commercialproductionol biopol by fermentation.
It is generally observed that when a PHA
producingorganismis grown on a carbon source
containingn carbons,3-hydroxymonomerswith n- Applications. of biopol
2 or n + 2 carbonsare produced.This suggests
the Biopol is approved for use as a food contact
involvementof p-oxidationof fatty acids (which material.lt is being used in the manufactureof
sequentiallyremoves2-carbon fragment)in the papercups and food trays.lt is also usedas a thin
biosynthesisof PHA. waterprooflayerin food packaging. Biopol,can be
The organism Pseudomonasoleovorans can mouldedto oroducebottlesand severalotheritems.
s y n th e s i z PH
e A w h e n o rg a n icaci ds and al kanes
a re s u p p l i e di n th e m e d i u m as carbon sources. GE N E TIC E N GIN E E R IN G FOR
For indtance, PHA with high content of PHA PRODUCTION
3-hydroxyoctanoatecan be produced from
n-octaneor n-octanoicacid. It i s possi bl eto cdmmerci al l yprod ucePHB,
PHBI/ and other polyhydroxyalkanoates by
t BIOPOL.A BIODEGRADABLE PLASTIC fermentation of R. eutropha. This organism,
II however,grows very slowly and utilizes only a
II Biopol was the trade name used by lCl plc in limited number of carbon soureesfor growth.
Ilr UK for the biodegradableplasticscomposedof For these reasons,the production of PHA by
I
P H Ba n d P H B ryT . h eC ra m -n e gati ve
soi lbacteri um, R. eutrophais expensive.
i: ; Ralstonia eutropha was employed for the
ll ma n u fa c tu re of bioool. Fortunately,the genes responsiblefor the
I
I synthesis of importanttypesof PHA by R. eutropha
II have been characterized and cloned.Thesegenes
i
Production of biopol
I have been transferredto E, coli (a bacteriumthat
I The ferrnentation processfor the productionof does not normallysynthesizePHA).The resultant
I biopol involves two distinct phases-biomasstransformants,E. coli cells, grow rapidly and
productionphaseor growth phase,and polymer produced l arge quanti ti es of P H B . But t he
a c c u mu l a ti o np h a s e .T h i s i s comparabl eto the major limitation is that about half of the E. coli
productionof PHB (ReferFig. 30.4), cells lose the plasmid constructsin about 50
When the bacterium Ralstoniaeutropha is generati ons. Thi s posesa bi g probl em,p ar t icular ly
c u l tu re do n a s i mp l eme d i u mcontai ni nggl ucose for large scale continuous cultures. In recent
and salts,growth phaseis observed.By restricting years, some workers have tried to genetically
the essentialnutrientphosphate(preferredsince it mani pul ate and producestabl epl asmi ds wit h som e
is expensivecomparedto other nutrients),growth success.
phasecan be stopped.Now, glucoseand propionic Recombinant strainsof E. coli have been useo
acid are continuously fed for largescaleproduction for the productionof PHB, PHBI/ and other types
of PHA i.e. PHBlri.The relativeconcentrationof of PHA. There are some other advantagesof
g l u c o s ea n d p ro p i o n i ca c i d i n the cul turemedi um producing PHA by E. coli instead of R. eutropha.
d e te rmi n e sth e p ro p o rti o no f 3-H B and 3-H V
i The polymersare producedin a crystallinestate
lr
(I
monomersin biopol. Biopol containingaround
10% 3-HV is found to possessthe desired
by E. coli (rhey are in amorphous state in
R. eutropha),and their extraction and recovery are
characteristics. much easier.
Recoveryof biopol: The cellscan be disrupted
and subjectedto solubilisation
of components other PHA production by plants
than biopol. The polymer can be washed and
The major limitation of producing PHA by
recoveredby centrifugation.
it is
fermentationis the cost factor.Approximately,
l-imitationsin the production of biopol : The ten times more expensiveto produce PHA by
manufactureo( biopol by fermentationis very bacteria compared to the manufacture ol
expensivewhen comparedto the productionof petrochemical plastics.
 POLYHYDROXYALKANOATES,
Chapter30 : POLYSACCHARIDES, AND LIPIDS
Plantsare attractivesourcesfor a lessexpensive
production of PHA. A transgenic plant of
Arabidopsis thaliana, containing the bacterial
genes for PHB genes, was developed in 1992.
However, the yield of PHB was rather low.
Oil seedplantswhich haveplentyof acetylCoA
(for oil biosynthesis)
are attractivesourcesfor PHA
production.This is becausethe acetylCoA is the
key substratefor PHA synthesis.No significant
success has been achieved so far for PHA
productionby oil seedplants.
Among the microorganisms,the eukaryotescan ( D H A ) . M i c r o b i a l D H A i s u s e d f o r i n c l u s i o n , i n
accumu Iate triacylglycerols. In contrast,prokaryotes baby foods.
predominantly accumulate polyhydroxyalkanoates
{as described above).
Microorganisms as oil factories
In general, commercial oils (and fats) used in
foodstuffsand cosmotics are obtained from plants
and a nima ls. Thu s , t he t r adit ional s our c es of oil s
are plants and anirnals. In recent years, microbial
oroduction of oils is gaining importance. There is a
:rend to use microorganisms as oil factories for the
iollowin g re ason s .
1 Plant oils contain residuesof pesticidesand
rerbicides that are used in the cultivation of oil
.eed olants.
2 Fats and fatty acids from animal sources are
-racceptable by some sections of the society on
-oral and/or religious grounds. Further,animal fats
"ave the risk of transfer of disease-causingagents
e q . p rion s) to h um ans .
3. The eukaryotic microorganismsare capable
rf producing a wide range of polyunsaturatedfatty
acids. Certain microorganismscan synthesizehuge
q ua ntitie s o f a s ingle f at t y ac id whic h is quite
advantageousfor large scale production and easy
recovery of a particular fatty acid.
4 . Microb ially pr oduc ed oils whic h ar e dev oi d
of harmful contaminants will be acceptable to the
p u blic on mo ral and et hic al gr ounds als o.
PRODUCTION O F SI NG LE. CELL O I LS
Oils rich in a particular fatty acid arc regarded
as single-cell oils. So far, researchers have been
391
successfulin producing single-celloils rich in three
polyrtnsaturatedfatty acids. These developments
have occurred due to the interest taken by
m u l t i n a t i o n a l p h a r m a c e u t i c a lc o m p a n i e s .
I i n o l e n i c a c i d : S i n g l e - c e l lo i l r i c h i n l i n o l e n i c
acid was the first oil produced by the fungus Mucor
circinelloides. Its production however, was later
discontinued due to cost factor.
Arachidonic acid : The soil fungus, Mortierella
alpina has been used for the production of oils rich
in arachidonic acid.
Docosahexaenoic acid : The ,.narine alga,
Crypthecodinium cohnii has been employed for
commercial production of decosahexaenoic acid
Limitations in the production of
single-cell oils
The fermentation technology used for the
p r o d u c t i o n o f s i n g l e - c e l lo i l s i s v e r y e x p e n s i v e .
It is visualisedthat the day may not be far off to
t r a n s f e rt h e g e n e s p r o d u c i n g s i n g l e c e l l o i l s f r o r n
m i c r o o r g a n i s m st o p l a n t s . T h e s e t r a n s g e n i c p l a n t s
can produce large quantitiesof single-celloils more
cheaply. But these oils will be genetically
engineered, and therefore, it is doubtful whether
they will have wide-spread public acceptance.
Naturalrubberis producedby a largenumberof
plants.Chemically,it is a biopolymer composedof
cis-l, 4-polyisoprene.The biosyntheticpathwayfor
rubberhas been worked out. About l7 enzymatic
reactionsoccur for the formationof rubber.The
substratesare simplesugars.The final stepinvolves
the polymerisationof isopentenylpyrophosphate
on to allylic pyrophosphate,catalysedby the
enzymerubberpolymerase.
Attempts are on to synthesize rubber in
genetically engineered microorgnaisms. For this
purpose, the rubber producing plant Hevea
brasiliensiswas selectedand a cDNA library by
using mRNA was contructed.lt may be possiblern
the nea!" future, to synthesize rubber from
geneti cal l mani
y pul ated
mi croorgani sms.
392 B IOTE CHNO LO G Y

The biopolymer,adhesiveproteinproducedby
the blue musselMytilus edulis is very strong and lndole
water proof. lt is usefultbr the musselto attacn
lndole2-3-
I
itself to different surfaces.As the adhesive is
dihydrodiol Ixyene
secreted,it gets randomlycross-linked,hence it oxidase
cannot be sequenced.Fortunately, the immediate I
precursorfor the formationof adhesiveproteinhas
+
Indoxyl
been isolatedand characterized. lt is a 130-kDa
p ro te i n ri c h i n c e rta i n a mi n o aci ds l i ke seri ne
I
lo z
threonine,tyrosine,lysineand proline.Most of the J
proline and tyrosine residuesare hydroxylated. Indigo
F u rth e r,b i o c h e mi c a la n a l y s i sreveal edthat thi s
precursorprotein contains repeatingunits of a Fig. 30.6 : Biosynthesis of indigo from tryptophan by
rlprrnoniirlp

The cDNA for the precursoradhesiveprotein

has been constructedby usingmRNA. This cDNA
was introduced into yeast cells and adhesive
protein molecules were synthesized by the
recombinant veast.
T h e g e n e e n c o d i n g th e repeati nguni t of
decapeptidehas been identifiedand chemically
synthesized. This genewas incorportedinto E. coli
c e l l sfo r s y n th e s i z i nagd h e s i veprotei n.B uttherers
a limitation-E. coli cannot carry out post-
translationalreactionsi.e. hvdroxvlationof the
adhesiveprotein.This deficiencycan be overcome
by carryingout in vitro hydroxylation. lt is possible
to hydroxylateamino acidsby employingbacterial
enzytnes (e.9. tyrosinase)in the presence of
v i ta mi nC .
It is expectedthat adhesivebiopolymerswill be
soon producedfor widespreaduse in dentistryand
me d i c i n e .
lndigo is a blue pigment, extensivelyused fo
dye cotton and wool. There is a heavydemandfor
indigo worldover,and it is at presentthe largest
sel l i ngdye i n the w orl d. Ori gi nal l y,i n digo was
isolatedfrom plants.Now, it is being synthesized
by usi ngthe hazardous compounds sucha aniline,
formaldehydeand cyanide.
In the recent years, it has been possibleto
synthesizeindigo from tryptophan,by genetically
engineeredE. coli. The biosyntheticpathwayfor
the productionof indigo is given in Fig. 30.6.
However,a largescalecommercialmanufacture of
indigo by geneticallyengineeredE. coli is yet to
commence.
-l- he capacity to do work is referredto as energy.
I Energy may be considered as a form of matter
',rh ich is inter c onv er t ible. The m oder n m a n i s
rostly d ep en dent on t hr ee s our c es f or his en e r g y
^eecls coal, natural gas and oil-s, collectively
-eierred to as fossil fuels or fossil energy sources.
-re
fea r of d eplet ion of global f os s il f uels h a s
-:rrced man to look f or s uit able alt er nat iv een e r g y
:curces su ch a s s olar , hy dr o, t idal and wind po w e r ,
.-rcl more recently nuclear energy. In addition to
.'ese, a dvan c es in biot ec hnology hav e helpe d t o
'-:itfully utilize the energy from biological systems.
Biomass is the fofal cellular and organic mass,
produced by the living organisms. lt is the primary
product of photosynthesrs and is a good source of
en erg y i.e. bi oener gy . Br oadly s peak ing, bio m a s s
-=:r'esents all forms of matter derived from
- oq ica l ac t iv it ies . Thes e inc lude plant s a n d
-- u ltura l pr oduc t s , m ic r oor ganis m s , an i m a l
.,.iesan d manur e. The t er m biom as s is als o u s e o
rolle ctively des c r ibe t he was t e m at e r i a l s
- r:rrced in f ood and agr ic ult ur al indus t r i e s .
::: r€S b ein g a good s our c e of ener gy ,biom a s s t s
--: 'ta nt for t he pr oduc t ion of several
^ - -re rcia lly im por t ant pr oduc t s .Thus , biom a s s i s
. --p riate ly r egar ded as a r enewable s our c e o f
which c an be dir ec t ly c onv er t ed t o e n e r g y
--erg y
,r en erg y ca r r ier c om pounds by v ar ious m ea n s .
I n m o s t d e v e l o p e d c o u n t r i e s ,b i o m a s s i s u t i l i z e d
for the production of industrial and commercial
products (ethanol, oils, methane, single-cell
protein) In contrast, in the developing countrres
( l n d i a , L a t i r rA m e r i c a , A f r i c a ) , a m a j o r p a r t o f t h e
b i o m a s s i s d i r e c t l y u s e c la s a s o u r c e o f e n e r g y ( a s
firewood).
I t i s e s t i m a t e dt h a t t h e a n n u a l n e t v i e l d o f o l a n t
biomass is around 175 billion tons of dry matter
(-i25 billion tons on land and 50 billion tons on
oceans) Forests significantly contribute to the
p r o d u c t i o n o f l a n d b a s e d b i o m a s s ( a r o u n d 4 5 %) .
Agricultural crops on the cultivated land account
f o r a b o u t 6 % o f t h e p l a n t b i o m a s s .T h e a g r i c u l t u r a l
b i o m a s s p r o d u c t s ( c e r e a l s ,p u l s e s ,o i l s , a n i m a l f e e d
etc.) adequately meet requirements of foods for
humans and animals, besides other basic needs
( f u e l s ,c h e m i c a l s e t c ) . F r o m t h e c h e m i c a l p o i n t o f
view, about 50% of the land produced biomass is
in the conrplex form of lignocellulose.
Fossil fuels.derivatives of biomass
The modern society is dependent of on the non-
r e n e w a b l e s o u r c e so f e n e r g y n a m e l y o i l , g a s a n d
c o a l . T h e s e f o s s i l f u e l s a r e a c t u a l l y d e r i v a t i v e so f
ancient biomass. lt took millions of years for the
fossil fuels to be deposited beneath the earth and
oceans. However, in just within a cer-rtury of
e x p l o r a t i o n , t h e m a j o r f u e l r e s e r v e s( p a r t i c u l a r l y
gas and oil) are depleted, and at the present rate,
they are not likely to last long. As such, there exists
393
394 B IOTE C HNO LO G Y

Naturalvegetation Combustion
(grass,timber,foliage) (electricity,
steam)

Energycrops -----+ Dry chemical Processes

(sugarcane,cassava) (methane,ammonia)
FGassification
IL
Pyrolysis(gas,oils)

Wastes --------------) Aqueous processes

(agriculture,industries) I Fermentation(alcohol,methane)
L Chemicalreduction(oils)

SOURCES UTILIZATION

Fig,3l.1 : An overview of the sources and utilizationof biomass,

an energy crisis throughout the world. The biomass produced by photosynthesiscan be

Consequently, continuetheir searchfor
researchers a p p r o p r i a t e l y u t i l i z e d f o r t h e p r o d u c t i o n o f fu e l s
alternateand renewablesourcesof energy. (alcohol, methane) and various other commercial
products.
Photosynthesis-the ultimate source
Ghemical nature of biomass
of energy
T h e p l a n t b i o m a s s i s m a i n l y c o m p o se d o f
Photosyntheticorganisms are the ultimate cellulose, hemicellulose, Iignin, starch, proteins,
sources for trapping the solar energy. In the w a t e r s o l u b l e ( s u g a r s ,a m i n o a c i d s ) a n d f a t so l u b l e
presenceof photosyntheticpigment chlorophyll, (oils, pigments) compounds. ln fact, majority of
carbon dioxide is converted into complex these constituents are present in the plant cell
carbohydrateswith the evolutionof oxygen. w a l l s . T h e r e i s a w i d e v a r i a t i o n i n t h e c h e m i ca t
' composition of the biomass, depending on the
C h l o ro o h v l l
C Q , + H rO .;. + ( C H ,O) + O, source. For instance, the biomass obtained from
Lr ent
s u g a r c a n e a n d b e e t s u g a r i s r i c h i n s u g ar s w h i l e
In the reactionsthat follow later, solar energy is the biomass of potato and topioca is rich in starch.
trapped into molecules such as fat and proteins, On the other hand, cotton has high content of
besides other complex carbohydrates (cellulose, c e l l u l o s e .
h em ic ellulos e,lignin) .
The chemical nature of biomass derived from
Photosynthetic organisms are the true solar i n d u s t r i a l a n d m u n i c i p a l w a s t e s i s h i g h l y va r i a b l e
energy converters. lt is estimated that at present which mostly depends on the sources that
more than 10 times more energy is generated by contribute to the biomass.
photosynthesis annually than consumed by the
SOURCES AND UTILIZATION OF
world's population. Unfortunately, the role of
BIOMASS
photosynthesisis not well recognized to solve the
present day problem of energy crisis. This, despite The maior sources of biotnass are natural
the , fact that it is onlv the photosynthetically vegetation,energy crops, and agricultural,industrial
produced biomass that is available today in the a n d u r b a n o r g a n i c w a s t e s ( F i g . 3 1 . 1 ) . Th e r r
form of fossil fuels. p r o d u c t i o n i n t u r n i s d e p e n d e n to n t h e s o l a r e n e r g y.
Chaot er3 1 : B IO MA SSA N D BIOE N E RC Y
The natural vegetation (growing natural forests
a nd a qu at ic weeds ) s ignif ic ant ly c ont r i b u t e s t o
biomass. Wood-rich plants are grown in many
cou ntrie s ( par t ic ular ly dev eloping c oun t r i e s ) t o
generate fire for cooking and other purposes In
recen t year s / well planned and o r g a n i z e d
ola nta tion s ar e c ar r ied out in s om e c ou n t r i e s t o
pro du ce biom as s t o m eet ener gy dem a n d s . F o r
instan ce , s ugar c ane and c as s av a plant a t i o n s i n
Brazil and Australia are used for ethanol
pro du ctio n. Plant sr ic h in lignoc ellulos ea r e g r o w n
in Ame ric a and Sweden whic h ar e us ef u l f o r t n e
pro du ctio n of liquid f uels ( et hanol, m et ha n o l ) .
Agricult ur al, indus t r ial and m unic ipa l w a s t e s
we re ea rlier c ons ider ed as us eles sand d i s c a r d e o .
But irr the recent past, many countries have
developed methods for converting these wastes
into biofuels and commercially important
products. The successfullyused agricultural wastes
include straw; bagasse,bran, cotton wastes.Among
th e ind ust r ial was t es , m olas s es , whey , d i s t i l l e r y
wastes and sevr'ageare the important ones.
As already stated,the bionrassis utilized for the
p rod uction of biof uels and v ar iou s o t h e r
co mpo un ds . The t ec hnique m ainly depen d s o n t h e
chenrical nature and moisture content of biomass.
Combustion : Low moisture containing biomass
(n,ood, straw, bran) can be directly burnt l:y a
process referred to as combustion to Benerate
electricity.
Dry chemical processes: The biomasswith little
moisture content can be subjected to various dry
chemical processes-pyrolysis, gassification to
pro du ce m et hanol, oil and am m onia biom a s s .
Aqueous processes : The biomass with high
',\ater content is used in aqueous processessuch as
'ermentationto produce ethanol, oils and methane.
An orrerview of the sources and utilization of
rio mass is depic t ed in Fig. 31 . 1 . T h e
riotechnological strategiesfor the production of
riofu els ( alc ohol, hy dr ogen) f r om biom a s s a r e
r.ie flv de s c r ibed in t he f ollowing pages .
PRODUC TI O N O F ALCO HO L FRO M
BIOMASS
\lcoh ol, c hem ic ally et hanol ( C2Hs O H) h a s b e e n
:'oduced by fermentation for thousands of years.
\ though the developed countries these days prefer
395
to manufactureethanol bv chemical means.the
developingcountriescontinue to produce it by
microbial fermentation.Alcohol is the liquid fuel
which is mostly produced from the biomass.Ihe
raw materials (biomass) used for alcohol
productionincludestarchy materials(wheat,rice,
maize, potato) and cellulosic materials (wood,
agriculturalwastes)
The readermust refer the Chapter23 {or full
detailson alcohol fermentation.
Biogas is a mixture of gases composed of
methane (50-80%), carbon dioxide (15-40%),
nitrogen (4'h) and hydrogen sulfide (1%) with
traces of hydrogen, oxygen and carbon monoxide.
There are many other common names for biogas-
gobar gas, sewage gas, klar gas and sludge gas.
Biogas is a gaseous fuel and serves as a good
source of energy for various purposes.
1. lt can be used for cooking purposes (on
combustion).
2. Cobar gas can generate electricity.
3. lt can be purified to yield good grade
methane. Methane gas is extensively used as a fuel
for domestic and industrial purposes.lt is employed
for the generation of electrical, mechanical and
heat energy.
I t i s e s t i m a t e dt h a t u n d e r i d e a l c o n d i t i o n s , 1 0 k g
o f b i o m a s sc a n p r o d u c e 3 m 3 o f b i o g a s .T h i s b i o g a s
can provide 3 hour cooking, 3 hour lighting or 24
hour refrigeration (with suitable equipment). The
calorific value of biogas (with B0% methane) is
around 8.500 callm3.
Biogas production in different
countries
Biogas production significantly contributes to
world's energy source. China has the largest
number of biogas or gobar gas systems, with an
estimated 7 million units. Covernment of China
encouragesand offers subsidiesfor construction of
biogas units. As a result, the cost of a biogas plant
is cheaper than a bicycle in China!
 396 B IOTE C H NO LO CY

C o v e rn me n ts i n ma n yd e v e l o p i ng countri es
al so Cellulose Starch Proteins Lipids
o f b i o g a spl ants.In Indi a,
en c o u ra g ei n s ta l l a ti o n
Covernment provides 25% subsidy, besides Hydrolytic phase
encouraging banksto offerloansfor construction of
. h e y a re b e c o m i ngpopul ari n rural
bio g a sp l a n ts T
areas.Pakistanalso has a good numberof gobar
gas plants.
Acidifying phase

Substrates for biogas

Organic H2 COz Alcohol
acros
The usual substrates for biogasproductionare \ ,/ ,/
the wasteproductsof animalhusbandry, industries, Y ,/ Acetogenic phase
a n d mu n i c i p a l i ti e sIn. Indi a and other
ag ri c u l tu re n"!t^t"/
i
I
developingcountries,cattle dung (gobar) is most
tw"thanogenicphase
commonly used.The major raw materialfor biogas I
in C h i n a i s p i g d u n g . METHANE
I
i The concentration of organicdry matteror total
I Fig. 31.2 : Microbial production of methane
I s o l i d s (T S) i s u s e fu l fo r g ra d i n g the i ndustri al , (methanogenesis from biomass).
iil l a s te s.Thus,l ow grade
ag ri c u l tu raaln d m u n i c i p aw
( < 1 % T S ),m e d i u mg ra d e(1 -5 %TS ),hi ghgrade(5-
l' l
il 20% TS) and solid (2O-4O% TS) wastes are 2. Acidifying phase : This phase rs
:i
available.Solidor high gradewastesare preferred characterized by more formation of organic acids,
l' as substrates for biogasproduction. besidesHz, CO2, and alcohol.
I
I
I In general,mostof the substrates usedin biogas 3. Acetogenic phase : Acetogenic bacteria
I pl a n tsc o n ta i na d e q u a te
q u a n ti ti eof
s al mostal l the convert alcohol into acetate. These bacteria also
I essentialnutrientsrequiredfor microbialgrowth.lf generate acetate from H, and COr.
necessary, nitrogen,phosphorus and traceelements 4. Methanogenic phase : This is the actual
are added.A carbonnitrogenratio (C/N) lessthan phase of methane gas formation. The methanogenic
40 : 1 is preferredfor optimal biogasformation. bacteria (e.g. Methanobacterium omelianskii, M.
Water hyacinth (Eichorniacrasipes)an aquatic formicicum, M. bryantii, Methanosarcina barkeri)
weed with huge biomassis a good source (raw convert acetate, and CO, and H, into methane.
material)for methaneproduction.With high C/N CH3COOH -------J CHo + CO,
ratio and low lignin content,the yield of methane
is high. Another aquatic weed Azolla is equally 4H, + CO, -----) CHo + 2HrO
importantfor methanegeneration. Some other substrateslike formate and methanol
can also be converted to methane.
Microbial production of methane
4HCOOH ------+ CH, + 3CO, + 2HrO
(biogasl
4CH3OH -_> 3CHo + CO, + 2HrO
Methane is the most abundantconstituentof
bi o g a s .l t c a n a l s o b e d i re c tl y used for vari ous The overall reaction of methane formation from
p
do m e s ti ca n d i n d u s tri a l u rp o ses.The mi crobi al glucose as the starting material may be represented
generationof methane,appropriately referredto as a s f o l l o w s .
methanogenesis from biomassoccursin four phases C6H1206 -----) 3CHo + 3CO,
( F i g .3 t.2 ).
T h e c o m p l e x p o l y s a c c h a r i d e sp a r t i c u l a r l y l i g n i n
1. Hydrolytic phase : Certain facultative a n d c e l l u l o s e d u e t o t h e i r i n e f f i c i e n t c o n v e r si o n ,
an a e ro b i cb a c te ri ah y d ro l y s eth e compl exorgani c l i m i t m e t h a n e p r o d u c t i o n . I n t h e n o r m a l pr o ce ss
m a te ri a losf th e b i o ma s s(c e l l u l o se,
starch,protei ns, of methanogenesis, approximately 50% of
lip i d s )to l o w m o l e c u l a rw e i g h t sol ubl eproducts the complex polysaccharides contribute to
an d s o m eo re a n i ca c i d s . methanogenesis.
 3 1 : BIOMA SSAN D B IO E N E RC Y
ChA O t Cr 397

=--+Gas outletpipe
Pulley

Usedslurry
(manure)

Fig.31.3: Diagrammatic representation of a biogas plant (gobar gas plant).

PROCESS OF BIOGAS PRODUCTION
(BIOGAS PLANTI
Biogas poduction from biomass is an anaerobic
process.The anaerobic digestion is usually carried
o ut by us ing air t ight c y lindr ic al t ank s w h i c h a r e
referred to as anaerobic digesters. A digester may
be made up of concrete bricks and cement or steel,
u su ally bu ilt under gr ound.The diges t erha s a n i n l e t
attached to a mixing tank for feeding cow dung
(Fig. 31 .3). The methanogenic bacteria from
another digester .are also added with cow dung.
The digester is attached to a movable gas holding
or storage tank with a gas outlet. The used slurry
(spent cow dung) comes out from the digester
through an outlet. This can be used as a manure.
The anaerobic digester described above, is a low
technology gobar gas plant, commonly used for
do mestic pur pos es in r ur al ar eas in I n d i a . T h e
processof digestion usually takes about 2-3 weeks
lvhen cow dung is used as the substrate.
Landfill sites for methane production
w el l s i nto the top of the l andfi l l .(Formore detai l s
ReferChaoter5B).
Factors affecting biogas (methanef
production
The factorsaffectingmethaneproduction,with
special referenceto biogas plant, are briefly
descri bed.
1. Temperature and pH : The idealtemperature
i s 30-40' C , w hi l e the pH i s 6-8, for good yi el o.
2. Slurrycomposition: The ratiobetweensolid
and w ater composi ti oni n the sl urry shoul d be
around 1 : 1. A carbon nitrogenratio of 30 : 1 in
the sl urryresul tsi n opti mal methaneproducti on.
C ood mi xi ng and sol ubi l i zati onof the organi c
constituents is reouired.
3. Anaerobicconditions: The digestershould
be completelyairtight, so as to create suitable
anaerobi ccondi ti ons.

Landfill sites are low cost digesters huilt
underground for the digestion of solid wasfes (of
ind ustries and m unic ipalit ies ) . As t he a n a e r o b i c
d ige stio no f s olid or ganic m at er ial oc c ur s , m e t h a n e
gas is generated.lt can be recovered by boring gas
4. Presenceof inhibitors: Ammonium sulfate
and anti bi oti cs i nhi bi t methane producti on.
A gri cul turalw astes, pi g and chi cken manure
(generating ammonia)and wastesfrom paper(rich
i n sul fate)i nhi bi tbi ogasproducti on.
 398 BIOTECHNOLOGY

Advantages of biogas production and 8,000 caloriesper gram of gasolineand coal

respectively. Further, use of hydrogen is
By u s i n g a s i m p l e te c h n o l o g y,agri cul tural ,
environmental friendly,since it is a non-pollutant.
irrdustrialand municipalwasfescan be converted
into a biofuel. The left over residue after biogas Hydrogenis truely a versatilefuel. lt can be
formation can be used as fertilizers. Thus, the used {or automobiles,aeroplanes,helicopters,
waste materialsthat would cause environnrental buses, cars and scooters.Liquid hydrogen is
pollu ti o n a re fru i tfu l l y u ti l i z e d fo r bi ogas and consideredto be an ideal fuel for subsonicano
fertiIizer production. supersonicaircraftsworldover.

Limitations for large scale production PRODUCTION OF BIOHYDROGEN

of methane
There are mainly two ways of generating
Althoughproductionof methaneas a constituent biohydrogen - by photosynthetic bacteriaand by
of biogasis ideally suitedfor domesticpurposes. fermentation.
Many people argue against the large scale
commercial production of methane for the By photosynthetic bacteria
following reasons.
Biological production of hydrogen can be
1. M e th a n e i s a b u n d a n tl ya v a i l abl e i n the achievedby photolysisof water by photosynthetic
naturaloil and gas fields (producedby the same algae and bacteria,a phenomenonreferredto as
mechanismof methanogenesis, over a period of biophotolysis. Certain microalgae, and cyano-
years). bacteria (e.8. Chlorella, Chlamydomonas,
2. Microbial production of methane is more Scenedesmus, M i crocystis, Osci IIatoria, A neboena\
expensivethan its isolationfrom the naturalgas. can generatemolecular hydrogen.Water is the
sourceof raw material.
3. Methaneproductionby gassification of coal
is more economical than its production from jE9ltl---
H2o o, + H++ e-
bioma s s .
H vdrosenase
s e l , i t i s q ui te di ffi cul tas
4. Be i n ga Ba s e o ufu
H+ + H+ ---:------:----+ H2
well as expensiveto store,transportand distribute
methane. The action of hydrogenase
can be inhibitedby
creatingoxygen pressure.This condition favours
5 . M e th a n ei s u n s u i ta b l efo r u se as a fuel i n
releaseof free hydrogen.
automobiles.This is becauseit is very difficult to
convertthe gaseousmethaneinto liquid state. lsolatedchloroplastsalong with the bacterial
Despitethe limitationslistedabove,production enzyme hydrogenasehave also been used for
of methanefrom a wide range of biodegradable production of hydrogen.
materials(particularly the wastes) is still attractive.
This is due to the fact that the biomassused for By fermentation
methanegenerationis renewable,in contrastto the It is possibleto producehydrogenfrom glucose,
permanent depletion of naturally produced by bacterialaction.However,the yield is lessand
i
I
m et h a n e(i n th e g a sa n d o i l fi e l d s ). uneconomical. Hydrogencan also be generated by
anaerobicfermentation,by a processcomparable
I to that of methaneproduction.This is also not
economical, besides being low in efficiency.
Photosynthetic bacteriumRhodospirilliumcan be
usedto producehydrogenfrom organicwastes
H y d ro g e ni s a s i mp l em o l e c u l ew hi ch can be
easily collected,stored (as a gas or liquid) and
By legume crops
t r an s p o rte dl t. i s h i g h l y c o mb u s ti bl eand can be
used as a fuel or for the production of electricity. The l egumi nous pl antsconvertN , to N H , and
Hydrogen,on mixingwith oxygen,providesaround Hr. This reaction is catalysedby the enzyme
30, 0 0 0c a l o ri e sp e r g ra m a s c o m p aredto 11,000 nitrogenase.In the normal circumstances,this H,
 Chapte r3 1 : BIOMA SSAN D B IO E NE R C Y 399

gas, a byproduct of nitrogen metabolismis lost in
the soil. lt is estimatedthat from a soybeancrop in
one hectarefield, about 30 billion m3 hydrogenis
generatedand lost annually.As such,thereare no
methodsavailableto trap such huge quantitiesof
hydrogenproducedin agriculturalfields.
rubber produced from petroleum is preferredfor
use in automobiliesand planes,due to low-cost
and high elasticity.BesidesHevearubber,there are
some other plants for the productionof naturar
rubber e.g. Parthenium agrentatum (guayule)
Taraxacumkoksaghyz(Russiandandelion) grown
in Mexico and some partsof USA.

Euphorbialathyrusand E. terucal/icontain high

contentsof terpenoids(complexhydrocarbons) that
can be directly convertedto gasoline/petrol.lt is
Some of the plants are very efficient in estimatedthat E terucelli can vield about 5-1 0
converting CO, into biomassand such plants are barrefsoI oil/acrelvear.
collectivelyreferredto as energy-richcrops.
Aak plant (Calotropis procera) secretes latex
which is very rich in hydrocarbons.These
Sugar and starch crops
hydrocarbons,and the yield are comparableto
Certain plants Iike sugar cane, sugar beet, Euphorbialathyrus,and they also serve as good
cerealsand tuber cropsproducehigh quantitiesof substitules o{ petroleum.
starchand fermentation sugars.Thesecropssupply
energy-rich foodsand feeds.Suchplantsare useful Forobviousreasons,the cultivationof petroleum
for productionol biofuels,particularlyethanoloften plantsis encouragedthroughoutthe world.
referred to as bioethanol.

Wood.rich plants
Some plants grow very fast and they serve as
good suppliers of wood. e.g. Eucalyptus,Butea,
Besidesits utility for the generationbiofuels
Melia, Casurina.Theseplantsare importantsources (alcohol,
methane),biomassis also used for the
of firewood.lt is estimatedthat approximately 50% production of butanol, acetone, single-cell
protein
of the total wood harvestedannuallyis utilizedfor and many
other producfs (Chapter29).
the purposeof firewood.Wood is also usefulfor
the supplyof pulp for papermanufacture. As such, the contributionof biomassto the
world's requirements of energy is very low. lt rs
Petroleum plants around5% i n the U .S .A .,and may be a l i ttl ehi gh er
Thereare certainplantswhich can accumulate in the developingcountries.However, being a
high molecular weight hydrocarbons. They are renewable source of energy, biomass will have
referred to as petro-crops or gasoline plantations. immensevalue in future. This is particularlytrue as
The productsof thesehydrocarbon-rich the world's non-renewable fuels (gasand oil) get
plantscan
depleted.There is a growing realisation
on the fuel
serve as good substitutes of conventional
petroleum and petroleumproducts. value of biomass. In the coming years, biomass
productionand utilizationstrategies will be fully
The rubber plant (Hevea rubber), grown in exploited.In addition,furtherimprovements in the
South"East Asia is the principal sourceof rubber. biotechnological processes for bettermanagement
Rubberis collectedin the form of latex from the and uti l i zati on of i ndustri al ,agri cul turalan o
stemsof trees.This plant meetsaboutone third of domesticwasteswill also solve the problem of
the total world's demandof rubber.However,tne world energycrisis.
 o il m ic r oor ganis m sar e v er y c lose l y i n v o l v e d a s
Q
J cat aly t ic agenl. sin m any geolog i c a l p r o c e s s e s .
Th es e inc lude m iner al f or m a t i o n , m i n e r a l
d eg r adat ion, s edim ent at ion and g e o c h e m i c a l ln microbial leaching (bioleaching),metals can
cycl ing. be extracted from iarge quantities of low grade
ores Although recovery of metals (e.g. copper)
In r ec ent y ear s , a new dis c ipl i n e o f m i n e r a l
from the drainage water of mines has been known
science namely biohydrometallurgy or microbial
f o r c e n t u r i e s ,t h e i n v o l v e m e n t o f m i c r o b e s i n th i s
mining (mining with microbes) is rapidly growing.
process was recognized about 40 years ago.
Broadly s peak ing, biohy dr om et allu r g y d e a l s w i t h
the application of biotechnology in mining T h e b a c t e r i aw h i c h a r e n a t u r a l l y a s s o c i a t e dw i th
industry. In fact, microorganisms can be t h e r o c k s c a n l e a d t o b i o l e a c h i n g b y o n e o f th e
successfully used for the extraction of metals (e.9., following ways.
co pper ,z inc , c obalt , lead, ur anium ) f r o m l o w g r a d e
1 . Direct action ol bacteria on the ore to extract
ore s . M ining wit h m ic r obes is bot h e c o n o m i c a l a n d
metal.
e nv ir onm ent al f r iendly .
2. Bacteria produce certain substancessuch as
The term metal is used to any substancethat is
s u l f u r i c a c i d a n d f e r r i c i r o n w h i c h e x t r a c tt h e me ta l
hard, possessing silvery lusture, and is a good
(indirect action).
conductor of heat and electricity. Some of the
metals, however, are relatively soft, malleable and ln practice, both the methods may work
d uct ile e. g. s ulf ur . An or e is a nat u r a l l y o c c u r r i n g together for efficient recovery of metals.
sol id m iner al aggr egat ef r om which o n e o r m o r e
minerals can be recovered by processing. Organisms for bioleaching

M ajor it y of m ic r oor ganis m s c a n i n t e r a c t w i t h The mosf commonly used microorganisms for

metals. The metals can be recovered by the
microorganismsby two Processes.
1. Bioleac hing or m ic r obial le a c h i n g : T h i s
bro adly inv olv es t he ex t r ac t ion or s o l u b i l i z a t i o no f
miner als f r om t he or es by t he m icr o o r g a n i s m s '
2. Bios or pt ion : I t deals wit h t h e m i c r o b i a l c e l l
surface adsorption of metals from the mine wastes
or dilut e m ix t ur es .
bioleaching are Thiobacillus ferrooxidans and
Thiobacillus thiooxidans.
Thiobacillus ferrooxidans is a rod-shaped,
motile, non-spore forming, C r a m - n e g a ti ve
bacterium. lt derives energy for growth from the
oxidation of iron or sulfur. This bacterium is
c a p a b l e o f o x i d i s i n g f e r r o u s i r o n ( F e 2 *) t o fe r r tc
f o r m ( F e 3 +) , a n d c o n v e r t i n g s u l f u r ( s o l u bl e o r

400
 Chaot er32 : MIC R OB IAL
MIN IN C A N D M E TA LTE C H N OLOC Y 4(|1

insolu ble su lf ides , t hios ulf at e,elem ent al s ul f u r ) t o

sulfate (SO?-). Thiobacillus thiooxidans is
comparable with jl ferrooxidams,and grows mostly
on sulfu r co m oounos .
Several studies indicate that the two bacteria
T. ferrooxidans and T. thiooxidans, when put
together, work synergistically and improve the
extraction of metals from the ores.
Besidesthe above two bacteria, there are other
microo rga ni s m s inv olv ed in t he pr oc e s s o f
bioleaching. A selected few of them are briefly
Fig. 32,1 : A flow diagram for microbial bioleaching-
described below.
a general picture.
Sulfolobus acidocaldarius and 5. brierlevi are
the rmop hilic and ac idophilic bac t er ia which c a n
production)from iron or sulfurto oxygen.That is
gro w in acidic hot s pr ings ( > 60' C) . Thes e ba c t e r i a
can be used to extract copper and molybdenum these organisms can obtain energy from the
oxidationof Fe2"to Fe3+or from the oxidationof
re sp ective ly f r om c halc opy r it e ( CuFeSr ) a n d
mo lyb de nite ( M oSz ) . sul furand reducedsul furcompoundsto sul fareas
A conrbination of two bacteria Leptospirillum illustratedbelow.
ferrooxidans and Thiobacillus organoparpus can 4FeSO,+ 2HrSOo+ O, -----+2Fer(SOa)3 + 2HrO
effectively degrade pyrite (FeS2)and chalcopyrite
tCu Fe Sr).The indiv idual or ganis m salone ar e o f n o 2So + 30, + 2HrO ------+2HrSOo
u se in extrac t ing m et als . 2FeS,+ 70, + 2HrO -> 2FeSOo+ 2HrSOo
Pseudomonasaeruginosa can be employed in
As is evident from the third reaction given
min ing lo w gr ade ur anium ( 0. 02% ) or e . T h i s
above,iron is extractedin the solubleform the rron
org an ismh as been s hown t o ac c um ulat eabo u t 1 0 0
ore pyrite (FeSr).
mg u ran ium per one lit er s olut ion in les s t ha n t e n
seconds. Another organism, Rhizopus arrhizus is lndirect bioleaching: In this indirect method,
also effective for extractine uranium from waste the bacteria produce strong oxidizing agents such
water. as ferric iron and sulfur.icacid on oxidation of
Certain fungi have also found use in sol ubl ei ron or sol ubl esul fur respecti vel y.
Ferri c
bioleaching. Thus, Aspergillus niger can extract i ron or sul furi c aci d, bei ng pow erful oxi di zi ng
copper and nickel while Aspergillus oryzae is used agents reacf with metals and extract them. For
ior extracting gold. i ndi rect bi ol eachi ng, aci di c envi ronment i s
absol utelessenti
y al
i n orderto keepferri ci ron and
As already stated, among the various
other metal s i n sol uti on. l t i s oossi bl e ro
microorganisms, T. ferrooxidans and T. thiooxidans
conti nuousl ymai ntai naci di c envi ronmentby the
are th e mo s t widely us ed in bioleac hing . T h e
oxi dati on of i ron, sul fur, metal sLtl fi desor by
u tiliza tion of m any of t he ot her oiganis m s is s t i l l a t
dissolutionof carbonateions.
the experimental stage.

Mechanism of bioleaching COMMERCIAL PROCESS

The me ch anis mof bioleac hing is r at herc om p l e x OF B IOLE A C H IN G
a rd n ot well under s t ood. The c he m i c a l The natural l yoccurri ngmi nerall eachi ngi s very
t'a nsforma tion of m et als by m ic r oor ganis m s m a ysl ow . The mi crobi albi ol eachi ngprocesscan be
cccu r b y d irec t or indir ec t bioleac hing. opti mi zed by creati ng i deal condi ti ons-
Direct bioleaching : ln this process, there is a temperature,pH, and nutrient,O, and CO, supply
direct enzymatic attack on the minerals (which are etc. A diagrammaticrepresentation' of general
;-s,:eotible to oxidation) by the microorganisms. bioleachingprocessis depicted in Fig. 32.1. The
:'tr -:';':a cedatn baclerta /e.9., Z,/erraas'tVarzd denTedmtcroorganrms wrtri nulrtenl4 aad efq. are
iliiiillr-rii,r<i:' e ectrons (coupled with ATP pumped into the ore bed. The microorganisms

ffilttr4nEnrffiurrr,:i'
402
(A)
(B)
(c)
Metal
recovery growth of the microorganisms.
Fig. 32.2 : Commercial bioleaching processes (A)
Slope leaehing (B) Heap leaching (C) In situ leaching.
grow and produce more acid. The extracted leach
Iiquor is processed for the metal recovery. The
leach liquor can be recycled again and again for
further metal extraction.
In commercial bioleaching, three methods are
com m only us ed- s lopeleac hing, h e a p l e a c h i n g a n d
in sifu leaching (Fig. 32.2).
Slope leaching : The ore is finally ground and
d um ped in lar ge piles down a m o u n t a i n s i d e
( Fig. 32. 2A) . This or e is t h e n s u b j e c t e d t o
c ont inuous s pr ink ling of wat e r c o n t a i n i n g t h e
desired microorganism (T. ferrooxidans).The water
collected at the bottom is used for metal extraction.
The water can be recycled for regeneration of
bacteria.
Heap leaching : ln this case, the ore is arranged
in large heaps (Fig. 32.28) and subjected to
treatmentsas in slope leaching.
In sifu leaching : The ore, in its original natural
place is subjected to leaching (Fig. 32.2C). WaIer
c ont aining t he m ic r oor ganis m sis p u m p e d t h r o u g h
B IOTE C HNO LO CY
drilled passages.In most cases,the permeability of
blasting of the rock.
rock is increasedby subsr-rrface
As the acidic water seeps through the rock, it
collects at the bottom which is used for metal
extraction. This water can be recycled and reused.
S e l e c t e de x a m p l e s o f m i c r o b i a l b i o l e a c h i n g a r e
briefly described below.
B'OLEACHING OF COPPEB
C o p p e r o r e s ( c h a l c o p y r i t e , c o v e l l i te a n d
chalcocite) are mostly composed of other metals,
b e s i d e s c o p p e r . F o r i n s t a n c e ,c h a l c o p y r i t e m a i n l y
contains 26"h copper, 26"h iron, 33olosulfur and
2.5"/" zinc.
B i o l e a c h i n g o f c o p p e r o r e ( c h a l c o p yr i te ) i s
widely used in many countries. This is carried out
by the microorganism Thiobacillus ferrooxidans
w h i c h o x i d i s e s i n s o l u b l e c h a l c o p y r i t e( C u F e Sr )a n d
converts it into soluble copper sulfate (CuSOo).
S u l f u r i c a c i d , a b y p r o d u c t f o r m e d i n t h i s r e a cti o n ,
m a i n t a i n sa c i d i c e n v i r o n m e n t ( l o w p H ) r e q u i r e d fo r
Copper leaching is usually carried out by heap
and in situ process (details given above). As the
c o p p e r - c o n t a i n i n g s o l u t i o n ( i . e . , c o p p e r i n th e
dissolved state) comes out, copper can be
precipitated and the water is recycled, after
a d j u s t i n gt h e p H t o a r o u n d 2 .
Extraction of copper by bioleaching is very
c o m m o n s i n c e t h e t e c h n i q u e i s e f f i c i e n t , b e si d e s
b e i n g e c o n o m i c a l . l t i s e s t i m a t e dt h a t a b o u t 5 % o f
t h e w o r l d 's c o p p e r p r o d u c t i o n i s o b t a i n e d vi a
m i c r o b i a l l e a c h i n g . I n t h e U S A a l o n e , a t l e a st 1 0 %
of the copper is produced by bioleaching process.
B'OLEACHING OF UNAN'UM
Bioleaching is the method of choice for the
large-scale production uranium from its ores.
U r a n i u m b i o l e a c h i n g i s w i d e l y u s e d i n I n d i a , U SA,
C a n a d a a n d s e v e r a lo t h e r c o u n t r i e s .l t i s p o ssi b l eto
r e c o v e r u r a n i u m f r o m l o w g r a d e o r e s ( 0 . 0 1 to 0 .5 %
uranium) and low grade nuclear wastes.
In situ bioleaching technique is commonly used
f o r e x t r a c t i n gu r a n i u m . I n t h e t e c h n i q u e e m p l o ye d ,
t h e i n s o l u b l et e t r a v a l e n tu r a n i u m i s o x i d i z e d ( i n th e
presence of hot HrSOo/Fe3* solution) to soluble
h e x a v a l e n tu r a n i u m s u l f a t e .
UO, + Fer(SOa)3--------) UO2SO4 + 2FeSO.
 Ch APICT32 : M I CRO BI AL M I NI NC AND M ET A L T E C H N O L O C Y 403

Bio lea ch ing of ur anium is an indir ec t pr o c e s s ADVANTAGES OF BIOLEACHING

sin ce the micr obial ac t ion is on t he ir on ox id a n t ,
a nd no t d irec t ly on t he ur anium . The or ga n i s m
Thiobacillus ferrooxidans is capable of producing
sulfuric acid and ferric sulfatefrom the pyrite (FeS2)
within th e u ranium or e.
For o ptim al ex t r ac t ion of ur anium b y
bio lea ch ing , the ideal c ondit ions ar e t em per a t u r e
45 -50 "C, pH 1 . 5- 3. 5, and CO , ar ound 0 2oh o f t h e
rncomin g arr.
The soluble form of uranium from the leach
liquor can be extracted into organic solvents (e.9.,
tributyl phosphate)which can be precipitated and
then recovered.
Heap leaching process is sometimes preferred p o s s i b l e .
insteadof the in sifu technioue. This is becausethe
recovery of uranium in much higher with heap
lea chin g.
B|OLEACHING OF OT'IER METALS
Wh e n c o m p a r e d t o c o n v e n t i o n a l m i n i n g
techniques, bioleaching offers several advantages.
Some of them are listed below.
.l
. Bioleaching can recover metals from low
grade ores in a cost-effective manner.
2. lI can be successfully employed for
c o n centrating metals from wastes or dilute
mixtures.
3. Bioleaching is environmental friendly, since
it does not cause any pollution (which is the case
with conventional mining techniques).
4. lt can be used to produce refined and
expensive metals which otherwise may not be
5. Bioleaching is a simple process with low
cost technology.
6. lt is ideally suited for the developing
cou ntfles.

Beside s copper and ur anium , bioleac h i n g The major limitation or disadvantage of

technioue is also used for extraction of other metals bioleaching is the slowness of the biological
such as nickel, gold, silver, cobalt, molyhdenum process.This problem can, however, be solved by
and antimony. lt may be noted that removal of rron undertaking an in depth research to make the
is de sira ble pr ior t o t he ac t ual pr oc es sof leac h i n g process faster, besides increasingthe efficiency.
for other metals. This can be done by using the
organism Thiobacillus ferrooxidans which can
precipitate iron under aerobic conditions.

Bioleaching is also useful for the removal of

certain impurities from the metal ric.h ores. For Biosorptionprimarilydeals with the microbial
instance,the microorganismssuch as Rhizobium sp cell surface adsorption of metals from the mine
and Bradyrhizobium sp can remove silica from w astesor di l utemi xtures.
The mi croorgani sms
can
b au xite (alu minium or e) . be used as biosorbents or bioaccumulators of
metals.The processof biosorptionperformstwo
Bioleaching in desulfurization of coal i mportantfuncti ons.

The process of removal of sulfur containing
pyrite (FeS2) from high sulfur coal by
microorganisms is referred to as biodesulfurization.
Hig h su lfur c oal, when us ed in t her m al po w e r
stations, emits sulfur dioxide (S02) that causes
en viro nme nta lpoll ut ion.
By using the microorganisms Thiobacillus
ierrooxidans and T. thiooxidans, the pyrite which
contains most of the sulfur (80-90%) can be
remo ve d. Thu s , by em ploy ing bioleac hing, h i g h
.u lfur co al can be f r uit f ully ut iliz ed in a n
:-.''onment friendly manner. ln addition, this
;-., - :.': i; o ui t e ec onom ic al als o.
1. Removalof toxic metalsfrom the industrial
effluents.
2. R ecoveryof val uabl ebut toxi c metal s.
Boththe aboveorocesses are concernedwith a
poi soni ng/pol l uti on.
reducti oni n envi ronmental
A wide rangeof microorganisms(bacteria,algae,
yeasts,moulds) are employed in biosorption.In
fact, some workers have developed biosorhent-
based granules for waste water/industrial effluent
treatment, and metal recovery.
ln general, the microbial cell membranesare
negativelychargeddue to the presenceof carboxyl
40'4 B IOTE C HNO LO CY

(COO ), hydroxyl (OH-) phosphoryl (POi-) and instance,Chlorella vulgaris and C. regularis can
s ulf hy dr y l ( HS- ) gr oups . This ena b l e st h e p o s i t i v e l y certai nmetal sl i ke P b,H g, C u, M o and
accumul ate
charged metal ions (from solutions)to be adsorbed U. The green algae Hydrodictyon reticulatum
on t o t he m ic r obial s ur f ac es . adsorbsand accumul ates hi gh quanti ti es of Pb, Fe
and Mn.
The different groups of microorganismsused in
biosorption processesare briefly described below. Someworkersare in fact trying to use marine
algae (e.9., Luminaria, Ulva, Codium sp) as
Bacteria bioaccumulatorsto reducethe metal pollution in
fl vers.
Several bacteria and actinomyceles adsorb and
accumulate metals such as mercurv cadmium, lead,
Higher plants in control of
z inc , nic k el, c obalt and ur aniu m . F o r e x a m p r e ,
metal pollution
Rhodospirullumsp can accumulate Cd, Pb and Hg.
Bacillus circulans can adsorb metals such as Cu, Besidesihe microorganisms describedabove,
Cd, Co, and 7n. By use of electron microscopy, thereare somehi gheraquati cpl ants(i .e.,aquat ic
deposition of metals on the bacterial cell walls was macrophytes) that can accumulatepotentialtoxic
recorded. lt appears that the cell wall composition wastes including many metals. Water hyacinth
plays a key role in the metal adsorption. (Eichornia crassipes),duck weeds (Spirodel sp),
water lettuce (Pistia stratiotes)and ceriain ferns
Fungi (Saiviniasp) are importantin the control of metal
pollution.For more details,the readermust refer
There is a large scale productior.r of fungal C haoter54.
biom as s in m any t . er r nent at io ni n d u s t r i e s . T h i s
bior,nasscan be utilized for metal biosorption from
industrial effluents. lmmobilized fungal bionrass rs
more effective in biosorpticln due to increased
density, mechanical strength and resistance to
c hem ic al env ir onr ner r t . Fur t h e r , i m n t o b i l i z e d
B y t h e c o n v e n t i o n a l t e c h n i q u e u s e d i n th e o i l
biomass can be reused after suitable processing.
f i e l d s , a p p r o x i m a t e l y o n e - t h i r d o f t h e o i l ca n b e
The fungus Rhizopus arrhizus can adsorb several recovered. However, the oil recovery can be
m et allic c at ions e. g. ur anium , t h o r i u m . P e n c i l l i u m e n h a n c e d b y u s i n g s o l v e n t s , a n c j s u r fa cta n ts.
lapidorum, P. spimulosum are useful for the Certain polymers produced by microorganisms
biosorption of metals such as Hg, Zn, Pb, Cu. (e.g., xanthan gum), when added to oil wells are
Several fungi were tried with some degree of capable of increasing oil recovery Xanthan gum
s uc c es s t o s elec t iv ely ads or b u r a n i u m e . g . can pass through small pore spaces and promote
Aspergillus niger, A. oryzae, Mucor haemalis, the releaseof more trapped oil.
Penici II i u m chrysogenum.
In recent years, oil technologists are trying to
Edible mushrooms were also found to adsorb directly use microorganisms in situ Ior increasing
the oil recovery. In this process, there is no
certain metals. For instance,fruit bodies o{ Agaricus
bisporus can take up mercury while Pleurotus sajor-formalised use of a bioreactor. The naturar
caju can adsorb lead and cadmium. geological site itself is the bioreactor. This allows
the water and microorganismsto flow over the ore
Many yeasts, commonly used in fermentation which are collected after seepageand outflow. The
industries, are capable of adsorbing and microorganisms,through surfactantproduction, gas
accumulating metals. For instance, Saccharomyces formation or by other microbial activities reduce
cerevisae and Sporobolomyces salmonicolour can the viscosityof the oil so as to enhance its recovery.
respectivelyadsorb mercury and zinc. However, the success in this direction is not very
commendable. -
Algae
Continued further researchmay one day help to
Severalspecies of algae (fresh.water or marine) use microbes for commercial releaseof oil from oil
can serve as bioaccumulators of metals. For w e l l s o r t a r s a n d s .
 (t)
33 A ni mal C el l C ul ture -
Fundamental s,Faci l i ti esand
A ppl i cati ons 4O7
34 C ul ture Medi a for
A ni mal C el l s 4' 15
35 C ul tured C el l s- B i ol ogy and
Characterization 423
36 P ri mary C ul ture and
C el l Li nes 437
37 A ni mal C el l C ul ture-C eneral
C onsi derati onsand S cal e-up 450
3B C el l V i abi l i ty and C ytotoxi ci ty 459
ANIMAL CELL/ 39 C el l Transformati onand C el l
ANIMAL C l oni ng 463
40 Organ and H i stotypi c C ul tures,and
BIOTEGHNOLOGY Ti ssueE ngi neeri ng 468
41 Transgeni cA ni mal s 480

405
 I nim al c e l l c u l tu re b a s i c a l l yi n v o l v e sthe i n
/ | vitro (in the laboratory)maintenanceand their reaggregation
pr opagat ioonf a n i m a lc e l l s i n a s u i ta b l en utri ent represents
media.Thus,culturing is a processof growing cells
artificially.Cell culturehasbecomean indispensible
t ec hnologyin v a ri o u sb ra n c h e o
s f l i fe s c i e n ces.
His t or ic al b a c k g ro u n d
I t was in 1 9 0 7 ,R o s sH a rri s o nfi rs td e v e l opeda
. e p ro b a b l ychose
f r og t is s uec u l tu rete c h n i q u e H
f rog for two reasons-beinga cold-bloodedanimal,
no inc ubat i o ni s re q u i re da n d ti s s u ere g e n e rati on
f as tin f r og.In 1 9 4 0 ' sc h i c k e m b ry oti s s u eb e came
a f av our it e fo r c u l tu re te c h n i q u e s .In te restrn
c ult ur inghu ma n ti s s u e ss ta rte di n 1 9 5 0 ' safter i t
was demonstrated(by HeLa; Cey) that human
t um orc ellsc o u l dg i v e ri s eto c o n ti n u o u cs e l l l i nes.
A m ongt he v a ri o u sa n i ma lc e l l c u l tu re smo
, usecel l
c ult ur esar e th e m o s t c o mmo n l y u s e d i n the
laboratory.
Histotypicculture: The culturingof the cellsfor
to form a tissue-like structure
histotypicculture.
Organotypi ccul ture : Thi s cul turetechni que
i nvol vesthe recombi nati on
of di fferentcel l tvoesto
form a more defi nedti ssueor an organ
Primaryculture : The culture producedby the
freshl y i sol atedcel l s or ti ssuestaken from an
organi smi s the pri mary cul ture.Thesecel l are
heterogenousand slow growing, and represent
rs the ti ssue of thei r ori gi n w i th regardto thei r
properties.
C el l l i ne: The subcul turi ngof the pri mary
culturegivesriseto cell lines.The term continuous
cell linesimplies the indefinitegrowth of the cells
i n the subsequent subcul turi ng.
On the otherhand,
finite cell lines representthe death of cells after
severalsubcul tures.

T er m inolog y i n c e l l c u l tu re
The term tissue culture is commonly used to
inc ludebot h o rg a nc u l tu rea n d c e l l c u l tu re.
Organ culture : The cultureof nativetissue(i.e
undisaggregated tissue)that retainsmost of the in While designing the laboratory for animal cell
t,ivo histological features is regarded as organ
culture technology, utmost care should be taken
c uit ur e. with regard to the maintenance of aspetic
Celf cufture : This refers to the culture of conditions. The facilities required with regard to
- .::i'rsed(or disag,g,regated) cells obtainedfrom the infrastructure and equipment are /isted in the next
. : ^al t is s ueo, r fr6 m a c e l l l i n e . pa8e.

4|J7
408 B IOTE C H N O LO CY

MINI M AL REQ UI REM ENTS cultures.The most commonly used plasticsare

FOR CELL CULTURE polystyrene, polyvinyl chloride (PVC), poly-
carbonate,metinexand thermonex(TPX).
,NFRASTRUCTUNE
. C lean and quit e s t er ile ar ea
Types of culture vessels

. P r epar at ionf ac ilit ies The fol l ow i ngare the commontypesof cult ur e
vessets.
. A nim al hous e
. Mul ti r,velpll ates
. M ic r ohiology labor at or y
o P etri di shes
. St or age f ac ilit ies ( f or glas s w a r e , c h e m i c a l s ,
l iquids , s m all equipm ent ) . . Fl asks
o S ti rrerbottl es.
EOUTPMENT
The actualchoi ceof sel ecti nga cul turevessel
Laminar-flow, sterilizer, incubator, refrigerator
dependson severalfactors.
and f reezer (-.20'C), balance, COz cylinder,
centrifuge, inverted microscope, water purifier, 1. The w ay cel l s grow i n cul ture-mon olayer
hemocytonreter, liquid nitrogen freezer, slow- or suspensron.
cooling device (for freezing cells), pipette washer,
2. The quanti tyof the cel l srequi red.
de ep was hing s ink .
3. The frequencyof sampl i ngfor the desir ed
Bes ides t he bas ic and m inim a l r e q u i r e m e n t s
W OT K
listed above, there are many more facilities that
may be benef ic ial or us ef ul f or t i s s u e c u l t u r e s . 4 . T h e p u r p o s e f o r w h i c h t h e c e l l s a r e 8 r o \ /n .
Th es e inc lude air - c ondit ionedr oom s , c o n t a i n m e n t
5. The cost factor.
room for biohazard work, phase-contrast
microscope, fluorescence microscope, confocal ln general,for monolayer cultures, the cell yield
microscope, osmometer, high capacity centrifuge is almost proportional to the surface area of the
an d t im e laps e v ideo equipm ent . c u l t u r e v e s s e l .T h e f l a s k s a r e u s u a l l y e m p l o ye d fo r
this purpose.
CULTURE YESSEI.S
Any type of culture vessel can be used to grow
I n t he t is s ue c ult ur e t ec hnology,t h e c e l l s a t t a c h suspension cr-rltures.lt is necessaryto slowly and
to the surface of a vessel which serves as the c o n t i n u o u s l y a g i t a t e t h e s u s p e n d e d c e l l s in th e
substrate, and grow. Hence there is a lot of vessel.
importance attached to the nature of the materials
used and t he qualit y of t he c ult ur e v e s s e l s . Treatment of culture vessel surfaces
The term anchorage dependent cells is used For improving the attachment of cells to the
wh en t he c ells r equir e an at t ac h m e n t f o r t h e i r surfaces, arrd for efficient growth, some devices
g rowt h. O n t he ot her hand , s o m e c e l l s have been developed. lt is a common observation
undergo transformation, and become anchorage that the growth of the culture cells is better on the
independent. surfaces for second seeding. This is attributed to
matrix coating of the surfaces due to the
Materials used for culture vessels accumulation of certain compounds like collagen
and fibronectin releasedb,7the cells of the previous
Glass : Although glasswas the original substrate
c u l t ur e .
used f or c ult ur ing, it s us e is alm o s t d i s c o n t i n u e d
no w. This is m ainly bec aus e of t h e a v a i l a b i l i t y o f T h e r e a r e n o w c o m m e r c i a l l y a v a i l a b l e m atr i ce s
more suitakrleand alternate substrates. (e.g. matrigel, pronectin, cell-tak).

Disposable plastics : Synthetic plastic materials F e e d e r l a y e r s : S o m e o f t h e t i s s u e c u l tu r e s

with good consistency and optical properties are require the support of metabolic products from
no w in us e t o pr ov ide unif or m a n d r e p r o d u c i b l e l i v i n g c e l l s e . g . m o u s e e m b r y o f i b r o b l a s t s .In th i s
Cha pte r 3 3 : ANI M AL CELL CULTURE- FUNDAMENTALF
SA, C I L I T I EASN D A P P L I C A T I O N S 409

case, the growing fibroblasts release certain

oroducts which when fed to new cells enhance
their erowth. a tlssueculturelaboratory
Equipment
and facilities
Alternate substrates as substitutes
of culture vessels Laminar-flow
hoods
ln recentyears,certainalternatives for culture Dryincubators
vessels have been developed. The important CO,incubators
alternative are microcarriers
artificialsubstrates ano
Humidified
incubators
metallicsubstrates.
Wooden
lurniture,
benches
Microcarriers: They are in bead form and are
m ade up o f c o l l a g e ng, e l a ti n ,p o l y a c ry lami de
and Other
instruments
polystyrene. Microcarriersare mostlyusedfor the
propagation of anchorage-dependent cells in Glasswareand reagents
s us pens ro n . Pipettes
Metallicsubstrates : Certaintvpesof cellscould Screwcaps
be s uc c e s s fu lgl yro w no n s o meme ta l l i cs u rfaces
or glasses
Culture
even on the stainlesssteel discs. For instance,
Mediabottles
f ibr oblastsw e re g ro w n o n p a l l e d i u m .
Mediaandvarious
solutions
USE OF NON.ADHESIVE SUBSTNATES
,N TISSUE CULIUBE Biologicalmaterials

The growthof anchorageindependent cellscan Infected

tissuesamples
be c ar r ie do u t b y p l a ti n gc e l l s o n n o n -adhesi ve Celllines
substratelike agar,agaroseand methyl cellulose.ln
this situation,as the cell growthoccurs,the parent Operatingtechniques
and daug h te rc e l l s g e t i mmo b i l i z e da n d form a Operaiorhands,hair,clothing,
breathing
colony,althoughthey are non-adhesive.
Workspaces
Pipetting,
dispensing
Operating,nanipulations

Thereare severalroutesof contaminationin the
tissueculturelaboratory(Table33.1).Theseinclude
the various materials (glassware, pipettes),
equipment (incubators,refrigerators,laminar-flow
hoods), reagents(media, solutions),contaminated
cell lines anclpoor technigues.
The routes of contamination are mosfly
associatedwith the laboratorvenvironment,and
operatingtechniques.
Types of microbial contamination
Severalspeciesof bacteria,yeasts,fungi, molds
and mycoplaimas,besidesvirusesare responsible
for contamination.Major problems of conta- o Strict adherenceto standardsteriletechniques
minationare linkedto the repeatedrecurrence of a
single species. Despite utmost care taken, no
laboratory can claim to be totally free from
contami nati on.l t i s necessaryto conti nuousl y
monitorfor contaminationand eliminatethe same
at the earliest.
ASEPTIC CONDITIONS
Maintenanceof proper aseptic conditions is
necessarvto eliminate various cantaminants (due
to different microorganisms and viruses). The
fol l ow i ngmeasures
are suggestedfor mi ni mi zi ng
contami nati on,and mai ntenance of asepti c
condi ti ons.
and code of oractices.
410 B IOTE C H NO LO CY

. Checking of reagentsand media for steri l i ty

before use. Tlru 33.2 Sterlllzation of naior equipment,
apparatusand llquids used in tissue culture
. Checking of cultures by eyes, and microscopes
(phase contrast) every time they are used. Sterilization device Items sterilized
. Use of media and separate bottles for each cell I For equipment and apparatus
line is adv is ed. Dry heal Glassslides
o M aint enanc e of c lean and t id y c o n d i t i o n s a t Pipettes
work places. Ampoules (glass)
Pasteur pipettes
. Personal hygiene of the staff, is very important.
Instruments
STERI LI ZATI O N Testtubes
Autoclave Ampoules (plastic)
The s t er iliz at ionpr oc edur esar e d e s i g n e d t o k i l l
Apparatus withsilicone
the microorganisms,besides destroying the spores.
tubing
Th er e ar e t hr ee m aior dev ic es f or s t e r i l i z a t i o n .
Filters(reusable)
1 Dry heat Glassbottles withscrew
2. Moist heat (autoclave) caps
Glasssyringes
3. Filt er s .
Magnetic stirrerbases
ln the lable 33.2, the sterilization of major Screwcaps
e quipm ent , appar at usand liquids i s g i v e n . Stoppers
Sterilization by dry heat : This is carried out at (rubber silicone)
a m inim um t em Der at ur eof 160' C f o r a b o u t o n e
ll For liquids and nutrients
nou r.
Autoclave Saltsolutions
Sterilization bv moist heat : Certain fluids and Glucose-20%
o er is hable it em s c an be s t er iliz e d i n a n a u t o c l a v e
Agar
at 121"C { or 15- 20 m inut es . Fo r e f f e c t i v e m o r s t
Bacto-peptone
heat sterilization, it is necessary that the
steam penetratesto all the parts of the sterilizing Glycerol
m at er ials . Lactalbuminhydrolysate
Phenolred
Sterilization by filters : The use of filters for
Tryptose
sterilization of liquids often becomes necessary,
sinc e t he c ons t it uent s of t hes e l i q u i d s m a y g e t HEPES
destroyedat higher temperatures(dry heat or moist EDTA
heat). Water
Sterile filtration is a novel technique for heat- Filter Serum
labile s olut ions .The s iz e of m ic r o p o r e so f t h e f i l t e r s Aminoacids
is 0. 1- 0. 2 pm . Filt er s ,m ade f r om s e v e r a l m a t e r i a l s Vitamins
a r e in us e. Thes e m at er ialsinc lud e n y l o n , c e l l u l o s e Antibiotics
acetate, cellulose nitrate, polycarbonate, Bovineserumalbumin
polyethersulfone(PES)and ceramics The filters are l l nl l anonaeo
made in different designs-discfilters, cartridgesand
Glutamine
hollow f iber .
Drugs
I n t ac t , m any c om m er c ial c o m p a n i e s ( e . 9 . NaOH
Millipore. Durapore) supply reusable and Trypsin
disposable filfers, designed for different purposes of
Transferrin
s t er iliz at ion.
Cha pte r 3 3 :AN I M AL CELL CULTURE- FUNDA M E N T A L S F
, A C I L I T I EASN D A P P L I C A T I O N S 411

4. Control of the environmental factors (pH,

temperature, dissolved gases, disposal of
biohazards) is not easy.
5. The native in yivo,cells exist in a three-
ADVANTAGES OF TISSUE CULTURE dimensional geometry while in in vitro tissue
culture, the propagation of cells occurs on a two
Tissue cu ltur e t ec hnique has a wide r ange o f
dimensional substrate.Due to this, the cell to cell
applications.The most important advantagesof this
interactive characters are lost.
techn iqu e a re lis t ed below.
.l 6. The cell lines may represent one or two
. Control of physico-chemical environment- types of cells from the native tissue while others
pH, temperature, dissolved gases (O, and COr), may go unrepresented.
osmolarity.
7. Tissueculture technioues are associatedwith
2. Regulation of physiological conditions- the differentiation i.e. Ioss of the charactersof the
n utrie nt con ce nt r at ion, c ell t o c ell int er ac t i o n s , t i s s u e c e l l s f r o m w h i c h t h e y w e r e o r i g i n a l l y
ho rmon al co ntr ol. isolated.

3. Th e cu ltur ed c ell lines bec om e hom ogen o u s B . T h i s h a p p e n sd u e t o a d a p t a t i o na n d s e l e c t i o n

(i.e . cells a re ide nt ic al)af t er one or t wo s ubc ult u r e s . p r o c e s s e sw h i l e c u l t u r i n g .
This is in co ntr as t t o t he het er ogenousc ells o f 9. Continuous cell lines may result in genetic
tissue samples. The homogenous cells are highly i n s t a b i l i t yo f t h e c e l l s . T h i s m a y u l t i m a t e l y l e a d t o
useful for a wide range of purposes. heterogeneityof cells.
4. lt is easy to characterizecells for cytological 10. The comoonents of homeostalic in vivo
a nd immun olo gic al s t udies . regulation (nervous system, endocrine system,
metabolic integration) are lacking in in vitro
5. Cu lture d c ells c an be s t or ed in liq u i d
cultures. Addition of hormones and growth factors
nitrogen for several years.
has been started recently.
6. Due to direct accessand contact to the cells,
bio log ica l stud ies c an be c ar r ied out m o r e
con ve nie ntly. T he m ain adv ant age is t he l o w
quantities of the reagents required in contrast to
in vivo studies where most of the reagents (more
than 90% in some cases)are lost by distribution to
various tissues,and excretion. There is a widesoreadconcern that extensive
use of animalsfor laboratoryexperimentsis not
7 . Utility o f t is s ue c ult ur es will dr as t ic a l l y morally and
ethically justifiable.Animal welfare
reduce the use of animals for various experiments.
groups worldover are increasingly criticising the
use of animals.Some researchworkersthesedavs
Limitations of tissue culture prefer to utilize animal cell cultures wherever
possihle for variousstudies.The major applications
Thereare severallimitations of tissueculture, of laboratoryarrimal cell cultures are given in
some of them are given below. Table33.3, and listedbelow.
1. Needof e x p e rti s ae n d te c h n i c asl k i l l fo r the o S tudi eson i ntracel l ul aracti vi tye.g. cel l cycl e
dev elopm enta,n d re g u l a ru s eo f ti s s u ec u l tu re . and differentiation,metabolisms.
2. Cost factor is a maior limitation. r E l uci dati onof i ntracel l ul arfl ux e.g. hormonal
E s t ablis hm enotf i n fra s tru c tu ree,q u i p me n t a nd receptors,signaltransduction.
ot her f ac ilit esa re e x p e n s i v e . . Studiesrelatedto cell to cell interactione.g. cell
3. lt is estimatedthat the cost of productionof adhesionand motility,metaboliccooperation.
c ells is about 1 0 ti me s h i e h e rth a n d i re c t u s e of o E val uti on of envi ronmentali nteracti onse g.
anim alt is s ues . cytotoxicity, mutagenesis.
412 B IOTE C H NO LO CY

b i o l o g i c a l c o m p o u n d s b y t h e m i c r o o r g a ni sm s i s
,3 Summaryof the applications described in Chapters 23-30.
of animal cell cultures
A s e l e c t e dl i s t o f a n i m a l c e l l c u l t u r e p r o du cts o f
Category Applications cornnrercial importance is given in Table 33.4.

Intracellularactivity Studies
relatedto cellcycle
Production of vaccines
anddifferentiation,
transcription,
translation,
energy metabolism, M o n k e y k i d n e y o r c h i c k e m b r y o c e l l s, o r
1,,,^
ur uv il lvtquuilJil
-^r^L^l;^- t. r e c e n t l y h u m a n d i p l o i d c e l l s a r e i n u s e fo r th e
o r o d u c t i o n o f v a c c i n e s . T h e v a c c i n e m a n ufa ctu r e
lntracellular
flux Studies involving
hormonal i n a n i m a l c e l l c u l t u r e s i s r a t h e r c o m p l e x wi th r i sk
receptors,metabolites,
signal of contarnination, and safetv aspect. For these
transciuction,
membrane reasons, production of vaccines by recombinant
trafficking D N A t e c h n o l o g y e m p l o y i n g b a c t e r i a o r v e a sts r s
.l
Cell to cell interactionStudies
dealingwithcell preferred (Refer Chapter 6).
adhesion
andmotility,
matrix
Interaction,
morphogenesis, Production of high value therapeutics
paracrine metabolic
control, M a n y h u m a n p r o t e i n s w i t h h i g h t h e ra p e u ti c
cooperation potential are often in short sLrpply e.g tissue
p l a s m i n o g e na c t i v a t o r ,c l o t t i n g f a c t o r s ( V l l l a n d l X) ,
E n V i ro n me n ta l Studies relatedto drugactions,
infections, e r y t h r o p o i e t i n . T h e r e i s a m a j o r l i m i t a ti o n to
interaction cytotoxicity,
produce human proteins that undergo post-
mutagenesis, carcinogenesis
t r a n s l a t i o n a lm o d i f i c a t i o n s ( g l y c o s y l a t i o n ,c a r b o xy-
Genetics Studies dealing withgenetic l a t i o n e t c . ) i n b a c t e r i aa n d y e a s t s .T h i s i s d u e to th e
analysis, transfection, fact that these organisms do not possess the
transformation, immortalization,m a c h i n e r y t o p e r f o r m p o s t - t r a n s l a t i o n a cl h a n g e s.
q A N A E 'A N A A
However, pharmaceutical proteins that do not
Cell products Widerangeof applications of r e q u i r e p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s ca n b e
thecellular productsformed p r o d u c e d b y b a c t e r i a o r y e a s t s e . g . i n su l i n ,
(Referlable 33.4)e g. vaccines, l b u m i n , g r o l v t h h o r m o n e .
a
hormones,
interferons
etc. Animal cell cultures (particularly mammalran
c e l l c u l t u r e s )a r e u s e f u l f o r t h e p r o d u c t i o n of m a n y
. St udies dealing wit h genet i c s e . g genetic p h a r m a c e u t i c a l l y / m e d i c a l l y i m p o r t a n t p r o te r n s
(Table 33.4) These include the following.
analy s is ,im m o( aliz at ion, s ene s c e n c e
r Laboratory production of o Plasminogen
medical/pharma-
ceutical compounds for wide range of .lnterferons
applic at ionse. 8. ' r ac c ines ,int er f e r o n s ,h o r m o n e s . . Blood clotting factors
. Hormones
There are however, several limitations on the
us e of anim al c ell c ult ur es . This i s m o s t l y d u e t o . Monoclonal antibodies
the differencesthat exist between Ihe in vivo and in . Ervthroooietin
vit r o s y s t em s , and t he v alidit y o f t h e s t u d i e s
conducted in the laboratory. Purification of pharmaceutical products

MEDICAL / PHABMACEUTICAL
PRO DUCTS O F ANI M AL CE L L
C ULTURES
The m os t im por t ant applic at io n o f a n i m a l c e l l
cult ur es is t he pr oduc t ion of a w i d e r a n g e o f
com m er c ial c om pounds f or m edi c a l a n d p h a r m a -
ceut ic al us e. The c om m er c ial pr o d u c t i o n o f s e v e r a l
A s t h e d e s i r e d p r o d u c t i s p r o d u c e d i n th e ce l l
c u l t u r e m e d i u m , i t s p u r i f i c a t i o n , i s o l a t i on a n d
storage (collectively re{erred to as downstream
p r o c e s s i n g )a s s u m e ss i g n i f i c a n c e .T h e f i n a l pr o d u ct
for therapeutic applications is expected to satisfy
the following criteria.
. T h e o r o d u c t s h o u l d h a v e a s t a b l e s t r u c t u r ew i th
ootimal activitv
Ch ap ter 33 : ANI M AL CELL CULTURE- FA
FU N D A M E N T A L S , C I L I T I EASN D A P P L I C A T I O N S 413

The product should be free from other

biomoleculesthat may interferewith its activity
and/orcausei mmunol ogi cal
compl i cati ons.
medlcal/phalnaceutical inportance
It shoul dbe free from al l pathogensi ncl udi ng
Product(s) Application(s) vtruses.

Vaccines GENETIG ENGINEERING OF ANIMAL

Poliovaccines prophylaxis
Poliomyelitis CELLS AND THEIR APPLICATIONS
Measlesvaccine Measlesprophylaxis
It is now possible to genetically modify the
Rabiesvaccine Rabiesprophylaxis
animal (mammalian) cells to introduce the genes
Malaria
vaccines prophylaxis
Malaria
needed for the prodr,rctionof a specific protein or
HIVvaccine AIDSprophylaxis
and i m o r o v e t h e c h a r a c t e r i s t i c so f a c e l l l i n e . T h e
treatment following methods are used to introduce foreign
Plasminogen activators DNA into marnmalian cells.
Tissue{ype Acutemyocardial infarction,
1 . Electroporation
plasminogen activator ' pulmonary embolism, deep
Urokinase-type veinthrombosis, acutestroke. 2. Lipofection
plasminogen activator 3. Microinlection
Recombinant
4. Fusion of mammalian cells with bacteriaor
plasminogen activalor
V U SES
lnterferons
Interferon-g Anticancer, immunomodulator As the foreign DNA gets integrated into the
Interferon-B Anticancer, antiviral m a m m a l i a n c e l l u l a r g e n o m e , t h e g e n e e x p r e s s e st o
produce the desired protein. lt is however,
Interferon-y Anticancer, immunomodulator
necessaryto select the best producing recombinant
Blood clotting factors
cells by conventional methods using selectable
FactorsVll,Vlll,lX Hemophilia, as bloodclotting
marker genes.The following selectablemarkers are
andX agents
used for choosing the transfectedcells.
Hormones
Humangrowth Growthretardation in children . V i r a l t h y m i d i n e k i n a s e
n0rm0ne . Bacterial dihydrofolate reductase
Somatotropin Chronic renalinsufficiency . Bacterial neomycin phosphotransferase
Follicle
stimulating Treatrnentoi infertility
It has been possible to overproduce several
hormone
proteins in mammalian cells through genetic
Human chorionic Treatment of infertility
manipulations e.g., tissue plasminogen activator,
g0nad0tropin
erythropoietin, interleukin-2, interferon-B, clotting
Monoclonalantibodies factors Vlll and lX, tumor necrosis factors.
Anti-lipopolysaccharide Treatment of sepsis
Treatment of B-cell The recombinant mammalian cells are
Human B-cell
lymphoma conveniently used for the production of
Lympn0mas
monoclonal antibodies which have wide range of
AntiJibrin99 Diagnosis of bloodclotby
a p p l i c a t i o n s ( R e f e rC h a p t e r 1 7 ) .
rmaglng
Tcm-FAb (breast) Diagnosis oi breastcancer
Others
Erythropoietin Antianaemic agent
lnterleukin-2 Anticancer, HIVtreatment
Tumornecrosis lactor Anlicancer
There are severalrisks associatedwith tissue
Granulocyte stimulatingAnticancer
culturetechnology. Mostof the accidents that occur
factor
in culture laboratoriesare due to negligenceand
Carcinoembryonic Diagnosis andmonitoring of
casual approach while dealing with biological and
antigen nannar nationtc
radiologicalsamples,besidesimpropermaintenance
414 B IOTE C HNO LO CY

o Appropriatewaste disposalsystemfor biohazards,

Tneu 33.5 Risks in a tissue culture laboratory radioactivewastes,toxins and corrosives.

Category Contributing factor (s)

Maintenance
risks Ageandcondition of various
leakage
equipment, of disposals.
Personnel
risks Inadequate
training, lackof The accidents or the risks associatedwith the
concentration
andinterest. biological materials are regarded as biohazards or
biological hazards. There are two main systems
Physicalrisks Electric
shocks,fire,intense cold.
that contribute to the occurrence of biohazaros
Chemicalrisks dueto poisons,
Toxicity (Table 33.6.
carcinogens,
mutagens, irritants,
allergens. 1 . The direct sourcesof the biological materials.
Biohazards Pathogenic
organisms, viruses, 2. The processesor operations involved in their
geneticmanipulalions,culture h a n dIi n g .
cellsandDNA(quality and
quantity). Gontrol of biohazards
risks
Radioisotope Energy emissionand its Biohazards can be controlled to a large extent
penetration,
ionization. b y s t r i c t a d h e r e n c et o t h e r e g u l a t o r yg u i d e l i n e sa n d
maintenance programmes.Some important aspects
are listed.
of the laboratory.A broad categorizationof risksand
the contributory factors is given in Table 33,5. . Microbiological safety cabinet or biohazard
wood with pathogen trap filters have been
Safety regulations develooed.
o Vertical laminar-flow hood (insteadof horizontal
Some of the develooed countries have
l a m i n o r - f l o w h o o d ) i s r e c e n t l y i n u se . Th i s
formulated general safety regulations fo minimize
minimizes the direct exposure of the operator to
the risks associated with tissue culture
the samples/processes.
Iaboratories. Selected examoles :
.l . Pathogen containing samples are treated In
. "Bios af et yin m ic r obiologi c a la n d b i o m e d i c a l
separaterooms with separatefacilities (centrifuge,
labor at or ies ", U. S. Depar t m e n t o f H e a l t h a n d
incubator, cell counting etc.).
Hum an Sc ienc es( 1993) .
o Sterilization of all wastes, solid glassware etc.
2. "Safe working and the prevention of infection
and their proper disposal.
in c linic al labor at or ies " U. K. H e a l t h S e r v i c e s
Adv is or y Com m it t ee ( 1991) . . F a c i l i t i e s f o r c h a n g e o f c l o t h i n g w h i l e e n te r i n g
and leaving the rooms.
Some of the general precautionsfor the safetyof
o Strict adherence to the access of designated
a tissue culture laboratory are listed here.
oersonnel to the culture rooms.
Strict adherence to recommendations of
regulatorybodies.
Pe ri o d i c a lm e e ti n g sa n d d i scussi onsof local Tlrr.r 33.6 Sources that contribute to biohazards
safetycommittees.
Biological material(s)
a Regular monitoring of the laboratories.
Tissuesamples andcultures withhuman pathogens.
a Per iodic al t r aining of t he p e r s o n n e l t h r o u g h Human cellsinfected (including
withviruses retroviruses)
s em inar sand wc lr k s hops .
Cellssubjectedto variousgeneticmanipulations.
Print and make the standardoperating procedures
(SOPs)available to all staff. Operating pracesses
oJthe media.
Preparation
a Cood record keeping.
Developmentof primarycullures,cell linesand other
a Limited access to the laboratory (only for the
laboratorv
works.
trained personnel and selected visitors).

-f h e se lect ionof an appr opr iat e gr owt h m e d i u n r
E fo r th e in v it r o c ult iv at ion of c ells i s a r r
imp orta nt an d es s ent ials t ep The nr anr m alia nc e l l s
of a n o rga n in t he body r ec eiv e nut r ient s f r o n r
Irloo d circu lat ion For c ult ur ing,t hes e c ells in v i t r r , t ,
it is expected that they should be provided with
the components similar to those present in blood
In ge ne ral, t he c hoic e of t he m edium m o s t l y
de pe nd so n the t y pe of t he c ells t o be c r , r lt ur e da, n d
th e p urp ose of t he c ult ur e ( gr owt h, dif f er ent i a t i o n ,
p rod uction o f des ir ed pr oduc t s ) The c ult ur e m e d i a
may be na tur al or ar t if ic ial.
iIIATURAL M EDI A
. (glucose, amino acids, vitamins, growth factors,
:{ In the ea r ly y ear s , t he nat ur al m edia obta i n e d
s
:t from vario us biologic al s our c eswer e us ed
{ developed Addition of serumto the various media
Bod y fluid s : Plas m a, s er um , ly m ph, am n i o t i t
fluid , a scitic and pleur al f luids , aqueous hu m o u r
from e ye s an d ins ec t hem oly m ph wer e in c om m o n
use. Th ese fluids wer e t es t ed f or s t er ilit y a n c l
toxicity be for e t heir ut ilit y
Tissueextracts : Among the tissueextracts,chick
embryo extract was the most commonly employed.
The e xtra cts of liv er , s pleen, bone m ar r ow a n d
le ucocyte sw er e als o us ed as c ult ur e m edia
So me wo rk er s s t ill pr ef er nat ur al m edia f or o r g a n
cu lture
ARTIFICIAL M EDI A
The artifi c ial m edia ( c ont aining par t ly de f i n e d
co mpo ne nts)hav e been in us e { or c ell c ult ur e s i n c e
MCDB 402
MCDB 110
MC D B 131
MCDB 170
WAJC 404
LHC 9
MCDB 202
MCDB 402
1950 The nrinimal criteria needed for choosing a
r r e r l i r : n r f o r a n i m a l c e l l r . r - r l t u r casr e l i s t c d b e l o w .
. T h e n r c c l i t r r l s h o r - r l cpl r o v i d e a l l t h e n u t r i e n t s t o
the cells
o M a i n t a i n t h e p h y s i o l o g , i c ap
l H arouncl 7 0 with
acJequatebuffering
o T h e m e d i u m m u s t b e s t e r i l e ,a n d i s o t o n i c t o t h e
ceils.
The basis for the cell culture media was the
balanced salt solution which was originally used to
create a physiological pH and osmolarity required
to maintain cells in vitro For 1lromolinSJgrr>wth
and proliferation of cells, vartous constituents
antibiotics etc.) were added, and several media
is a common practice However, some workers in
r e c e n t y e a r s h a v e s t a r t e d u s i n g s e r u m - f r e em e d t a
The physicochemical properties of media
required for tissue cultures are briefly desc.ribed.
t n b a l a n c e ds a l t
T h i s i s f o l l o w e d b y a b r i e f a c c o L r no
solutions,commonly used culture media and the
s e r u m - f r e em e d i a
PHYS!COCHEMICAL PROPERTIES
OF GULTURE MEDIA
T h e c u l t u r e m e d i a i s e x p e c t e dt o p o s s e s sc e r t a i n
physicochemical properties (pH, O2, COz,
b u f f e r i n g ,o s m o l a r i t y ,v i s c o s i t y ,t e m p e r a t u r ee t c ) t o
support good growth and proliferation of the
cultured cells
415
 416 B IOTE CHNO LO CY

pH f a c t m a d e p o s s i b l eb y a c o n t i n u o u s s u p p l y o f O, to
t h e t i s s u e sb y h e m o g l o b r n .
Most of the cells can grow at a pH in the range
of 7. 0- 7. 4, alt hough t her e a r e s l i g h t v a r i a t i o n s T h e c u l t u r e d c e l l s m o s t i v r e l v o n t h e d i sso l ve o
depending on t he t y pe of c ells ( i . e . c e l l l i n e s ) T h e O , i n t h e m e d i u m w h i c h m a y b e t o xi c a t h i g h
indicator phenol red is most commonly used for concentrationdue to the generationof fi'eeradicals
v is ible det ec t ion of pH of t he m e d i a . l t s c o l o u r a t i o n T h e r e f o r e , i t i s a b s o l u t e l y n e c e s s a r y to su p p l y
at the different pH is shown below. a d e q u a 'r e q u a n t i t i e s o f O , s o t h a t t he ce l l u l a r
AI pH 7.4 ReC requirementsare met, avoiding toxic effects.Some
workers add free-radical scavengers (glutathione,
At pH70 - Orange
m e r c a p t o e t h a n o l t)o n u l l i f y t h e t o x i c i t y . A d d i ti o n o f
At pH 6. 5 Ye l l o w s e l e n i u m t o t h e m e d i u m i s a l s o a d v o ca te d to
At pHZB - Pu r p l e r e d u c e O . t o x i c i t v T h i s i s b e c a u s e s e l en i u m i s a
c o f a c t o r f o r t h e s y n t h e s i so f g l u t a t h i o n e.
CO, bicarbonate and buffering
I n g e n e r a l , t h e g l y c o l y s i s o c c u r r i n g i n cu l tu r e d
Car bon diox ide in t he m ed i u m i s i n a d i s s o l v e d c e l l s r s m o r e a n a e r o b i c w h e n c o m p a r e d to i n vi vo
state, the concentration of which depends on the cells. Since the depth of the culture nredium
atmospheric CO, ter.rsionand temperature.CO, in i n f l u e n c e st h e r a t e o f O . d i f f u s i o n , i t i s a d vi sa b l e
t he nr edium ex is t s as c ar boni c a c i d ( H 'C O . ) , a n d to keep the depih of the'medium in the range 2--5
bic ar bonat e( HCO 3) and H+ io n s a s s h o w n b e l o w . mm.

CO r + H2O HH2CO 3 ( | H+ + HCO;

Temperature
As is ev ident f r om t he a b o v e e o u a t i o n , t n e
I n g e n e r a l , t h e o p t i m a l t e m p e r a t u r ef o r a g i ve n
c onc ent r at ions of CO z , HC O ; a n d p H a r e
cell culture is dependent on the body temperature
int er r elat ed. By inc r eas ing t he a t m o s p h e r i c C O r ,
of the organism, serving as the source of the cells.
t he pH will be r educ ed m ak ing t h e m e d i u m a c i d i c .
A c c o r d i n g l y , f o r c e l l s o b t a i n e d f r o m h um a n s a n d
I Addit ion of s odium bic ar bo n a t e( a s a c o m o o n e n r w a r m b l o o d e d a n i m a l s , t h e o p t i m a l t e m p e r a tu r ei s
of bicarbonate buffer) neutralizesbicarbonate ions 3 7 'C . l n v i t r o c e l l s c a n n o t t o l e r ate h i g h e r
NaHCO, {---J Na+ + HCO; t e m D e r a t u r e a n d m o s t o f t h e m d i e i f th e
temperature goes beyond 40oC lt is therefore
I n f ac t , t he c om m er c ial l y a v a i l a b l e m e d i a
a b s o l u t e l y n e c e s s a r y t o m a i n t a i n a co n sta n t
c ont ain a r ec om m ended c o n c e n t r a t i o n o f
temperature (+ 0.5"C) for reproducible results.
bic ar bonat e,and CO , t ens ion f o r t h e r e q u i r e d p H .
In recent years HEPES (hydroxyethyl piperazine l f t h e c e l l s a r e o b t a i n e d f r o m b i r d s , t he o p ti m a r
2-sulfonic acid\ buffer which is more efficient than t e m p e r a t u r ei s s i i g h t l y h i g h e r ( 3 8 . 5 'C ) f o r cu l tu r i n g .
bicarbonate buffer is being used in the culture F o r c o l d b l o o d e d a n i n i a l s ( p o i k i l t h e r m s )th a t d o n o t
media. However, bicarbonate buffer is preferred by r e g u l a t e t h e i r b o d y h e a t ( e . 9 . c o l d - w a t e r fi sh ) , th e
most workers becauseof the low cost, less toxicity c u l t u r e t e m p e r a t u r e m a y b e i n t h e r a n g e o f
and nut r it ional benef it t o t he m e d i u m . T h i s i s i n 15-25"C.
c ont r as t t o HEPES whic h is e x p e n s i v e , b e s i d e s B e s i d e s d i r e c t l y i n f l u e n c i n g g r o w t h o f ce l l s,
being t ox ic t o t he c ells . t e n r p e r a t u r ea l s o a f f e c t s t h e s o l u b i l i t y o f C O, i .e .
The pr es enc eof py r uv at e in t h e m e d i u m r e s u l t s h i g h e r t e m p e r a t u r ee n h a n c e s s o l u b i l i t y
in the increasedendogenousproduction of CO, b',,
t he c ells . This is adv an t a g e o u s s i n c e t h e Osmolality
dependenc e on t he ex ogenou s s u p p l y o f C O , a n d
I r r g e n e r a l , t h e o s m o l a l i t y f o r m ost o f th e
HCO S will be les s .I n s uc h a c a s e ,t h e b u f f e r i n gc a n
c u l t u r e d c e l l s ( f r o m d i f f e r e n t o r g a n i s m s) i s i n th e
be achieved by high concentration of amino acids.
range of 260-320 mosm/kg. This is comparable to
the osmolality of human plasma (290 mosm/kg).
Oxygen
O n c e a n o s m o l a l i t y i s s e l e c t e d f o r a cu l tu r e
A great majority of cells in vivo are dependent n i e d i u m , i t s h o u l d b e m a i n t a i n e da t t h a t l e ve l ( w i th
on t he C) , s upply f or aer obic r e s p i r a t i o n .T h i s i s r n an allowance of + 10 mosm,/kg)
 Chaot er34 : C U L T T J RMEED IAF O RA N IMA LC E LLS 417

vitamins, serum etc.) to promote proliferation of

cells in culture. Eagle was a pioneer in media
salt solutlons (BSS) formulation. He determined (during 1950-60) the
n u t r i e n t r e q u i r e m e n t sf o r m a m m a l i a n c e l l c u l t u r e s .
Ingradient Earle's BSS Hank's BSS
Many developments in media preparation have
NaCl 6.68 8.0 occurred since then. There are more than a dozen
KCI 0.4 04 media now available for different types of cultures.
CaCl, 0.02 0.14 Some of them are stated below.
(anhydrous) E M E M - E a g l e 's m i n i m a l e s s e n t i a lm e d i u m
MgSOr.THrO 0.2 0.1
DNlEM-Dulbecco's modification of Eagle's
NaHCO, 2.2 0.35 medium
NaHrPOo.HrO 0.14
C M E M - G l a s g o w 's m o d i fi c a t i o n of E a g l e 's
NarHPOo.THrO 0.09 medium
KH"PO, 0.06
RPMI 1630 and RPMI 1640-Media from
D-Glucose 1.0 1.0
Rosewell Park Memorial lnstitute.
Phenol
red 0.01 0.01
T h e o t h e r i m o o r t a n t c u l t u r e m e d i a a r e H a m 's
HEPES, Nasalt 13.02 2.08
F10, and /2, rC 199 and CMRL 1060.
(buffe0
The detailed composition of three commonly
used media namely Eagle'sMEM, RPMI 1640 and
Whenever there is an addition of acids, bases,
Ham's F12 is given in Table 34.2. fhe complete
drugs etc. to the medium, the osmolality gets
media, in g,eneral, contains a large number of
affected. The instrunrent osmometer is employed
components amino acids, vitamins, salts, glucose,
tbr nreasuringosmolalities in the laboratory.
other organic supplements, growth factors and
hormones, and antibiotics, besides serum.
BALANCED SALT SOLUTIONS
D e p e n d i n go n t h e m e d i u m , t h e q u a l i t y a n d q u a n t i t y
The balanced salt solutions (859 are primarily of the ingradientsvary. Some important aspects of
composed of inorganic salts. Sornetimes, sodium t h e m e d i a i n g r a d i e n t sa r e b r i e f l y d e s c r i b e d .
bicarbonate,glucose and HEPESbuffer may also be
added to BSS. Phenol red servesas a pH indicator. Amino acids
The imo orta nt f unc t ions of balanc ed s alt s o l u t i o n s
All the essentialamino acids (which cannot be
a re liste d h er eunder .
synthesizedby the cells) have to be added to the
. Sup ply e s s ent ialinor ganic ions . medium. In acidition,even the non-essentialamino
. Provide the requisite pH. acids (that can be synthesizedby ihe cells) are also
usually added to avoid any limitation of their
o Main tain t he des ir ed os m olalit y .
c e l l u l a r s y n t l r e s i sA
. m o n g t h e n o n - e s s e n t i aa
l mino
. Supply energy from glucose. acids, glutamine and/or glutamate are frequently
added in good quantities to the media since these
In fact, balanced salt solutionsform the basis for
amino acids serve as good sources of energy and
the preparation of complete media with the
carbon.
requisite additions. Further,BSS is also useful for a
sho rt p erio C ( up t o 4 hour s ) inc ubat ion of c e l l s .
Vitamins
The composition of two most widely usecj BSS
T h e q u a l i t y a n d q u a n t i t y o f v i t a m i n s d e p e n d so n
na mely Ea r le' s BSS and Hank ' s BSS is giv e n i n
t h e m e d i u m . F o r i n s t a n c e , E a g l e 'sM E M c o n t a i n s
Table 34.1.
only water soluble vitamins (e.g. B-complex,
c h o l i n e , i n o s i t o l ) .T h e o t h e r v i t a m i n s a r e o b t a i n e d
GOMPL ETE CULTURE M EDI A
from the serum added. The medium M 199
In the e ar ly y ear s , balanc ed s alt s olut ion s w e r e c o n t a i n sa l l t h e f a t s o l u b l e v i t a m i n s ( A , D , E a n d K )
supplemented with various nutrients (amino acids, also. In general, for the media without serum,

Biotechnology [27]
418 B IOTE CHNO LO CY

Component Eagle's RPMI Ham's

Taelr 34.2 Compositionof three conmonly
MEM 1640 F 12
used culture media
Inorganicsalts
Component Eagle's RPMI Ham's
MEM 1610 F 12 CaClr2HrO 200 44.1
Amino acids CaNOr.4HrO 100
L-Alanine 8.91 CuSOo.5HrO 0.0025
L-Arginine
HCI 105 200 211 FeSOo.THr0 083
L-Asparagine
l-tO 50 IJ .U
KCI 400 400 223
I A^^^,+i^
^^ii
L-nDPqr uu duru 20 IJ J

MoSO,.7H"O 220 100 133

L-Cystine 24 50 24.0
L-Glutamic
acid 20 14.7 NaCl 6800 6000 7599
L-Glutamine 292 300 146.2 NaHCO, 2000 2000 1176
Glycine 10 /.c I
NarHPOo.THrO 1512 268
L-Histidine
HCIH20 JI tc 21.0
NaHrPOo.2HrO 150
L-lsoleucine 52 50 3.94
L-Leucine 52 50 13.12 Other components
L-Lysine 58 40 36.54 D-Glucose 1000 2000 1801
L-Methionine tc tc 4.48
red
Phenol 5.0 t.1
L-Phenylalanine 32 t3 496
pyruvate
Sodium 110
L-Proline 20 345
L-Serine 30 10.51 Lipoic
acid 0. 21
L-Threonine 48 20 11.91 Linoleic
acid 0.084
1T,,,^+^^h^^
L- | | yPruPr rqr I 10 5 2.042 Hypoxanthine 4.08
L-Tyrosine 36 20 543
Putrescine
2HCl 0. 16
L-Valine 46 20 117
(red)
Glutathione 1
L-Hydroxyproline 20 more vitamins in higher concentrations are
required
Vitamins
D-Biotin 02 0.007 Salts
Ca D-pantothenate 1 0 25 0.26 The salts present in the various media are
Cholinechlcride 1 3.0 13.96 basically those found in balanced salt solutions
Folicacid 1 10 1.32 (Eagle'sB5S and Hank's BSS).The salts contribute
(Na+, K+, Mg2*, Ca2* etc.) and anions
i-lnositol 2 18.02 to cations
(Cl-, HCOI, SOtr-, POI), and are mainly
Nicotinamide 1 35 0.037 r e s p o n s i b l e f o r t h e m a i n t e n a n c e o f o sm o l a l i ty.
p-Aminobenzoic
acid '1.0
There are some other important functions of certatn
HCI
Pyridoxine 1 0.062 ions contributed by the salts.
Pyridoxal
HCI 'I
. C a 2 * i o n s a r e r e q u i r e df o r c e l l a d h e s i o n ,i n si g n a l
Ribof
lavin 0.1 02 0038 t r a n s d u c t i o n , b e s i d e s t h e i r i n v o l v e m e n t i n ce l l
Thiamine
HCI 1 1.0 0.34 prol iferation and d ifferentiation.
Vitamin
8,, 0.005 1.36 . N a +, K + a n d C l - ions regulate membrane
Table 34.2 contd. next column potential.
 ME D IAF O RAN IM A LC E LLS
Chaot er34 : C U L T U R E
. POI-, SOI- and HCO5 ions are involved in the
main ten anc e of int r ac ellular c har ge, b e s i d e s
serving as precursorsfor the production of certain
important compounds e.g. POj- is required for
ATP synthesis.
Glucose
Ma iority of c ult ur e m edia c ont ain gluc os ew h i c h
serves as an important source of energy. Clucose
is degraded in glycolysis to form pyruvate/lactate.
Th ese co moounds on t heir f ur t her m et abolis me n t e r
citric acid cycle and get oxidized to COr. However,
exoe rimen t al ev idenc e indic at es t ha t the
contribution of glucose for the operation of citric
a cid cycle is v er y low ln v it r o ( in c ult ur e c e l l s )
compared to in vivo situation. Glutamine rather
than glucose supplies carbon for the operation of
citric acid cycle. And for this reason, the cultured
ce lls req uir e v er y high c ont ent of glut am in e .
Hormones and growth factors
For th e m edia wit h s er um , addit ion of ho r m o n e s
and growth factors is usually not required. They are
fre qu en tly added t o s er um - f r eem edia.
Other organic supplements
Several additional organic compounds are
u su ally a dd ed t o t he m edia t o s uppor t c u l t u r e s .
Th ese in clu de c er t ain pr ot eins , pept ides, l i p i d s ,
nu cle oside sand c it r ic ac id c y c le int er m edia t e sF
. or
serum-free media, supplementation with these
comp ou nd s is v er y us ef ul.
Antibiotics
In the e ar ly y ear s , c ult ur e m edia inva r i a b l y
co nta ine d a nt ibiot ic s . The m os t c om m onl y u s e d
an tibio ticswer e am pic illin, penic illin, genta m y c i n ,
erythro mycin, k anam y c in, neom y c in and
tetracycline. Antibiotics were added to reduce
contamination. However, with improved aseptic
conditions in the present day tissue culture
laboratories, the addition of ' antibiotics is not
required. In fact, the use of antibiotics is associated
with several disadvantages.
. Possib ilit yof dev eloping ant ibiot ic - r es is t a nct e l l s
in cultu re.
. May cause antimetabolic effects and hamper
proliferation.
. Possibilityof hiding severalinfectiohstemporarily.
r Ma y en c our age poor as ept ic c ondilions .
419
The oresent recommendation is that for the
routine culture of cells, antibiotics should not be
added. However, they may be used for the
development of primary cultures.
SERUM
Serum is a naturalbiological fluid, and is rich in
various components to support cell proliferation.
The major constituentsfound in different types of
sera are listed in Table 34.3. The most commonly
used sera are calf serum (CS), fetal bovine serum
(FBS),horse serum and human serum. While using
human serum, it must be screenedfor viral diseases
( h e p a t i t i sB , H l v ) .
Approximately 5-20% (v/v) of serum is mostly
used for supplementingseveral media. Some of the
imoortant features of the serum constituents are
briefly described.
Proteins
The ln vitro functions of serum protein are nor
very clear. Some of them are involved in promoting
cell attachment and growth e.g. fetuin, fibronectin.
Proteins increase the viscosity of the culture
medium, besides contributing to buffering action.
Nutrients and metabolites
Serum contains several amino acids, glucose,
phospholipids, fatty acids, nucleosides and
metabolic intermediates(pyruvic acid, lactic acid
etc.). These constituents do contribute to some
e x t e n t f o r t h e n u t r i t i o n a l r e o u i r e m e n t so f c e l l s . T h i s
may however, be insignificantin complex media
with well supplemented nutrients.
Growth factors
There are certaih growth factors in the serum
that stimulate the oroliferation of cells in the
cu lture.
r Platelet-derivedgrowth factor (PDCF).
o Fibroblastgrowth factor (FGF).
o Epidermal growth factor (ECF).
. Vascular endothelial growth factor (VECF),
. lnsulin-like growth factors (lCF-1, ICF-2).
ln fact, almost all these growth factors are
commercially availablefor use in tissueculture.
 42f, B IOTE C HNO LO CY

Hormones
Tasle34.3 Major constituentsof serum
Hydrocortisone promotescell attachment,while
Proteins insulinfacilitatesglucoseuptakeby cells. Crowth
hormone,in association with somatomedins (lGFs),
Albumin
Globulins promotescell proliferation.
Fetuin
Fibronectin lnhibitors
Translenin S erum may al so contai n cel l ul ar gr owt h
Proteaseinhibitors inhibitingfactors.Majorityof them areartefacts e.g.
(cr''
-antitrypsin) bacterialtoxins,antibodies. The natural serum also
grow th i nhi bi to rnam ely
contai nsa physi ol ogi cal
Amino acids
transforming growth factor p (TCF-P). Most of
Almostall the20
thesegrowthinhibitoryfactorsmay be removedby
tipids heat inactivation(at 56'C for 30 minutes).
Cholesterol
Phospholipids S E LE C TION OF ME D IU M A N D S E RUM
Fattyacids As already stated,there are around a dozen
Carbohydrates media for the cell cultures.The selectionof a
h
rh Glucose parti cul armedi umi s basedon the cel l l i ne and t he
lh purpose of cul turi ng.Fori nstance, for chi ckem br yo
Hexosamine
fi brobl astsand H eLa cel l s, E ME M i s used. The
lP
I
Otherorganiccompounds medium DMEM can be usedfor the cultivationof
Lactic
acid neurons. A sel ected l i stof cel l sand cel l l i n esalong
Pyruvicacid w i th the medi a and sera used i s given I n
h
rb Polyamines Table34.4. In fact, informationon the selectionof
4, Urea appropri atemedi um for a parti cul arcell line is
avai l abl efrom l i terature.
Vitamins
A
Vitamin The selectionof serumis alsobasedon the type
Folicacid of cells being cultured.The following criteriaare
w hi l e choosi ngser um .
takeni nto consi derati on
Growth factors
r Batchto batch variations.
Epidermalgrowthfactor
growthfactor
Platelet-derived . Quality control.
growthfactor
Fibroblast to promotegrowthand preservation
. Efficiency of
Hormones cel l s.
Hydrocortisone r Sterility.
Thyroxine r Heat inactivation.
Triiodothyronine
In recent years, there is a tendency to
lnsulin
discontinuethe use of serum,and switch over to
Inorganics more clearlydefinedmedia (describedlater).
Calcium
Sodium S U P P LE ME N TA TION OF TH E ME DI UM
Potassium WITH TISSUE EXTRACTS
Chlorides B esi desserum,the cul turemedi a can also be
tr0n supplementedwith certain tissue extracts and
Phosphates The examplesare-chick
microbialcultureextracts.
Zinc embryo extract, proteolytic digestsof beef heart,
Selenium bactopeptone, lactalbuminhydrolysate, tryptose.
ChA P I CT
34 : C U L T U R E
M ED IAF ORAN IM ALC E LLS 421

The chick embryo extract was found to contain Contamination : lt is rather difficult to get serum
bot h high m o l e c u l a rw e i g h t a n d l o w m o l ecul ar totally free from all pathogens,particularly viruses.
weight compounds that support growth and
proliferatior.t of cells. - Presenceof growth inhibitors : In general, the
concentration of growth promoters in the serum is
m u c h h i g h e r t h a n t h e i n h i b i t o r s . B u t s o n 'r e t i m e s ,
the growth inhibitors such as TCF-B may dominate
and inhibit cell oroliferation.
'serum
Addition of to the culture media has been
, Availability and cost : There is a dependenceon
an age-old practice. However, in recent years, t h e c a t t l e f o r t h e s u p p l y o f s e r u m . H e n c e t h e
certain serum-free media have been develooed. lt availability may be restrictedon several occasions
is worthwhile to know the disadvantagesassociated f o r p o l i t i c a l a n d e c o n o m i c r e a s o n s F . u r t h e r ,c o s t a l s o
with the the use of serum, and the advantagesand i s a n o t h e r f a c t o r f o r d i s c o u r a g i n gt h e u s e o f s e r u m .
disadvantagesof serum-free media.
Downstream processing: The presenceof serum
Disadvantages of serum in media in the culture medium interferes with the isolation
and purification of cell culture products. For this
Variable composition : There is no uniformity rn
reason,severaladditional steps may be required for
the co mpo sition of t he s er um . lt is highly v ar i a b l e
(source, batch, season/ collection method, t he isolationof the desired oroduct.
p rocessin g).Suc h dif f er enc es in t he c om po s i t r o n
ADVANTAGES AND DISADVANTAGES
sig nifican tly i nf luenc e t he c ells in c ult ur e.
OF SERUM.FREE MEDIA
Quality control : To maintain a uniform quality
Advantages
of the serum, special tests have to be performed
with each batch of serum, before its use. T h e l i m i t a t i o n sa s s o c i a t e dw i t h t h e u s e o f s e r u m
i n t h e m e d i a ( d e s c r i b e da b o v e ) a r e e l i m i n a t e d i n
the serum-free media. In addition, there are two
more distinct advantages.
lines along with the media and seyum rscd
Selection of media with defined composition :
The main advantage of serum-free medium is to
Cells or cell line Medium Serum control growth of the cells as desired, with a well
defined medium This is in contrast to the use of
Chickembryo E M EM CS
serum wherein the growth frequently proceeds in
fibroblasts
an uncontrolled fashion.
Chinese hamster EMEM,
Ham'sF12 CS
ovary(CHO) Regulation of differentiation : lt is possible to
HeLacells E M EM CS use a factor or a set of factors to achieve
Human leukemia RPMI1640 FB differentiation of cells with the desired and
s o e c i a il s e d f u n c t i o n s .
Mouse leukemia Fischer's
medium, FB,HoS
RPMI1640
Disadvantages
Neurons D M EM FB
Mammary epithelium R PMI1 6 4 0D, ME M FB Slow cell proliferation : Most of the serum-free
Hematopoietic
cells RPMI1640, FB media are not as efficient as serum added media rn
medium
Fischer's the growth promotion of cells.
muscle
Skeletal D M EM,F 12 F B ,H OS Need for multiple media : A large number of
Glialcells ME MF, 1 2 ,D ME M FB serum-freemedia need to be developed for different
3T3cells ME M , M EM
D CS cell lines. This may create some practical
d i f f i c u l t i e s i n a l a b o r a t o r ys i m u l t a n e o u s l yh a n d l i n g
(EMEM--Eagle's minimalessentialmedium;RPMI 1640-Medium
cell lines. Another limitation of serum-free
from to BosewellPark Memoriallnstitute;DMEM-Dulbecco'ss e v e r a l
of Eagle'snediun; CS-Calfserum;FB-Fetalbovine m e d i u m i s t h a t a g i v e n m e d i u m m a y n o t b e a b l e t o
modification
serum:HoS-Horse serum) support the different stages of development even
 422 BIOTECHNOLOCY

for a given cell line. Hence, sometimesseparate

mediamay be requiredevenfor the samecell line. Tmrr 34.5 A selccied llst of cell llnes along
wlth serun-free nedla
Purity of reagents : The native serum does
possesssomeamountof protectiveand detoxifying Cell line Medium
machinarythat can offer a cleansingeffecton the embryo
Chick fibroblasts MCDB 202
apparatusand reagents.And therefore, in the MCDB402
hamster
Chinese ovary(CHO)
absence of serum, pure grade reagents and
Humanlungfibroblasts MCDB110
completelysterileapparatusshouldbe used.
Humanvascularendothelium MCDB131
Availabilityand cost : In general,the serum-free
Mammary epithelium MCDB170
media are costlierthan the serum added media.
This is mainlydue to the factthat manyof the pure Prostatic
epithelium WAJC 404
chemicals added to the serum-freemedia are Bronchial
epithelium LHC 9
themselvesexpensive.Further,the availabilityof Fibroblasts MCDB 202
serum-free media is also anotherlimitation. 3T3cells MCDB 402

D EV EL OP M EN T O F SE R U M.FR E E ME D IA Almost all the growth factors are now

Wh i l e d e s i g n i nsge ru m-fremedi e a,i t i s desi rabl e commercially available for the preparationof
to identify the various serum constituents(Refer serum-free media.
rl Table34.3)and theirquantities. The mostimportant
rI constituents of naturalserumwith reference to their Hormones
use in cell culturesmay be categorized as follorvs. Crowth hormone,insulinand hydrocortisone
are
e Crowth regulatoryfactorse.g. PDCF,TCF-p. the mostcommonlvaddedhormonesintothe serum-
free media. A combinationof steroidhormones-
i, . C e l l a d h e s i o nfa c to rse .g . v i t ami ns.
hydrocortisone, estrogen/ androgen and
I r E s s e n ti anl u tri e n tse .g . v i tami ns,metabol i tes, progesterone are used in formulatingserum-free
minerals,fatty acids. mediafor the maintenanceof mammaryepithelium.
r H o rm o n e se .g . i n s u l i n ,h y d ro corti sone.
Nutrients
F o r re p l a c i n gth e s e ru m a nd devel opmentof
ethanol-
Addition of certain nutrients-choline,
serum-free media, several constituentsshould
amine, linoleic acid, iron, copper,seleniumetc.,
supplementthe media.Some highlightsare given
are added in most of the serum-freemedia.
below.
Proteins
Growth factors
B ovi ne serum al bumi n (B S A ) i s the m ost
A large numberof growth factors(nearly100)
commonlyaddedprotein.It promotescell survival
that promote in vitro cell proliferation and
and growth.
differentiationhave been idenfitied.Besidesthe
factorsdescribedalready(above),someothersare Polyamines
listedbelow.
Putrescine is the most widely addedpolyamine
o Erythropoietin(EPO). to the serum-freemedia. Polyaminespromote
. Eye-derived growthfactors(EDCF1 and EDGF2). cellulargrowth and differentiation.
o In te rl e u k i n(lsL -1 ,l L -2 ). Protease inhibitors
o Hepatocytegrowth factor (HCF). Addition of protease inhibitors (e.9. soy bean
. Brain-derived
neurotrophicfactor.(BDNF). trypsin inhibitor)is done to the serum-freemedia
r Ph y to h e m a g g l u ti n(P for the trypsin-mediatedsubcultures.
i nH A ).
r Lipopolysaccharide
(LPS). C OMMON LY U S E D S E R U M.FR E E MEDI A
The growth factorsmay act synergisticallyor Severaltypes of serum-freemedia have been
additivelvwith each other or with <itherfactors developedfor differentcell lines.A selectedlist of
(e.g.hormones,prostaglandins). cell linesand the mediais given in the Table34,5.
 I t is an acceptedfact that the in vitro cultured r The nature of the substrate or phase in which
I cells do not possess the samecharacteristics
as cells grow. For monolayer cultures, the substrate
the in vivo cells.This is mostlydue to the changes is a solid (e.g. plastic) while for suspension
in t he c ellu l a re n v i ro n m e n r. cultures, it is a liquid.
. The compositionof the medium used for culture_
CHA RA CTER IST IC S O F
nutrients and physicochemical properties.
CULT URE D C E L L S
o Addition of hormones and growth factors.
Some of the important distinguishing properties
The composition of the gas phase.
of cultured cerrsare given below. '
o T h e t e m p e r a t u r eo f c u l t u r e i n c u b a t i o n .
1. Cells w h i c h d o n o t n o rm a l l y p ro l i ferate
in vivo can be grown and proliferatedin cultures. fhe biological and other aspects of cultured
cells with special reference to the following
2. Cell to cell interactionsin the cultured cells parametersare brieflv described.
are very much low.
1. Cell adhesion.
3. The threedimensionalarchitecture of the in
2. Cell proliferation.
vivo cells is not found in culturedcells.
3. Cell differentiation.
4. T he h o rmo n a la n d n u tri ti o n ailn fl u enceon
4. Metabolism of cultured cells.
the culturedcells differsfrom that on the in vivo
c ells . 5. lnitiation of cell culture.

5. Culturedcells cannotperform differentiated 6. Evolutionand development of cell lines.

and specializedfunctions.
CELL ADHESION
6. The environment of the culturedcellsfavours
pr olif er at i oann d s p re a d i n g
o f u n s p e c i a l i zed
cel l s. Most of the cells obtained from solid tissues
g r o w a s a d h e r e n tm o n o l a y e r si n c u l t u r e s .T h e c e l l s ,
derived from tissue aggregation or subculture,
Environmental influence
attach to the substrateand then start proliferating.
on cultured cells
In the early days of culture techniques, slightly
The environmental factorsstronglyinfluencethe negativelycharged glasseswere used as substrates.
cells in culture-The major routesthroughwhich In recent years, plastics Such as polystyrene, after
env ir onm e n tai nl fl u e n c eo c c u rsa re Ii s te d . treatment with electric ion discharge, are in use.

423
424 B IOTE C HNO LO CY

The cell adhesion occurs through cell surface

receotors for the molecules in the extracellurar
matrix. lt appears that the cells secrete nratrix
proteins which spread on the substrate.Then the
cells bind to matrix through receptors. lt is a
common observation that the substrates(glass or
p las t ic ) wit h pr ev ious c ell c ult ur e a r e c o n d i t i o n e d
to provide better surface area for adhesion.

Cell adhesion molecules

Three groups of proteins collectively referred
to as cell adhesion molecules (CAMs) are involved
in t he c ell- c ell adhes ion a n d c e l l - s u b s t r a t e
adhes ion. Fig. 35.1 : The cell cycle

Cell-cell adhesion molecules: These proteins are

pr im ar ily inv olv ed in c ell- t o - c e l l i n t e r a c t i o n proliferation
Gontrol of cell
between the homologous cells. CAMs are of two
types--calcium-dependent ones (cadherins) and F o r t h e c e l l s i n c u l t u r e , t h e e n v i r o nm e n ta l
c alc ium - indeoendentCAM s s i g n a l sr e g u l a t et h e c e l l c y c l e , a n d t h e r e b y th e ce l l
p r o l i f e r a t i o n .L o w d e n s i t y o f t h e c e l l s i n a m e d i u m
Integrins : These molecules mediate the cell
coupled with the presence certain growth factors
substrate interactions. Integrins possess receptors
(e I epidermal growth factor, platelet-derived
for m at r ix m olec ules s uc h as f i b r o n e c t i n a n d
growth factor) allows the cells to enter the cell
c ollagen
c y c l e . O n t h e o t h e r h a n d , h i g h c e l l d e n si ty a n d
Proteoglycans : These are low affinity c r o w d i n g o f c e l l s i n h i b i t s t h e c e l l c y c l e a n d th e r e b y
transmembrane receptors. Proteoglycanscan bind oroliferation.
to matrix collagen and growth factors.
B e s i d e s t h e i n f l u e n c e o f t h e e n v i r o n m e n ta l
Cell adhesion molecules are attached to the factors,certain intracellularfactorsalso regulatethe
cytoskeletonsof the cultured cells. cell cycle. For instance, cyclins promote while p53
and Rb gene products inhibit cell cycle.
CELL PRO LI FERATI O N
Pr olif er at ionof c ult ur ed c ells o c c u r s t h r o u g h t h e CELL DIFFERENTIATION
cell cycle, which has four distinct phases(Fi9.35. 1) T h e v a r i o u s c e l l c u l t u r e c o n d i t i o n s fa vo u r
M phase : In this phase (M = mitosis),the two m a x i m u m c e l l p r o l i f e r a t i o na n d p r o p a g a t i o no f ce l l
chromatids, which constitute the chromosomes, lines. Among the factors that promote cell
segregateto daughter cells. p r o l i f e r a t i o n ,t h e f o l l o w i n g a r e i m p o r t a n t .
G, phase : This gap I phaseis highly susceptible . L o w c e l l d e n s i t y
to various control orocessesthat determine whether
. Low Ca2+ concentration
cell should proceed towards DNA synthesrs,
re-enter the cycle or take the course towards . Presence of growth factors
d ifferentiation.
For the process of cell differentiation to occur,
S phase : This phase is characterized by DNA the proliferation of cells has to be severely limited
synthesiswherein DNA replication occurs. o r c o m p l e t e l y a b o l i s h e d C e l l d i f f e r e n t i a t i o nca n b e
G, phase : This is gap 2 phase that preparesthe promoted (or induced) by the following factors.
cell f or r eent r y int o m it os is . . High cell density
The integrity of the DNA, its repair or entry into
r High Ca2* concentration.
apoptosis (programmed cell death) if repair is not
possible is determined by two check points-at the . Presenceof differentiation inducers (e.g hydro-
beginning of DNA s y nt hes isand i n C , p h a s e . cortisone, nerve growth factor).
C haot er35 : CU L T U R E D AN D C H A R A C TE R IZA TION
C EL L S -BIOL OC Y 425

As is evident from the above, different and limited supply in the normal culture conditions(i e.
alm os toppos in gc o n d i ti o n sa re re q u i re dfo r cel l atmospheric oxygen and a submerged culture)
oroliferation. and for cell differentiation. Therefore creating an anaerobic situation. Lactate, secreted
is requiredtwo distinctsetsof
if cell differentiation i n t o t h e m e d i u m , a c c u m u l a t e s .S o m e a m o u n t o f
conditionsare necessary pyruvate produced in glycolysis gets oxidized
.l through Krebs cycle. A small fraction of glucose
. To optimizecell proliferation.
(4-9%) enters pentose phosphate pathway Io
2. T o oot im i z ec e l l d i ffe re n ti a ti o n .
supply ribose S-phosphate and reducing
e q u i v a l e n t s( N A D P H ) f o r b i o s y n t h e t i cp a t h w a y se . g .
Maintenance of differentiation
s y n t h e s i so f n u c l e o t i d e s .
lt is now recognizedthat the cells retain their
Clutamine is an important source of energy for
native and original functions for long when their
the cultured cells. By the action of the enzyme
three dimensional structures are retained. This is
g l u t a m i n a s e ,g l u t a m i n e u n d e r g o e sd e a m i n a t i o n t o
possible with organ cultures. However, organ
p r o d u c e g l u t a m a t ea n d a m m o n i u m i o n s . C l u t a m a t e ,
culturescannot be propagated.In recent years,
on transamination (or oxidative deamination)
someworkersaretryingto createthreedimensional
forms cr-ketoglutarate which enters the Krebs
by pe rfu s i n m
st r uc t ur es g o n o l a y ecr u l tu re sF. u rther,
cycle. Pyruvate predominantly participates in
in v it r oc ult ur in go f c e l l so n o r i n s p e c i ama
l tri ces
t r a n s a m i n a t i o nr e a c t i o n t o o r o d u c e a l a n i n e , w h i c h
( e. g. c ellulos e , c o l l a g e n g e l , m a tri x of
is easily excreted into the medium In the rapidly
g ly c opr ot einsa) l s o re s u l ts i n c e l l s w i th th ree
g r o w i n g c u l t u r e d c e l l s , t r a n s a m i n a t i o nr e a c t i o n i s a
d im ens ional s t ru c tu
re s .
dominant route of glutamine metabolism.

Dedifferentiation Deamination of glutamine releases free

ammonium ions, which are toxic to the cultured
Dedifferentiationrefersto the irreversiblelossof
cells, limiting their growth. ln recent years,
specialized properties of cells when they are
dipeptides glutamyl-alanine or glutamyl-glycine are
cultured in vitro. This happens when the
being used to minimize the production of
differentiatedin vitro cells lose their properties
ammonia. Further,these dipeptides are more stable
(Fig. 35.2).
in the medium.
fn the in vivo situation, a small group of stem
As already stated,a-ketoglutarateobtained from
cells give rise to progenitor cells that are capable
glutamine (via glutamate) enters the Krebs cycle
of producing differentiated cell pool (Fig. 35.2A).
and gets oxidized to carbon dioxide and water. For
On the other hand, in the in vitro culturesystem, proper operation of Kerbs cycle, balancing of the
c ellsa re p re d o m i n a n tlpyro d u c e dw h ich
pr ogenit or
intermediates oI the cycle is required. Two
go on proliferating. Very few of the newly formed
metabolites of Kerbs cvcle namelv malate and
cells can form differentiated cells (Fig.35.2D. fhe
oxaloacetate leave the cycle and get converted
net resultis a blockeddifferentiation.
respectivelyto pyruvate and phosphoenol pyruvate.
As alreadydescribed,dedifferentiation implies The latter two comoounds can reenter the Krebs
an irreversiblelossof specializedpropertiesof the cycle in the form of acetyl CoA. Thus, the
cells.On the other hand, deadaptafionrefersto the c o n t i n u i t y o f K e r b s c y c l e i s m a i n t a i n e d .
of sp e c i a l i z epdro p e rti eosf th e c e l l sby
r einduc t ion Glucose as well as glutamine gets metabolised
c o n d i ti o n s .
c r eat ingappr op ri a te by the cultured cells to supply energy in the form
of ATP.
M E T A B O LI S M O F C U L T U R ED C E L L S
T he m et abo l i s mo f ma mma l i a nc u l tu re dc el l s INITIATION OF CELL CULTURE
with specialreference to energyaspectsis depicted The cell culture can be initiated by the cells
in Fig.35.3.The culturedcellscan useglucoseor derived from a tissue through enzymatic or
glutamine as the source of energy. These two mechanical treatments. For details on primary
c om pounds als o g e n e ra te i mp o rta n t a n a b ol i c culture, the reader must refer Chapter 36 Primary
orecursors. c u l t u r e i s a s e l e c t i v ep r o c e s st h a t f i n a l l y r e s u l t si n
As glucosegetsdegradedby glycolysis,lactate a r e l a t i v e l y u n i f o r m c e l l l i n e T h e s e l e c t i o n o c c u r s
is m ainly pr odu c e dT. h i s i s b e c a u s eo x y g e ni s i n by virtue of the capacity of the cells to survive as
426 B IOTE CHNO LO CY

(A) Stem cells Progenitorcells cells

Differentiated

(o)
C o
(c)
(c) C o
+)

( o) 0 o
Stemcell C C
regeneration
Differentiation

(B) cell
Differentiated
Stem cells
t9
----1e,/-A
-tr7
@
(o) @
o) @
( o) @
@
@
Stemcell
regeneration @ Differentiation

Fig. 35.2 : Differentiation of cetls (A) ln vivo differentiation of stem cells (B) Blocked differentiation in cultured cells'

m onolay er c ult ur es ( by adher i n gt o s u b s t r a t e so) r a s c e l l s , w h i c h a r e s e n s i t i v et o g r o w t h l i m i ta ti o n d u e

s us oens ionc ult ur es .
Am ong t he c ult ur ed c ells , s o m e c e l l s c a n g r o w
and pr olif er at e while s om e a r e u n a b l e t o s u r v i v e
under t he c ult ur e env ir onm e n t . T h e c e l l s c o n t i n u e
t o gr ow ih m onolay er c ult ur e s , t i l l t h e a v a i l a b i l i t y
of t he s ubs t r at eis oc c upied.
The term confluence is used when fhe cultured
cells make close contact with one another by fully
utilizing the available growth area. For certain
to density, the cells stop growing once confluence
is reached. However, the transformed cells are
insensitiveto confluence and continue to overgrow.
Wh e n t h e c u l t u r e b e c o m e s c o n f l u e n t, th e ce l l s
possessthe following characters.
1 . T h e c l o s e s tm o r p h o l o g i c a l r e s e m b l a n ceto th e
tissueof origin (i e. parent tissue).
2 T h e e x p r e s s i o no f s p e c i a l i z e df u n cti o n s o f th e
c e l l s c o m p a r a b l e t o t h a t o f t h e n a t i v e ce l l s
Chaot er35 : C U L T U R E D A N D C H A R A C TE R IZA TION
C EL L S -BIOL OC Y 427

GLUCOSE

+
I
Glucose6-phosphate G
lL
+y
Fructose
f-OhosRhate B
J
Fructose1, 6-bisphosphate

Glyceraldehyde
{- Dihydroxyacetone
3--phosphaie pnosfinate S
J
:nol pyruvate

+
ruvate.
J \
rylCoA

iitrate
iREBS \ \
)YCLE
)YCLE tT \
+ lsocitrate \
I t \ A LA N TN E
\ J \/
Fumarate o-Ketoglutarate'!-:
\ / - *---7- Glutamate
\, ^
sudcinate
/ coA +_ ?' * ";
succinlii NH; 6LUTAM1NE

Fig' 35.3 : An outline of the glucose and glutamine metabolism in cultured mammalian cells,

EVOLUTION AltiD DEVELOPMENT The evol uti on of a conti nuouscel l l i ne i s

O F CE LL L IN ES depictedin Fig. 35,4.The cumulativecell number
The primary culture grown after the first in a cultureis representedon Y-axison a log scare,
s ubc ult ur ei s re fe rre dto a s c e l l l i n e . A g i ven cel l while the X-axisrepresents the time in weeks.The
line may be propagated by furthersubculturing. As time for developmentof a continuouscell line is
the subculturesare repeated,the most rapidly vari abl e._ For i nstance, for human di pl oi d
pr olif er at i n gc e l l s d o m i n a te w h i l e th e non- fi brobl asts,
the conti nuouscel l l i ne ari sesat about
pr olif er at i n o
g r s l o w l y p ro l i fe ra ti n cg e l l s wi l l get 14 weekswhile the senescence may occur between
diluted,and consequently disappear. 10 to 20 rveeks;usually after 30 and 60 cell
doubli ngs.
Senescence : The geneticallydeterminedevent
of c ell div i s i o n sfo r a l i m i te dn u mb e ro f ti mes(i .e.
Development of continuous cell lines
populationdoublings), followedby their deathin a
normal trssue is referred to as senescence. Certain alterationsin the culture, collectively
Howev el g e rm c e l l s a n d tra n s fo rme dcel l s are referred to as transformation, can give rise to
c apableof c o n ti n u o u s lpyro l i fe ra ti n g
l n. th e i n vttro conti nuouscel l l i nes. Transformati on may be
c ult ur e, t r a n s fo rm e dc e l l s c a n g i v e ri se ro spontaneousloccurri
y ng,chemi cal l yor vi ral l y-
c ont inuou sc e l l l i n e s i nduced. Transformati on basi cal l v i nvol ves an
 424 B I O TECHNO LO CY

1020

1018 Continuous
C U IIU T C c e l ll i n e
o
(d 10 1 6
a

o
o 10 1 4
o
Senescenceand
E
J 1012 cell death
c
Subculture
q) interval
O
10 1 0

Explantation
2nd subculture
108 J
106

?
I

Fig. 35.4 : Diagrammatic rcpresentation ol evolution of a cell line.

I

alterationin growth characteristics such as lossof E pi dermalcel l s and l ymphoblast oid

cells ar e
c o n ta c ti n h i b i ti o nd, e n si tyl i mi tati onof grow thand capabl eof formi ngconti nuousce ll lines.
a n c h o ra gien d e p e n d e n ce. Theterm i mmortal i zati on
i s fre q u e n tl yu s e dfo r the acqui si ti on of i nfi ni tel i fe
s o a nto c u l tu re dc e l l s .
Genetic variations: The ability of the cells to
g ro w c o n ti n u o u s l yi n cel l l i nesrepresents geneti c
v a ri a ti o ni n th e c e l l s .M ost often,the del eti onor oncul turedcellsor cell linesis
C haracteri zati of
mutation of the ps3 gene is responsiblefor i mportantfor di ssemi nati on of cell lines t hr ough
c o n ti n 0 o u sp ro l i fe ra ti onof cel l s. In the normal cell banks, and to establish contacts between
c e l l s ,th e n o rm a l p s 3 g ene i s responsi blfor
e the researchlaboratoriesand commercial companies.
a rre s o t f cell cycle. of cel l l i nes wit h soecial
C haracteri zati on
Most of the continuouscell lines areaneuploid, referenceto the followingaspectsis generallydone.
possessing chromosomenumber betweendiploid 1. Morphol ogyof cel l s
a n d te tra p l o i dv a l u e .
2. S peci esof ori gi n.
3. Ti ssueof ori gi n.
N o rm a l c e l l s a n d conti nuous cel l l i nes
4. Whethercell line is transformed
or not.
A greatmajorityof normalcellsare not capable
t 5. l denti fi cati on
of speci fi ccell lines.
o f g i v i n gri s eto c o n ti n uous cel l l i nes.For i nstance,
I
normal human fibroblastsgo on proliferatingfor
I MOR P H OLOGY OF C E LLS
I a b o u t 5 0 g e n e ra ti o ns,and then stop di vi di ng.
.l
I Howel,er,they remainviablefor about B months. A si mpl eanddi recti denti fi ca t ion
of t he cult ur ed
: A n d th ro u g h o uth t e i r l ife span,fi brobl asts remai n cel l scan be doneby observi ng theirm or phological
e u p l o i d .C h i c kfi b ro b l a stsal sobehavei n a si mi l ar characteristics.However,the morphologyhasto be
fa s hi o n . viewed with cautionsince it is largelydependent
Chaot er35 : C U L T U R E D AN D C H A R A C TE R IZA TION
C EL L S -BIOL OC Y 429

o n the cultu r e env ir onm ent . For ins t anc e , t h e as cell markers for identification. Some examples
e pith elia l ce lls gr owing at t he c ent er ( of t he c u l t u r e ) are given below.
a re reg ula r p oly gonal wit h c lear ly def ined e d g e s ,
. Albumin for hepatocytes.
while tho se g r owing at t he per ipher y ar e ir r e g u l a r
. Melanin for melanocytes
an d disten de d ( s wollen) . The c om pos it ion o f t h e
cu lture me diu m , and t he alt er at ionsin t he s ub s t r a t e . Hemoglobin for erythroid cells
also influ en ce t he c ellular m or phology . I n a t i s s u e o Myosin (or tropomyosin)for muscle cells.
cu lture la bo r at or v . t he t er m s f ibr oblas t ic a n d Enzymes as tissue markers : The identificatron
e pith elia l are c om m only us ed t o des c r ibe t h e
of enzymes in culture cells can be made with
a pp ea ran ceo f t he c ells r at her t han t heir or ig i n .
referenceto the following characters.
Fibroblastic cells : For these cells, the length is r
C o n s t i t u t i v ee n z y m e s .
usua lly mo re t han t wic e of t heir widt h Fibr ob l a s t i c
. Inducible enzymes.
cells a re b ipo lar or m ult ipolar in nat ur e.
. lsoenzymes.
Epith elia l cells : Thes e c ells ar e poly gon a l i n
The comnronly used enzyme markers for cell
na ture with regulardim ens ionsand us ually gr o w i n
l i n e i d e n t i f i c a t i o n a r e g i v e n i n T a b l e 3 5 . 1.
mon ora ye rs.
Tyrosine aminotransferase is specific for
The terms fibroblastoid (fibroblast-like) and
h e p a t o c v t e s ,w h i l e t y r o s i n a s e i s f o r m e l a n o c y t e s .
epitheloid (epithelial-like)are in use for the cells
C r e a t i n e k i n a s e ( M M ) i n s e r u m s e r v e sa s a m a r k e r
that do not possessspecific charactersto identify as
for muscle cells, while creatine kinase(BB) is used
fibro bla stic o r eoit helial c ells
for the detection of neurons and neuroendocrrne
cells.
SPECIES OF O RI G I N O F CELLS
Filament proteins as tissue markers : The
The ide ntific at ionof t he s pec iesof c ell line s c a n
i n t e r m e d i a t ef i l a m e n t p r o t e i n sa r e v e r y w i d e l y u s e d
be done by.
as tissue or lineage markers.For example-
. Chro mosom al analy s is .
a Ele ctro ph or es isof is oenz y m es .
o A combination of both these methods.
Tlelr 35.1 Some common enryne narkers for
cell lin6 identification
ln re ce nt vear s , c hr om os om al ident if ic at i o n i s
be ing do ne b y em ploy ing m olec ular pr obes . I nzy i nc u-.ell ivpr:
Tyrosine
aminotransferase Hepatocytes
I DE NT I F I CA T ION O F Tysosinase Melanocytes
T I S S UE O F O R IG IN
Glutamylsynthase Brain(astroglia)
The identificationof cell lines with regardto kinase
Creatine Musclecells
tissueof origin is carriedout with referenceto the (isoenzymeMM)
following two characteristics.
kinase
Creatine Neurons,
1. T he line a g eto w h i c h th e c e l l sb e l o n g . (isoenzymeBB) neuroendocrine
cells
s f th e c e l l si .e .s te mc e l l s ,p re cursor
2. T hes t at u o Non-specific
eslerase Macrophages
c elIs . DOPA-decarboxylase Neurons
phosphatase
Alkaline typell
Enterocytes,
T is s ue m ark e rs fo r c e l l l i n e pneumocytes
ident if ic at io n Angiotensin-converti
ng Endothelium
S om eof t h e i mp o rta ntit s s u eo r l i n e a g emarkers enzyme
f or c ell line id e n ti fi c a ti oanre b ri e fl yd e s c ri b ed Sucrase Enterocytes
Differentiatedproductsas cell markers: The Neuron-specific
esterase Neurons
c ult ur edc ells,o n c o mp l e tee x p re s s i o n
a ,re c a p abl e DOPA-Dihydroxy
phenylalanine;
MM-Twopolypeptidesubunitsol
of producingdifferentiation markers,which serve muscle;BB-Twopolypeptide of brain
subunits
430 B IOTE C HNO LO CY

o Crowth rate
a Mode of growth
used for tte detertlon of cell types a Longevity

Antibody Cell type a Tumori geni ci ty

Cytokeratin Epithelium . Specializedproductformation.

Epithelial
membrane antigen Epithelium W hi l e characteri zi ngthe cel l l i nes, it is
Albumin Hepatocytes necessarvto consider the above charactersto
cr-Lactalbumin Breastepithelium determinewhetherthe cell line hasoriginatedfrom
Carcinoembryonic
antigen andlung
Colorectal tumor cells or has undergonetransformationin
(cEA) a0en0carcrn0ma culture.
Prostate
specificantigen Prostatic
epithelium For more details on transformation,Refer
(PSA) Chaoter6.
Intracellular
celladhesion T-cellsand
molecuie(l-CAM) endothelium ID E N TIFIC A TION OF
a-Fetoprotein Fetalhepatocytes S P E C IFIC C E LL LIN E S
Humanchorionic Placentalepithelium There are many approaches in a culture
gonadotropin(hCG) laboratoryto identifyspecificcell lines.
Human growth hormone Anteriorpituitary . Chromosomeanalysis
(hGH)
o DNA detection
Vimentin Mesodermal cells
. RNA and proteinanalysis
Integrins Ailcells
a Enzyme activities
Actin Allcells
a Antigenic markers

o Astrocytescan be detected by glial fibrillary Ghromosome analysis

a c i d i cp ro te i n(C F AP ).
. M u s c l ec e l l sc a n b e i d e n ti fi edby desmi n.
. E p i th e l i aal n d me s o th e l i acle l l sby cytokerati n. Further,it is also possibleto distinguishnormal and
Cell surfaceantigensas tissuemarkers : The
antigensof the cultured cells are useful for the
detectionof tissueor cells of origin. ln fact, many
antibodieshave been developed(commercialkits
a re a v a i l a b l e )fo r th e i d e n ti fi cati oncel l l i nes
(Table.35.2). Theseantibodiesare raisedagainst
cell surfaceantigensor other proteins.
The antibodiesraisedagainstsecretedantigen
c,-fetoprotein serves as a marker for the
identificationof fetal hepatocytes.Antibodiesof
c e l l s u rfa c ea n ti g e n sn a me l yi n tegri nscan be used
for the generaldetectionof cell lines.
T R AN SF OR M ED C EL L S
Transformation is the phenomenonof the change
in phenotype due to the acquirement of new
genetic material. Transformationis associatedwith
promotionof genetic instability.The transformed
a n d c u l tu re d c e l l s e x h i b i t a l t erati onsi n many
characters with referenceto
The soeciesand sex from which the cell line is
derivedcan be identifiedby chromosomeanalysis.
malignant cells by the analysisof chromosomes.lt
may be noted that the normal cells contain more
stable chromosomes.The important techniques
employed with regardto chromosomeanalysisare
brieflydescribed.
Chromosomebanding: By this technique,it is
possibleto identify individualchromosomepairs
when there is little morphological difference
betweenthem. Chromosomebandingcan be done
by usi ngC i emsastai ni ng.
Chromosome count : A direct count of
chromosomescan be done per spread between
50-100 spreads.A cameraLucidaattachmentor a
cl osedci rcui ttel evi si onmay be useful .
Chromosomekaryotyping: In this technique,
the chromosomesare cut, sorted into sequence,
and then pastedon to a sheet.The imagecan be
recordedor scannedfrom the slide.Chromosome
karyotypingis time consumingwhen comparedto
chromosome counti ng.
Chapt er35 : C U L T U R E D A N D C H A R A C TE R IZA TION
C EL L S -BIOL OC Y 431

DNA deteetion Enzyme activities

The fofal quantity of DNA per normal cell is Some of the in vivo enzyme activitiesare lost
quite constant,and is characteristicto the species when the cells are culturedin vitro. For instance,
of origin.e.g. normalcell linesfrom human,chick arginaseactivityof the liver cells is lost within a
and hamster fibroblasts. However, the DNA few days of culturing. However, certain cell lines
contentvariesin the normal cell lines of mouse, expressspecific enzymesthat can be employed for
and also the cell lines obtainedfrom cancerous their detection e.g. tyrosine aminotransferase for
t is s ues , hepatocytes,glutamylsynthaseactivity for astroglia
in brain. For more examplesof enzymesusefulin
As alreadystated,most of the transformedcells
cell line detection, refer Table 35.1.
ar e aneup l o i da n d h e te ro p l o i dD
. N A a n al ysi si s
particularlyusefulfor characterization lsoenzymes: The multipleformsof an enzyme
of suchcells.
catalysing the same reaction are referred to as
Analysisof DNA can be carriedout by DNA
isoenzymesor isozymes.lsoenzymesdiffer in many
hybridization and DNA fingerprinting. physical and chemical properties-structure,
DNA hybridization : The popular Southern electrophoretic and immunologicalproperties,K-
blotting technique (For details, Refer Chapter 7), and V-"* values.
can be used to detect unique DNA sequences. The isoenzymescan be separatedby analytical
Specific molecular probes with radioisotope, techniques
such as electrophoresis and
fluorescentor luminescentlabelscan be usedfor chromatography.
Most frequently, electrophoresis
t his pur pose . by employingagarose, celluloseacetate,starchand
The DNA from the desiredcell linesis extracted, polyacrylamideis used. The crude enzyme is
cut with restrictionendonucleases, subjectedto applied at one point on the electrophoretic
electrophoresis, blotted on to nitrocellulose,and medium.As the isoenzymes migrate,they distribute
then hybridizedwith a molecular(labeled)probe, in different bands, which can be detected by
or a set of probes. By this approach,specific stainingwith suitablechromogenicsubstrates.
sequences of DNA in the cell linescan be detected. lsoenzymes are characteristic to the speciesor
DNA fingerprinting: Thereare certainregions fissues.lsoenzymesof the following enzymesare
These
in the DNA of a cell that are not transcribed. commonly usedfor cell line detection.
regions, referredto as satellite DNA, have no o Lactatedehydrogenase
known functions,and it is believedthat they may
r Malate dehydrogenase
provide reservoirfor genetic evolution. Satellite
DNA regions are considered as regions of r Clucose6-phosphatedehydrogenase
hypervariability.These regionsmay be cut with . Aspartateaminotransferase
specificrestriction'endonucleases, and detectedby . PeptidaseB.
us ingc DNA p ro b e s .
lsoenzyme analysis is also useful for the
By using electrophoresis and autoradiography,detectionof interspecies
cross-contamination
of cell
the oatternsof satelliteDNA variationscan be lines. For instance,contaminationof mouse cell
detected. Such patterns referred to as DNA line with hamstercell line can be identifiedov
fingerprintsare cell line specific. usingpeptidaseB isoenzymes.
In recent years, the technique of DNA
fingerprintinghas become a very popular and a Antigenic markers
powerfultool to determinethe origin of cell lines. Cell linescan be characterizedby detectionof
antigenicmarkersthrough the use of antibodies.
RNA and protein analysis The antigenicmarkersmay be locatedon the cell
surfaceor secretedby the cells into the culture
The phenotypecharacteristicsof a cell line can
medi um.
be detectedby geneexpression i.e. identification
of
RNAsand/or proteins.mRNAscan be identifiedby Someof the antibodiesin common use for the
Northern blot technique while proteins can bte detection of different cell types are given in
detected by Western blot technique. Table 35.2 (Seep. 43O).
 432 B IOTE C HNO LO CY

Information on the growth state of a given

cult ur e is r equir ed t o :
. Des i8n c ult ur e ex per im ent s
o Rout ine m aint enanc eof c ult u r e .
. M eas ur em entof c ell pr olif er a t i o n .
. Know t he t im e f or s ubc ult ur e .
o Det er m ine t he c ult ur e r es pon s et o a p a r t i c u l a r
s t im ulus or t ox in.
Som e of t he c om m only us ed te r m s i n r e l a t i o n t o
the measurement of growth of cultured cells are 2468
Days from subculture
e x plained.
Populationdoubling time (PDT) : The tlme Fig. 35.5 : Growth curve of cultured cetls (Note : The
i n te rv a lfo r th e c e l l p o p u l a ti onto doubl e at tne ceil concentration is expressed in semilog plot),
m i d d l eo f th e l o g a ri th m i (l
c o g )phase.
Cell cycletime or generationtime : The interval
attachesto the substrateand spreadsout. There is
f ro m o n e o o i n t i n th e c e l l d i vi si onto the same
a n i n c r e a s e d s y n t h e s i s o f c e r t a i n e n z y m e s ( e .g .
p o i n t i n th e c y c l e , o n e d i v i s ion l ater.Thus cel l
cy c l e ti me i s m e a s u re fo D N A p o l y m e r a s e )a n d s t r u c t u r a lp r o t e i n s ,pr e p a r i n g
d rm o n e poi nt i n the cel l
I
t h e c e l l s f o r p r o l i f e r a t i o n T h e p r o d u cti o n o f
cy c l e u n ti l th e s a mep o i n t i s re achedagai n.
i s p e c i a l i z e d p r o d u c t s d i s a p p e a r s w h i c h ma y n o t
Confluence: lt denotesthe culturestagewhere-
r e a p p e a r u n t i l t h e c e l l p r o l i f e r a t i o n c e a s e s.
ll i n a l l th e a v a i l a b l es u b s tra te(grow th area) i s
u ti l i z e d ,a n d th e c e l l s a re i n c l ose contactw i th The lag phase representspreparativestageof the
each other. c e l l s f o r p r o l i f e r a t i o n f o l l o w i n g s u b c u l tu r e a n d
reseeding.
C o n ta c ti n h i b i ti o n: In h i b i t i onof cel l moti l i ty
a n d p l a s m ame mb ra n eru ffl i n gwhen the cel l sare
The log phase
i n c o mp l e tec o n ta c tw i th o th e ra dj acentcel l s.Thi s
mostlyoccursat confluencestate,and resultsin the The log phase is characterizedby an exponential
ceasationof the cell oroliferation. g r o w t h o f c e l l s , f o l l o w i n g t h e l a g p h ase . Th e
d u r a t i o n o f l o g p h a s e d e p e n d s o n t h e c e l l s w i th
C e l l d e n s i ty: T h e n u mb e ro f cel l sper ml of the
referenceto :
m e d i u m.
Saturationdensity : The density of the cells o S e e d i n g d e n s i t y .
(cells/mlzsurfacearea)in the plateauphase . Crowth rate.
r Density after proliferation.
G R O W T H C Y C L E O F C U L TU R E D C E LLS
During the log phase,the cultured cells are in
T h e g ro w th c y c l e o f cul tured cel l s i s t h e m o s t u n i f o r m a n d r e p r o d u c i b l e s t a t e wi th h i g h
y p re s e n tebdy t hree phases-the
c o n v e n ti o n a l lre viability. This is an ideal time for sampling.
lag phase, the log (exponential) phase and the
plateau phase (Fig. 35.5). The propertiesof the The log phase terminates after confluence is
c u l tu re dc e l l sv a ry i n th e p h a ses. reached with an addition of one or two population
d o u b li n g s .
Th e l a g p h a s e
The plateau phase
The lag phaserepresents a periodof adaptation
d u ri n g w h i c h th e c e l l fo rm s the cel l surfaceand As the cells reach confluence, the growth rate is
e x tra c e l l u l a rma tri x (l o s t d u ri ng trypsi ni zati on),m u c h r e d u c e d , a n d t h e p r o l i f e r a t i o n o f cu l tu r e d
 Chapt er35 : C U L T U R ED
C E L L S-B IO L OC Y
A N D C H A R A C TE R IZA TION 433

No of coloniesformed
Platingefficiency = x 100
N o. of cel l s seeded

The term cloning efficiency is used (insteadof

s"trdtion platingefficiency)when each colony growsfrom a
density
si ngl ecel l .
i
tr Seedingefficiency representingthe survival of
E ros Normalcells
cel l sat hi gherdensi ti es,
i s cal cul atedas fol l ow s.
0)
O
N o. of cel l s recovered
Seedingefficiency = x 100
No. of cellsseeded

2468 10
Days of subculture

Fig. 35.6 : Growth curues of transformed S ynchroni zati on

l i teral l ymeansto maketw o or
and normal cells (Note : The cell concentration is more thi ngs happen exactl y si mul taneousl y. For
expressed in semilag plot). instance,
two or morewatchescan be synchronized
to show exactlythe sametime.
The cells at different stagesof the cell cycle in
plateauor
cells almoststops.This stagerepresents
a culture can be synchronizedso that the cells will
stationaryphase,and is characterized
by
be at the samephase.Cell synchronyis requiredto
. Low m ot i l i tyo f c e l l s . studythe progressi on of cel l s throughcel l cycl e.
. Reducedrufflingof plasmamembrane. Severallaboratorytechniqueshavebeendeveloped
. Cellsoc cu p y i n gm i n i m u ms u rfa c ea re a . to achi evecel l synchroni zati on. They are broadl y
r Cont ac tin h i b i ti o n . categorizedinto two groups.
. Saturationdensity. 1. P hysi calfracti onati on
for cel l separati on.
r Depletionof nutrientsand growthfactors. 2. C hemi calbl ockadefor cel l seoarati on.
. Reducedsynthesis of structuralproteins.
. Increasedformationof specializedproducts. C E LL S E P A R A TION B Y
The majorityof normalculturedcells that form P H Y S IC A L ME A N S
monolayers stopgrowingas they reachconfluence. P hysi cal fracti onati on or cel l separati on
S om eof t he c e l l s h o w e v e r,w i th re p l e n i s h ment
of techni ques, basedon the fol l ow i ngcharacteri sti cs
mediumcontinueto grow (at a reducedrate)after are tn use.
c onf luenc efo
, rm i n gm u l ti l a y e rs of cells.
. C el l densi ty.
The transformedculturedcells usuallyreacha .
C el l si ze.
higherc ell d e n s i tyc o m p a re dto th e n o rm a lc el l si n
. Affinity of antibodieson cell surfaceepitopes.
the plateau phase(Fig, 35.6).
. Light scatteror fluorescentemissionby labeled
P LA T I NG EF F IC IEN C Y OF cel l s.
CULT URE D C EL L S The two commonly used techniquesnamely
Plating efficiency,representi ng colonyformation centrifugalelutriationand fluorescence-activated
at low cell density,is a measureusedfor analyzing cell separationare brieflydescribedhereunder.
c ell pr olif e ra ti oann d s u rv i v a l .
Centrifugal elutriation
W hen t he c e l l s ,a t l o w d e n s i ti e sa,re c u l t uredi n
the form of singlecell suspensions, they grow as The physical characteristics - cell size and
discretecolonies.Platingefficiencyis calculatedas sedimentation velocity are operative in the
follows. techni queof centri fugal
el utri ati on.

Biotechnology
[28]
434 B IOTE C I.I NO LO CY

Centrifugal elutriator (from Beckman) is an (A)

advanced device for increasing the sedimentation
rate so that the yield and resolutionof cells is better.
T he c ell s epar at ion is c ar r ied o u t i n a s p e c i a l l y
designed centrifuge and rotor (Fig. 35.V. The cells
i n t he m edium ar e pum ped in t o t h e s e p a r a t i n g
Pumpingof cells
cham ber while t he r ot or is t u r n i n g . D u e t o
Windows
cent r if ugal f or c e, t he c ell will b e p u s h e d t o t h e
e dges .As t he m edium is t hen pu m p e d t h r o u g h t h e Separating
cnamoer
chamber in such a way that the centripetal flow rs Rotor
equal to the sedimentation rate of cells. Due to Stroboscopic
differences in the cells (size, density, cell surface light
configuration),the cells tend to sedimentat different (B)
r at es ,and r eac h equilibr ium at dif f e r e n tp o s i t i o n sr n
the chamber. The entire operation in the elutriator
can be viewed through the port, as the chamber is
i llum inat edby s t r obos c opicI ight . A t t h e e q u i l i b r i u m
the fiow rate can be increasedand the cells can be
pumped out, and separatedin collecting vesselsin
different fractions. lt is possible to carrv out
separationof cells in a complete medium, so that Centrifugalforce
the cells can be directly cultured after separation.
Centripetalforce

Fluorescence.activated cell sorting

Fluorescence-activated cell sorting is a
technique for sorting out the cells based on the
differences that can be detected by light scatter
(e.g.cell size) or fluorescence emission (bV
pretreated DNA, RNA, proteins, antigens).
The pr oc edur einv olv espas s in go f a s i n g l es t r e a m
of cells through a laser beam so that the scattered
light from the cells can be detected and recorded. CELL SEPARATION
When the cells are pretreated with a fluorescenr
st ain ( e. g.c hr om om y c in A f or DNA ) , t h e f l u o r e s c e n t
emission excited by the laser can be detected.
There are two instruments in use based on the
principle of fluorescent-activatedcell sorting.
Fig. 35.7 : (A) Diagrammatic view of a centrifugal
elutriator, (B) Separation €hamber of elutriator.
hand, fluorescent-activatedcell sorti ngi s m ost ly
used to obtain high grade pure fracti onsof cells
f r o m s m a l l q u a n t i t i e so f c e l l s .
BY
C H E M I C A L B L O C K A D E
The cel l s can be separatedby b locking
metabolic reactions. Two types of metabolic
bl ockadesare i n use- i nhi bi ti onof D N A synt hesis
and nutri ti onaldeori l ' ati on.

1 . Flow cytometer : This instrumentis capable of Inhibition of DNA synthesis

sorting out cells (from a population) in different
phasesof the cell cvcle based on the measuremenrs
D uri ngthe S phaseof cel l cycl e,D N A synt hesis
can be i nhi bi ted by usi ng i nhi bi torss uch as
of a c om binat ionof c ell s iz e and D N A f l u o r e s c e n c e .
thymi di ne,ami nopteri ne, hydroxyurea and cyt osine
2. Fluorescent-activatedcell sorter (FACS): rn arabinoside.The effects of these inhibitors are
this ins t r um ent ,t he em is s ion s ign a l sf r o m t h e c e l l s vari abl e The cel l cycl e i s predomi nantly b locked
ar e m eas ur ed, and t he c ells s o r t e d o u t i n t o i n S ohasethat resul tsi n vi abl ecel l s.
c ollec t ion t ubes .
Nutritional deprivation
Gomparison between physical methods
E l i mi nati onof serum or i sol euci nefrom t he
For s epar at ion of a lar ge n u m b e r o f c e i l s , culture medium for about 24 hours resultsin the
centrifugal elutriator is preferred. On the other accumul ati on of cel l s at C . phase.Thi s e f f ectof
Chaot er35 : C U L T U R E D A N D C H A R A C TE R IZA TION
C E L L S-B IO L OC Y 435

nutritional deprivation can be restored by their MEASUBEMENT OF SENESCENCE

a dd ition b y whic h t im e t he c ell s y nc hr ony o c c u r s .
SOME HIG HLI G HTS OF
CELL SYNCHRONIZATION
. Ce ll sep ar at ion by phy s ic al m et hods is m o r e
effective than chemical procedures
. Ch emical bloc k ade is of t en t ox ic t o t he ce l l s .
. Transformed cells cannot be synchronized by
nu trition al deor iv at ion.
. A h igh d egr ee of c ell s y nc hr ony ( > 80% ) c a n b e
obtained in the first cycle, and in the second
cycle it would be < 6ooh f he c ell dis t r i b u t i o n
ma y occu r r andom ly in t he t hir d c y c le.
A s t he c e l l sg ro w i n c u l tu re ,th e y b e c omeol d
due t o aging a , n dth e yc a n n o tp ro l i fe ra te
a n y more. 1 . Wa s h t h e c e l l s a n d f i x t h e m u s i n g a f i x a t i v e
The end of the proliferative life span of cells is ( e . g . p a r a f o r m a l d e h y d e ) ,a n d w a s h a g a i n .
referred to as senescence.
CE LLULA R SE N E SC EN C E
The growth of the cells is usually measuredas
population doublings (PDs). The PDs refer to the
num berof ti me s th e c e l l p o p u l a ti o nd o u bl esi n
num ber du ri n g th e p e ri o d o f c u l tu re a nd i s
c alc ulat edb y th e fo l l o w i n gfo rm u l a
log (N o of cells har vested) logr o ( No of cells s eeded)
pn-
loBr
o:
The phenomenon of senescence has been
mostly studied with human fibroblast cultures.After
30 -60 p op ulat ions doublings , t he c ult ur e is m a i n l y
composed of senescentfibroblasts.These senescent
fibro bla st ar e unable t o div ide in r es po n s e t o
mitotic stim uli. lt m us t be not ed t hat t he c e l l s d o
n ot ap pe ar s uddenly ,but t hey gr aduallyac c u m u l a t e
an d in cre asein num ber dur ing t he lif e s pan o f t h e
cu lture.
The d ifferent parameters used f or the
me asure mentof c ell gr owt h in c ult ur es ar e l i s t e d
below.
. Direct me as ur e of c ell num ber .
. Determination of DNA'/RNA content.
o Estimationof protein/ATPconcentration.
T h e d i r e c t m e a s u r e m e n t o f s e n e s c e n tc e l l s r s
rather difficult. Some of the indirect measuresare
. Loss of metabolic activity
. Lack of labeled precursor 13H-thymidine)
incorooration into DNA.
o C e r t a i n h i s t o c h e m i c a lt e c h n i o u e s .
Senescence.associated
B.galactosidase activity assay
There occurs an overexpressionof the lysosomal
enzyme p-galactosidase at senescence. This
enzyme elevation is also associated with an
increase in the cell size as the cell enters a
permanent non-dividing state.
T h e n u m b e r o f s e n e s c e n tc e l l s i n a c u l t u r e c a n
be measured by senescence-associated B-galacto-
sidase (SA-p) assay. The assay consists of the
following stages.
2. Add the staining solution (X-gal powder rn
dimethylformamide dissolved in buffer) to the fixed
cells and incubate.
3 The senescentcells display a dense blue
colour which can be counted
APOPTOSIS
The process of programmed cell death (PCD) is
referred to as apoptosis. The cell death may be
initiated bv a soecific stimulus or as a result of
several signals received from the external
e n v r r o nm e n t .
A p o p t o s i so c c u r s a s a r e s u l t o f i n h e r e n tc e l l u l a r
m e c h a n i s m s ,w h i c h f i n a l l y l e a d t o s e l f d e s t r u c t i o n .
The cell activatesa series of molecular events that
cause an orderly degradation of the cellular
constituents with minimal impact on the
n e i g h b o u r i n gt i s s u e s .
Reasons lot in situ apoptosis
1 . For proper development : The formation of
fingers and toes of the fetus requiresthe removal of
t h e t i s s u e sb e t w e e n t h e m . T h i s i s u s u a l l y c a r r i e d
out by apoptosis.
2. Destruction of cells that pose threat to the
integrity of the organism : Programmed cell de-ath
is needed to destroy and remove the cells that may
436 B IOTECHNO LO CY

otherwise damage the organisms. Some examples w h i c h c a n b e i d e n t i f i e d u n d e r p h ase co n tr a st

are listed mrcroscope.
. Cells wit h dam aged DNA d u r i n g t h e c o u r s e o f The cells that have undergone apoptosis contain
em br y onic dev elopm ent . l f t h e y a r e n o t fragmented chromatin which can be detected by
destroyed,they may result in birth defects. conventional stainingtechniques.
. Cells of t he im m une s y s t e m , a f t e r t h e r r I n r e c e n t y e a r s , m o r e s e n s i t i v e an d r e l i a b l e
appr opr iat eim m une f unc t i o n , u n d e r g o a p o p t o s i s . techniques have been developed for measuring
This is needed to prevent autoimmune diseases apoptosis. Some of them are briefly described.
e. g. r heum at oid ar t hr it is
. Cells infected with viruses are destroyed by Determination ADP/ATP ratio
apopt os r s . Both the growth and apoptosis of cells requrre
3. Cell destruction due to negative signals : ATP But when there is growth arrest,an elevation
Ther e ar e s ev er al negat iv e s i g n a l s w i t h i n t h e c e l l s o f A D P o c c u r s . T h u s m e a s u r i n gA D P / A TP r a ti o w i l l
that promote apoptosis. These include thron, light on the dead cells. In fact, some assay
accumulation of free radicals,exposureto UV rays, systems for measuring ADP/ATP ratios are
X-rays and chemotherapeuticdrugs. commercially available.

Mechanism of apoptosis TUNEL assay

The pr ogr am m ed c ell dea t h m a y o c c u r d u e t o A s i g n i f i c a n tb i o c h e m i c a l e v e n t f o r t h e a p o p to si s

t hr ee dif f er ent m ec hanis m s . is the activation of endogenous nuclease activity.
.l This enzyme cleaves DNA into fragmentswith free
. Apopt is is due t o int er n a l s i g n a l s
3 - h y d r o x y l g r o u p s . T h e n e w l y f o r m e d sm a l l D N A
2. Apopt os is t r igger ed by e x t e r n a l s i g n a l s e . g f r a g m e n t s c a n b e e x t e n d e d b y e m p l o yi n g th e
t um or nec r os isf ac t or - c r( TNF - a ) , l y m p h o t o x i n . e n z y m e D N A p o l y m e r a s e . l f l a b e l e d n u cl e o ti d e s
3. Apoptosis triggered by reactive oxygen are used for DNA fragment extension, they can be
specres. detected

T U N E L i s a n a b b r e v i a t i o n f o r T dT- m e d i a te d
Role of caspases in apoptosis
d U T P n i c k e n d - l a b e l i n g a s s a y .T U N E L i s ve r y fa st
A group of enzymes namely activated proteases and effective for the determination of DNA
play a c r uc ial r ole in t he pr o g r a m m e d c e l l d e a t h . fragmentsformed by endogenous nucleaseactivity.
These proteases are actually cysteinyl aspartate The apoptotic nuclei can be identified by a
specific proteinasesor in short, commonly referred f l u o r e s c e n t technique using fl u o r e sce i n
to as caspases.There are about ten different types i s o t hi o c y a n a t e( F I T C )a n d 4 , 6 - d i a m in o p he n yli n d o l e .
of caspasesacting on different substratesultimately
leading t o c ell deat h For i n s t a n c e , c a p s a s e I DNA laddering test
.l
cleaves interleukin B.
During the courie of apoptosis,the genomic
D N A i s c l e a v e d t o m o n o - a n d o l i g o nu cl e o so m a l
Inhibition of caspase activities
DNA fragments.These fragmentscan be separated
Sinc e t he c as pas es ar e c l o s e l y i n v o l v e d i n by agarose electrophoresis, and detected. The
apoptosis, it is possible to prevent cell death by nucleosomal fragments of apoptotic cells give a
inhibit ing t heir ac t iv it ies Ce r t a i n s p e c i f i c p e p t i d e s characteristicladder pattern on electrophoresis.
that can inhibit caspases,and thus apoptosis have
been identified. Limitations of the test : DNA laddering test is
not verv soecific since several cells that have
undergoneapoptosismay not show DNA laddering.
MEASUREMENT OF APOPTOS'S
Further,some cells not subjected to apoptosis may
A simple and easy way of detecting dead or also show DNA ladders, For these reasons, DNA
dy ing c ells is t he dir ec t m ic r o s c o p i c o b s e r v a t i o n . laddering test is coupled with some other test for
fhe dying cells are rounded with dense bodies measurementof apoptosis.
 I
primary culture reters to the starting culture
/ \of cells, tissues or organsl taken directly from
an o rga nism .Thus , t he pr inr ar y c ult ur e is t lr e i n i t i a l
cu lture b efo r e t he f ir s t s ubc ult ur e The t er m c e l l
linc is used for the propagation of cultures alrer
th e first su bc ult ur e Som e bas ic and f unda m e n t a l
aspe (itso f p r im ar y c ult ur e and c ell lines ar e b r i e f l v
de scribe d.
As alre ady s t at ed, pr im ar y c ult ur e b r o a d l y
involves the culturing techniques carried following
the isolation of the cells, but before the first
subculture. Prinrary cultures are usually prepared
from la rge ti s s ue m as s es .Thus , t hes e c ult ur e s m a y
con tain a v ar iet y of dif f er ent iat ed c ells e . g .
fibro bla sts, l y m phoc y t es , m ac r ophages , epi t h e l i a l
cells.
With th e ex per ienc esof t he per s onnel wo r k i n g
in tissu ecu lt ur e labor at or ies t, he f ollowing cr i t e r i a /
cha racterist ic s ar e c ons ider ed f or eff i c i e n t
de ve lop men t of pr im ar y c ult ur es .
. Emb ryo nic t is s ues r at her t han adult t is s u e s a r e
p refe rredfor pr im ar y c ult ur es .This is due t o t h e
fa ct tha t t he em br y onic c ells c an b e
disag gre gat edeas ily and y ield m or e v iable c e l l s ,
besides rapidly proliferating in vitro.
o The qu an t it y of c ells us ed in t he pr im ar y c u l t u r e
sh ou ld b e higher s inc e t heir s ur v iv al r a t e i s
substanti al l y l ow er (when compared to
sr,rbcul tures)
. T h e t i s s u e ss h o u l d b e p r o c e s s e dw i t h m i n i m L r m
damage to cells for use in primary culture
F u r t h e r ,t h e d e a d c e l l s s h o u l d b e r e m o v e d .
. S e l e c t i o no f a n a p p r o p r i a t em e d i u m ( p r e f e r a b l ya
n u t r i e n t r i c h o n e ) i s a d v i s a b l e .F o r t h e a d d i t i o n o f
serum, fetal bovine source is preferred rather
than calf or horse serum.
r lt is necessaryto remove the enzymes used for
d i s a g g r e g a t i o no f c e l l s b y c e n t r i f u g a t i o n .
TECHNIQUES FOR PRIMARY CULTUBE
Among the various techniques devised for the
p r i m a r y c u l t u r e o f i s o l a t e dt i s s u e s ,t h r e e t e c h n i q u e s
are most comrnonly used.
l. Mechanical disaggregation.
2. Enzymalic disaggregation.
3. Primary explant technique.
An outline of these techniques is depicted in
F i g . 3 6 . 1, a n d t h e p r o c e d u r e sa r e b r i e f l y d e s c r i b e d .
MECHAN'CAL D'SAGGBEGATION
For the disaggregationof soft fissues(e 9,.spleen,
brain, embryonic liver, soft tLrmors),mechanical
technique is usually employed. This technique
basically involves careful chopping or slicing of
tissue into pieces and collection of spill out cells.
The cells can be collected bv two wavs.
437
438 B IOTECHNO LO CY

Primaryexplanttechnique

Fig. 36.1 : Different techniques used for primary culture.

r Pressing the tissue pieces through a series of effi ci ent w i th hi gher yi el d of cel l s by use of
s iev eswit h a gr adual r educ t i o n i n t h e m e s h s i z e enzymes.This is due to the presence of lessfibrous
connecti ve ti ssue and extracel l ular m at r lx.
. Forcing the tissue fragments through a syringe
Enzymaticdisaggregation can be carried out by
and needle.
usi ngtrypsi n,col l agenase or someotherenzym es.
Alt hough m ec hanic al di s a g g r e g a t i o n i n v o l v e s
the risk of cell damage, the procedure is less Disaggregation by tryPsin
ex pens iv e, quic k and s im p l e . T h i s t e c h n i q u e r s
The term trypsinization is commonly used for
par t ic ular lyus ef ulwhen t he a v a i l a b i l i t yo f t h e t i s s u e
disaggregationof tissuesby the enzyme,trypsin.
is in plenty, and the efficiency of the yield is not
Many worker;spreferto use crude trypsin rather
very crucial. lt must however, be noted that the
than pure trypsinfor the followingreasons.
v iabilit y of c ells obt aine d f r o m m e c h a n i c a l
t ec hniques is m uc h lower t h a n t h e e n z y m a t i c . The crude trypsin is more effectivedue to the
t ec hnioue. presenceof other proteases
. Cellscan toleratecrude trypsinbetter.
E NZYM AT'C D'SAG G R EG AT' O N
. The residualactivity of crude trypsin can be
Enzymatic disaggregationis mostly used when easi l y neutral i zedby the serumof t he cult ur e
high recovery of cells is required from a tissue. media (when serum-freemedia are used, a
Disaggregation of embryonic tissues is more trypsi ni nhi bi torcan be usedfor neut r alizat ion) .
ChA P I CT
36 : P R IM AR Y AN D C E L LL IN E S
CULTURE 439

Disaggregation of cells can also be carriedout ineffective for certain tissues (e.g. fibrous
b y us ingpur et r y p s i nw h i c h i s l e s sto x i c a n d m o re connective tissue). Hence other enzymes are also
soecificin its action. i n u s e f o r d i s s o c i a t i o no f c e l l s .
The desiredtissueis choppedto 2-3 mm pieces
and then subjectedto disaggregation by trypsin. Disaggregation by collagenase
There are two techniquesof trypsinization-warm Collagen is the most abundant structuralprotetn
trypsinizationand cold trypsinization(Fig.36.2). i n h i g h e r a n i m a l s . l t i s m a i n l y p r e s e n ti n t h e e x t r a -
Warm trypsinization(Fig. 36.2A) : This method cellular matrix of connective tissue and muscle.
is widely used for disaggregation of cells. The The enzyme collagenase (usually a crude one
choppedtissueis washedwith dissectionbasalsalt contaminated with non-specific proteases)can be
solution (DBSS),and then transferredto a flasr effectively used for the disaggregationof several
containingwarm trypsin(37oC). The contentsare tissues(normal or maligndnt) that may be sensitive
stirred,and at an intervalof every thirty minutes, to trypsin. Highly purified grades of collagenase
th e s uper nat an
co g e d i s s o c i a tecde l l sc an
t n ta i n i n th have been tried, but they are less effective when
be collected.After removalof trypsin,the cellsare compared to crude collagenase.
dispersedin a suitablemedium and preserved(by The important stages in collagenase dis-
keepingt he v ial o n i c e ). aggregation, depicted in Fig. 36.3, are briefly
The processof additionof freshtrypsin(to the described hereunder.
tissue pieces), incubation and collection of
The desired tissue suspended in basal salt
c ells(a t 3 0 mi n u te si n te rv a l si s) c a rried
d is s oc iat ed
solution, containing antibiotics is chopped into
out for about 4 hours.The disaggregated cells are
pieces. These pieces are washed by settling, and
pooled, counted, appropriatelydiluted and then
t h e n s u s p e n d e di n a c o m p l e t e m e d i u m c o n t a i n i n g
incubated.
collagenase. After incubating for 1-5 days, the
Cofdtrypsinizatlon(Fig.36.28): Thistechnique tissuepieces are dispersedby pipetting.The clusters
is more appropriatelyreferredto as trypsinization of cells are separated by settling. The epithelial
with cold preexposure.The risk of damageto the cells and fibroblastic cells can be separated.
cells by prolongedexposureto trypsinat 37oC(in
war m t r y ps iniz a ti o nc)a n b e mi n i mi z e d i n th i s Collagenase disaggregation has been
techn ioue. successfully used for human brain, Iung and
several other epithelial tissues, besides various
After choppingand washing,the tissuepieces human tumors,
and other animal tissues. Addition
are kept in a vial (on ice) and soakedwith cold o f a n o t h e r e n z y m e h y a l u r o n i d a s e ( a c t s
on
trypsin for about 6-24 hours. The trypsin is carbohydrate residues on
cell surfaces) promotes
removedand discarded.However,the tissuepieces d i s a g g r e g a t i o n .C o l l a g e n a s e i n c o m b i n a t i o n w i t h
l p s i n .T h e s eti s s u ep i e c e si n a hyaluronidase is found
c ont ainr es idua try to be very effective for
mediumare incubatedat 37oCfor 20-30 minutes. dissociatingrat or rabbit liver. This can be
done by
The cellsget dispersedby repeatedpipettings. The perfusing the whole
organ in situ.
dissociatedcells can be counted, appropriately
dilut edand t hen u s e d . Some workers use collagenase in conjunction
with trypsin, a formulation developed in chick
The cold trypsinization method usually results
serum, for disaggregationof certain tissues.
in a higher yield of viable cells with an improved
survivalof cellsalter24 hoursof incubation.This
Use of other enzymes
methoddoes not involvestirringor centrifugation,
in disaggregation
and can be convenientlyadoptedin a laboratory.
The major limitationof cold trypsinization is that it Trypsin and collagenase are the most widely
is not suitablefor disaggregation of cellsfrom large used enzymes for disaggregation.Certain bacterial
quant it ies
of t is s u e s . proteases (e.g. pronase, dispase) have been used
with limited success. Besides hyaluronidase
Limitations of trypsin disaggregation (described above), neuraminidase is also used in
Disaggregation by trypsin may damage some conjunction with collagenase for effective
c ells ( e. g. epit h e l i a lc e l l s ) o r i t ma y b e a l most degradation of cell surface carbohydrates.
 5
D
o

After 30 minutes
piecessettle Tissuepieces viai kept
in a vial

I
Add iresh
trypsinand
continueincubation

RemovetrYPSin
and incubate
at37"C for 20-30 min

Cellsin a medlum
Dilutein a
suitablemedium

o
-o
m

I
Z
o
-o
basa! salt solution)',
^^1, '+ianl o
(A) warm trypsinization (B) cotd trypsinization (DBs'-Dissectron
Fig. 36.2: preparation of primary curture by trypsin disaggregation
Chapt er36 : P R IM AR Y AN D C E L LLIN E S
CULTURE
Choppedinto
piecesand
washed by settling
Fibroblastic
cells
Epithelial
cells
Fig. 36,3 : lmporlant stages in collagenase disaggregationof tissue for primary culture (BSS-Basal salt solution)
PRIMARY EXPLANT TECHN'QUE
Th e prima ry ex plant t ec hnique was , ir r f ac t t h e
o rigin al me tho d, dev eloped by Har r is on in 19 0 7 .
This techn ique has under gone s ev e r a l
mod ifica tion s, and is s t ill in us e. The s im pli f i e d
pro ce du re ad opt ed f or pr im ar y ex plant c ult ur e i s
depicted in Fig. 36.4, and briefly described below.
Th e tissu e in' bas al s alt s olut ion is f in e r y
chopped, and washed by settlings.The basal salt
soluticn is then removed. The tissue pieces are
spread evenly over the growth surface. After
ad ditio n of appr opr iat e m edium , inc ubat io n i s
ca rried o ut fo r 3- 5 day s . Then t he m edium i s
ch an ge d at wee k ly int er v alsunt il a s ubs t ant ialo u t -
growth of cells is observed. Now, the explants are
removed and transferredto a fresh culture vesset.
The primary explant technique is particularly
useful for disaggregation of small quantities of
fissues(e.g.skin biopsies).The other two techniques-
mechanical or enzymatic disaggregationhowever,
are not suitablefor small amountsof tissues,as there
is a risk of lo si ng t he c ells . The lim it at ion of ex p l a n t
technique is the poor adhesiveness of certain tissues
to the growth surface,and the selection of cells rn
the outgrorvth. It is however, observed that the
primary explant techniquecan be usedfor a majority
of e mbryon ic c ells e. g. f ibr oblas t s , m y obl a s t s ,
ep ithe lial cells , glial c ells .
SEPARATION O F VI ABLE AND
NON.VIABL E G ELLS
It is a common practice to remove the non-
viab le cells wh ile t he pr im ar y c ult ur e is pr ep a r e d
441
--------------- f
I
Pind++6d t^
tissues
disperse
Separationby
settling
from the disaggregatedcells. This is usually done
when the first change of the medium is carried
ouf. The very ferv left over non-viable cells get
d i l u t e d a n c l g r a d u a l l y d i s a p p e a ra s t h e p r o l i f e r a t i o n
of viable cells commences.
Sometimes, the non-viable cells from the
p r i m a r y c u l t u r e sm a y b e r e m o v e d b y c e n t r i f u g a t i o n .
The cells are mixed with ficoll and sodiunr
metrizoate, and centrifuged.The dead cells form a
pellet at the bottom of the tube.
MEDICAL ETHICS AND SAFETY
MEASURES IN CULTURE TECI.INIQUES
S i n c e t h e c u l t u r e t e c h n i o u e si n v o l v e t h e u s e o f
a n i m a l o r h u m a n t i s s u e s ,i t i s a b s o l u t e l yn e c e s s a r y
to follow several safety measures anci medical
ethics. ln fact, in some countries there are
established legislation/normsfor selection and use
of tissues in cultures. For example, in United
Kingdom, Animal Experiments (Scientific
Procedures)Act of 1986 is followed.
fhe handling of human tissues poses several
problems that are not usually encountered with
animal tissues.While dealing with fetal materials
and human biopsies, the consent o{ the patient
and/his or her relatives.besidesthe consent of locar
e t h i c a l c o m m i t t e e i s r e q u i r e d . F u r t h e r ,t a k i n g a n y
tissue (even in minute quantities) from human
d o n o r s r e q u i r e st h e f u l l c o n s e n t o f t h e d o r r o r i n a
prescribed forrnat. The following issuesneed to be
f u l l y c o n s i d e r e dw h i l e d e a l i n g w i t h h u m a n t i s s u e s .
1. The consent of the patient and/or relatives
for using tissuesfor researchpurposes.
442 B IOTECHNO LO CY

2 Ownership of the cell lines developed and

their derivatives.

3 . C o n s e n t f o r g e n e t i c m o d i f i c a t i o n o f th e ce l l
Ii n e s .

6 . P a t e n t r i g h t s f o r a n y c o n r m e r c i al u se o f ce l l
Ii n e s .

I n t h e g e n e r a l p r a c t i c e o f c u l t u r e te ch n i q u e s
u s i n g h u m a n t i s s u e s ,t h e d o n o r a n d / o r re l a ti ve sa r e
asked to sign a disclaimer statement (in a prescribed
p r o f o r m a ) b e f o r e t h e t i s s u e i s t a k en . By th i s
a p p r o a c h , t h e l e g a l c o m p l i c a t i o n s a r e mi n i n r i ze d

Safety measures

Hancllirrgo{ human ffssuesis associatedwith a

heavy risk of exposure for various infections
T h e r e f o r e ,i t i s a b s o l u t e l yn e c e s s a r yt h a t th e h u m a n
m;rterialsare handled in a biohazard cabineL fhe
t i s s u e s s h o u l d b e s c r e e n e d f o r v a r i o r - r si n fe cti o n s
s u c h a s h e p a l i t i s , t u b e r c u l o s i s , H I V be fo r e th e r r
u s e F u r t h e r ,t h e m e d i a a n d a p p a r a t us,a fte r th e r r
u s e m u s t b e a u t o c l a v e d o r d i s i n f e c t e d ,so th a t th e
s p r e a d o f i n f e c t i o n s i s d r a s t i c a l l y r e d u ce ci .

Tissuepieceson
growthsurface

+
I
T h e d e v e l o p m e n t a n d v a r i o u s o t h er a sp e cts o f
Incubateand change o r i n r a r v c u l t u r e a r e d e s c r i b e da b o v e T he te r m ce l l
mediumat line refers to the propagation of culture after the
weeklyintervals first subculture, In other words, once the primary
c u l t u r e i s s u b c u l t u r e d , i t b e c o m e s a ce l l l i n e . A

J
I given cell line contains several cell lineages of
either similar or distinct phenotypes.
t _l-r
rxpranrs
i I I t i s p o s s i b l et o s e l e c ta p a r t i c u l a rc e l l l i n e a g eb y

J
I c l o n i n g o r p h y s i c a l c e l l s e p a r a t i o no r so m e o th e r
selection method Such a cell line derived by
selection or cloning is referred to as cell strain.
C e l l s t r a i n s d o n o t h a v e i n f i n i t e l i f e , a s th e y d i e
after some divisions.

FINITE CELL LINES

T h e c e l l s i n c u l t u r e d i v i d e o n l y a l i m i te d n u m b e r
of times, before their growth rate declines arrd they
Freshculture eventually die. The cell lines with limited culture
vessel Iife spans are referred to as finite cell lines. fhe
c e l l s n o r n r a l l yd i v i d e 2 0 t o 1 0 0 t i m e s ( i .e . i s 2 0 - 1 0 0
Fig. 36.4 : Primary explant technique for
p o p u l a t i o n d o u b l i n g s )b e f o r e e x t i n c t i o n Th e a ctu a l
Drimarv culture.
n u m b e r o f d o u b l i n g s d e p e n d s o n t h e sp e ci e s,ce l l
Chapt er36 : P R IMA R Y
CULTURE
AN D C EL LL IN E S 443

Property Finite cell line Continuous cell line

Growthrate Slow I dDt

Modeof growth Monolayer Suspension

or monolayer
Yield Low H i gh
Transformation Normal lmmortal,
tumorigenic
Ploidy (multiple
Euploid of haploid (notan exactmultiple
Aneuploid of haploid
cnr0m0s0mes) chromosomes)
Anchorage
dependence Yes No
inhibition
Contact Yes No
Cloning
efliciency LOW H i gh
Serumrequirement High LOW
Markers Tissue
specific Chromosomal,
antigenic
or enzymatic

lin ea ge diffe renc es , c ult ur e c ondit ions et c . T h e It must be noted that the passagenumber and
h uma n ce lls ge ner ally div ide 50- 100 t im es , wh i l e generation number are not the same, and they are
mu rine ceils di v ide 30- 50 t im es bef or e dv ine totallv different.

NOMENCLATURE OF CELL LINES

CONTINUOUS CELL LI NES
A fe w cells in c ult ur e m av ac ouir e a dif f e r e n t
mo rph olo gy an d get alt er ed.Suc h c ells ar e c apa b l e
o f gro wing fa s t er r es ult ing in an indepen d e n t
culture. The progeny derived from these altered
cells h as un limit ed lif e ( unlik e t he c ell s t r ainsf r o m
wh ich th ey or iginat ed) . They ar e des ignat e d a s
continuous cell lines. fhe continuous cell lines are
transformed, immortal and tumorigenic. The
tra nsforme dcells f or c ont inuous c ell I ines m ay b e
o bta ine d from nor m al pr im ar y c ell c ult ur es( or c e l l s
strain s)by trea t ingt hem wit h c hem ic al c ar c inog e n s
o r by infe ctin g wit h onc ogenic v ir us es .
In the lable. 36.1,' the different properties of
fin ite cell line s and c ont inuous c ell lines a r e
co m0a reo .
It is a common practice to give codes or
d e s i g n a t i o n st o c e l l l i n e s f o r t h e i r i d e n t i f i c a t i o n .F o r
i n s t a n c e ,t h e c o d e N H B 2 - 1 r e p r e s e n t st h e c e l l l r n e
from normal human brain. followed bv cell strain
(or cell line number) 2 and clone number l. The
u s u a l p r a c t i c e i n a c u l t u r e l a b o r a t o r yi s t o m a i n t a i n
a log book or computer database file for each of
the cell Iines.
Wh i l e n a m i n g t h e c e l l l i n e s , i t i s a b s o l u t e l y
n e c e s s a r yt o e n s u r e t h a t e a c h c e l l l i n e d e s i g n a t i o n
is unioue so that there occurs no confusion when
reports are given in literature.Further,at the time of
publication,the cell line should be prefixedwith a
code designatingthe laboratory from which it was
o b t a i n e d e . g . N C I f o r N a t i o n a l C a n c e r I n s t i t u t e ,Wl
for Wistar Institute.

Th e most co m m only us ed t er m s while dea l i n g lines

Gommonly used cell
with ce ll lin es ar e ex olained below.
There are thousands of cell lines developed
Split ra tio : The div is or of t he dilut ion r at io o f a from different laboratoriesworldover. A selectedlist
ce ll cultu re at s ubc ult ur e For ins t anc e,when e a c h of some commonly used cell lines along with their
su bcultu red ivid ed t he c ult ur e t o half , t he s plit r a t i o o r i g i n , m o r p h o l o g y a n d o t h e r c h a r a c t e r sa r e g i v e n
isl:2 . in Table. 36.2.

Passagenumber : lt is the number of times that SELECTION OF CELL LINES

the cultu re h as been s ubc ult ur ed.
Several factors need to be considered while
Ceneration number : It refers Io the numher of selecting a cell line. Some of them are briefly
doublings that a cell population has undergone. described.
 444 B IOTE C HNO LO CY

Tnslr 36.2 A selectedlist of commonlyused cell lines

Cell line Speciesof origin Tissueof origin Morphology Ploidy Characteristics

rMR-90 Human LUng Fibroblast Diploid to human
Susceptible viral
infections.
3T3-A31 Mouse Connective
tissue Fibroblast Aneuploid Contact readily
inhibited,
transformed
BHK21.C13 (Syrian) Kidney
Hamster Fibroblast 4.levplqtq I'a.tl9lTi9le.
lgeqtrv_
cHo.k1 hamster Ovary
Chinese Fibroblast lsrretvPe
PrPleig9lrPle
NRK49F Rat Kidney Fibroblast Aneuploid Induction
of suspension
growtl
w l-919,
P, ,
BR L 3 A Rat LIVC T Diploid Produces
IGF-2
Vero Monkey Kidney Aneuploid Viralsubstrate
andassay
HeLa-S, Human Cervical
carcinoma Aneuploid Rapidgrowth,highplating
efficiency
S K/H EP -I Human Hepatoma Vlll
EndothelialAneuoloid Factor
It C a c o -2 Human carcinoma Epithelial Aneuploid Formstightmonolayer
Colo-rectal
rli withpolarised
support.
M C F .7 Human EpirhelialAneuploid Estrogenreceptorpositive.
d F ri e n d Mouse SuspensionAneuploid Hemoglobin,growth
n0rm0ne.
u
|m
1 . Sp e c i e s: In g e n e ra l ,n on-humancel l l i nes o Saturationdensity (yield per flask)
h a v e -l e s s ri s k o f b i o h a z a rds,hence preferred. .
Cloning efficiency.
However,speciesdifferences needto be takeninto
accountwhile extrapolating the data to humans 6 . S t a b i l i t y : T h e s t a b i l i t y o f c e l l l i n e w i th
particular reference to cloning, generation of
2. Finiteor continuouscell lines: Cultureswith adequate stock and storage are important.
c o n ti n u o u sc e l l l i n e s a re p re f erredas they grow
faster,easy to clone and maintain,and produce 7 Phenotypic expression: lt is important that
h i g h e r y i e l d . Bu t i t i s d o ubtful w hether the t h e c e l l l i n e s p o s s e s sc e l l s w i t h t h e r i g h t p he n o typ i c
c o n ti n u o u s c e l l l i n e s e x p re ss the ri ght and exoressron.
appropriatefunctionsof the cells.Therefore,some
w o rk e rss u g g e sth t e u s eo f fi n i tecel l l i nes,al though MAINTENANCE O F C E L L C U L T U R ES
i t i s d i ffi c u l t. For the routine and good maintenance of cell
3. Normal or transformed cells : The l i n e s i n c u l t u r e ( p r i m a r y c u l t u r e o r s u b c ul tu r e )th e
transformed cells are preferred as tlrey are examination of cell morphology and the periodic
d n d g ro w ra p i d l y.
i mmo rta l i z e a change of medium are ver)' important.

4 . Av a i l a b i l i ty: T h e re a d y avai l abi l i tyof cel l C e l l m o r p h o l o g y : T h e c e l l s i n t h e c u l tu r e m u st

l i n e s i s a l s o i mp o rta n t.S o m eti mes,i t may be b e e x a m i n e d r e g u l a r l yt o c h e c k t h e h e a l t h sta tu so f
n e c e s s a ryto d e v e l o pa p a rti cul arcel l l i ne i n a the cells, the absence of contamination, and any
laboratory. other serious complications (toxins in medium,
i n a d e q u a t e n u t r i e n t se t c ) .
5. Growth characteristics: The followine
growth parameters
need to be considered. Replacement of medium l Periodic change of
t h e m e d i u m i s r e o u i r e d f o r t h e m a i n t e n a n ceo f ce l l
r P o p u l a ti o n
d o u b l i n gti m e
l i n e s i n c u l t u r e , w h e t h e r t h e c e l l s a r e p r ol i fe r a ti n g
o A b i l i ty to g ro w i n s u s p e n s i on or non-proliferating. For the proliferating cells, the
 A N D C EL LLIN E S
CULTURE
Chaot er36 : P R IM AR Y 445

medium need to be changed more frequently when

compared to non-proliferating cells. The time
inte rva l be twee n m edium c hanges depends on t h e
fi7
rate of cell growth and nretabolism For instance,
fo r ra pid ly gro wing t r ans f olm ed c ells ( e. g HeL a ) ,
the medium needs to be changed twice a rn6
"veek, -
E '.
wh ile for slow ly gr owing non- t r ans f or m ed ce l l s
a
(e.g . IMR-90 ),the m edium m ay be c hanged onc e a o
wee k. Furth er, f or r apidly pr olif er at ing c ells , t h e
105 Lag
su bcultu ring h as t o be done m or e f r equer r t lyt h a n
for the slo wly g r owing c ells .

The following factors need to be considered for

the replacement of the medium. 68

1 . Ce ll co ncent r at ion : The c ult ur es wit h h i g h

cell con ce ntra t ion ut iliz e t he nut r ient s in t h e Fig. 36.5 : Depiction of a standard growth of cells in
med ium faster than t hos e wit h low c onc ent r ati o n , a culture (Note : the cell concentration is
he nce th e me di um is r equir ed t o be c hanged m o r e exqressed in semilog Plot).
freouentlv for the former

2 A de cre as e in pH : A f all in t he pH of t h e propagationof a cell line. The term B?ssage

n red ium is a n indic at ion f or c hange of m ediu m
Mo st o f the ce lls c an gr ow opt im ally at pH 7. 0, a n d
the y a lmost stop gr owing when t he pH f alls t o 6 . 5 .
A fu rthe r dro p in pH ( bet ween 6. 5 and 6. 0) , t h e
cells may lose t heir v iabilit y . The r at e of f all in p l l
is ge ne rally es t im at ed f or eac h c ell line wit h a
cho se n me diu m . lf t he f all is les st han 0. 1 pH u n t t s
per day, there is no harm even if the medium is not
immed iate ly changed But when t he f all is 0. 4 p H
un its p er d uy , m edir - r m s hould be c han g e d
immed iate ly.
number is usedto indicatethe numberof times a
cul turehas beensubcul tured.
The standardgrowth curve of cells in a culture
is depicted in Fig. 36.5. The initial lag phase rs
or l og phaseand a pl ateau
fol l ow edby exponenti al
phase.Duringthe activegrowth period in the log
phase,the mediummustbe changedfrequently, or
else growth ceases.As the cell concentration
exceedsthe capaci tyof the medi um,the cul ture
hasto be di vi dedto subcul ture.

3. Ce ll typ e : Em br y onicc ells , t r ans f or m edc e l l s There are two types of subcultures-monolayer
an d co ntin uo us c ell lines gr ow r apidly and r eq u i r e subcul ture subcul ture.
and suspensi on
more freq ue nt subc ult ur ingand c hange of m edi u m
Th is is in co nt r as t t o nor m al c ells , whic h g r o w MON OLA Y E R C U LTU R E S
slowly. When the bottom of the culture vessel is
4. Morphological changes:Frequentexaminatton covered with a continuous layer of cells, usually
o f ce ll mo rph ology is v er y im por t ant in c ul t u r e one cell in thickness,they are referred to as
techniques. Anv deterioration in cell morphology monol ayer cul tures.The attachment of cel l samong
may lead to an irreversibleciamage to cells. Change themselves and to the substrate(i.e. culturevesser,l
of the medium has to be done to completely avoid is mediated through surface glycoproteins
(cell
the risk o f cell dam age. adhesi onmol ecul es) and cal ci um i ons (C az* )'For
subcul turi ng of monol ayercul tures, i t i s usual ly
necessary to removethe mediumand dissociate the
cel l s i n the monol ayerby degradi ngthe cel l
adhesi onmol ecul es, besi desremovi ngC az+ .

Subculture(or passage) refersto the transferof

Methods of cell dissociation
cells from one culture vessel to another culture
yesser.Subcultureusually(not always)involvesthe There are physical and enzymatic methodsfor
s ubdiv is ionof p ro l i fe ra ti ncge l l s th a t e n a b l esthe of monol ayercul tures(Tabl e.36' 3\'
di ssoci ati on
 446 B IOTE C HNO LO CY

subcultureof each cell line. For a majorityof cell

cultures,the mediumchangeis usuallydone after
methods for monolayercultures 3-4 days,and subculturingafter 7 days.

Method Applicable to Purposeof subculture: The purposefor which

the cells are required is another important
Physical
of subculturing.
criteriafor consideration Cenerally,
Mechanical
shaking Looselyadherentcells if the cells are to be used for any specialized
Scraping Cellssensitive
to purpose, they have to be subcultured more
nrniatcaq
frequently.
Enzymatic
Trypsin Mostof thecontinuous Techniques of monolayer subculture
celllines The subcul tureof monol ayercel l s b asically
Trypsin+ collagenase andmultilayer consistsof the following steps(Fig. 36.6)
Dense
cullures 1. R emovalof the medi um
Dispase Removal in
of epithelium 2. Briefexposureof the cells to trypsin.
sneets
Removal of trypsin and dispersion in a
Pronase suspensions 3.
Single-cell
medi um.
4. lncubationof cells to round up.
M e c h a n i c aslh a k i n ga n dc e l l s c ra pi ng
areempl oyed
5. Resusoension of the cells in a medium for
for cultureswhich are looselyadhered,and the use
counti ngand reseedi ng.
of proteaseshas to be avoided. Among the
enzymes, trypsin is the most frequently used. For 6. Cells reseededand grown to monolayers.
ce rta i n c e l l mo n o l a y e rs , w h i ch cannot be Cell concentrationat subculture : Most of the
d i s s o c i a te db y try p s i n , o th e r enzymessuch as at a seeding
conti nuouscel l l i nesare subcul tured
pronase,dispaseand collagenase are used.Priorto concentrationbetween1 x 104and 5 x 104 cells/
cell dissociationby enzymes,the monolayersare ml. Howeverfor a new culture,subculturehas to
i usuallysubjectedto pretreatment of EDTAfor the be startedat a high concentrationand gradually
removal ol Ca2+. reduced.
t
Criteria for subculture of moriolayers S U S P E N S ION C U LTU R E S
The subculturingis ideallycarriedout between Majority of continuous cell lines grow as
the middle of the log phaseand the time before monolayers.Some of the cells which are non'
they enter plateauphase(Fig.36.6).Subcultureof adhesivee. g. cells of leukemiaor certain cells
cells should not be done when they are in lag which canbe mechanically kept in suspension,can
phase.The otherimportantcriteriafor subcultureof be propagatedin suspension.The transformedcells
monolayersare brieflydescribed. are subculturedby this method. Subcultureby
Culturedensity: lt is advisableto subculture the suspensionis comparableto culturingof bacteria
normalor transformed monolayercultures,as soon or yeast.
as they reachconfluence.Confluencedenotesthe
culturestagewhereinall the availablegrowth area Advantages of cell propagation by
i s u ti l i z e da n d th e c e l l s m a k e cl osecontactw i th suspension
each other. . The processof propagationis faster.
Medium exhaustion: A drop in pH is usually . The lag period is usuallyshorter.
a c c o mp a n i ebdy a n i n c re a s e
i n c ul turecel l densi ty. . of cells.
suspensi on
R esul tsi n homogeneous
Th u s , w h e n th e p H fa l l s , th e medi um must be
changed,followed by subculture. o Treatment with trypsin is not required.
. S cal e-upi s conveni ent.
Scheduledtimings of subculture : lt is now
possibleto have specifiedscheduletimings for . No needfor frequentreplacement
of the medium.
Chapt er36 : P R IMA R Y AN D C E L LL IN E S
CULTURE 447

Confluent
monolayercells

Cellsrounding
Resuspended up
cells

Fig. 36.6 : TEchniqueof monolayersubculture.

r Maintenanceis easy.
o B ulk pr oduc ti o no f c e l l s c a n b e c o n v e n i entl y
ac hiev ed. The cells that retain their proliferative capacity
throughout life are regarded as sfem cells. When
Criteria for suspension subculture the stem cells divide, they can generate
The criteriaadopted for suspension subculture differentiatedcells and/or some more stem cells.
are the same as that already described for Thesestemceilsare capableof regenerating tissue
m onolay ers ub c u l tu re(Se
s ep . 4 4 6 ).T h e fo l l o wi ng after injury. The lack of tissue-specific
asDectshaveto be considered. differentiationmarkersis a characteristic featureof
stemcel l s.
o Culturedensity.
. pH changerepresenting mediumexhaustion. E MB R Y ON IG S TE M (E S I C E LLS

. S c hedulet imi n g so f s u b c u l tu re . A s the embryoni cdevel opment occurs,cel l sof

the inner massof embryo (i.e.thosecontributingto
r Purooseof subculture. future fetus)representembryonicstem (ES)cells.
They conti nueto di vi de and remai n i n an un-
Technique of suspension culture differentiated to totipotentstate.lt hasbeenpossible
and mai ntai ncel l l i nesfor E Scel l s.The
to establ i sh
The cellscan be suspendedin a cultureflask(a
ES cells isolatedfrom mouse blastocystare the
s t ir r er f las k ) c o n ta i n i n g th e d e s i re d m e d i um most commonly used in the laboratory.
( F ig, 36. V T . he me d i u mi s c o n ti n u o u s lsyti rre dw i th
a magneticpendulum rotatingat the baseof the The mostwidely usedembryonicstemlinesare
f las k .T he c ells h a v eto p e ri o d i c a l l ye x a mi n e dfor the vari ous3T3 l i nes,W l -38, MR C -5 and other
contaminationor signsof deterioration. human fetal lung fibroblasts.
 444 B IOTE C HNO LO CY

MAINTENANCE OF STEM CELLS

IN CULTURE

T h e b a s i c c r i t e r i a t o m a i n t a i n s t e m c e l l i n vi tr o
is to ensure that they possess the same
Stirrerflask
c h a r a c t e r i s t i c sa n d d i f f e r e n t i a t i n g a b i l i ti e s w h e n
they are present in the tissue in vivo. fhe
maintenance of epidermal and non-epidermal
e p i t h e l i a l c e l l s i n t h e i n v i t r o c u l t u r e s i s b r i e fl y
loaa
alo described.
. a .. Cellsin
-aoa
aa
suspensron
o ro a Epidermal stem cells in culture
t ._---{t Rotating
. Y. pendulum The epidermal stem (or keratinocyte stem) cells
a//\a
c a n b e s u c c e s s f u l l y m a i n t a i n e d b y c o - cu l tu r i n g
w i t h 3 T 3 f e e d e r l a y e r . B y t h i s t e c h n i qu e , i t i s
p o s s i b l et o a c h i e v e l o n g t e r m m a i n t e n a n ceo f ce l l s,
b e s i d e s r e t a i n i n g t h e i r c a p a c i t y fo r b o th
Fig. 36.7 : Suspension culture in a stirrer flask. p r o l i f e r a t i v e a n d d i f f e r e n t i a t i n g c h a r a c te r i sti cs.l t
has been demonstratedthat the so maintained stem
||i c e l l s w h e n p l a c e d i n t o n u d e m i c e c ou l d fo r m
s Advantages of ES cells
stratified and differentiatedepithelium.
;
In general, the cultures fron'r enrbryonic tissues
Serum-free meciia rvith added growth factors
t survive, and proliferate better than those from the
w e r e f o u n d t o b e m o r e e f f i c i e n t i n n r a i n ta i n i n gth e
adult . This is due t o t he f ac t t h a t E S c e l l s a r e l e s s
eoidermal stem cells in culture.
s pec ializ edwit h higher pr olif e r a t i v ep o t e n t i a l .

Epithelial cells in culture

Limitations of ES cells
S e v e r a l t y p e s o f n o n - e p i d e r m a l e p i th e l i a l
In some cases,the EScells will be different from
c e l l s c a n b e g r o w n a n d m a i n t a i n e d i n cu l tu r e s.
t he adult c ells , and t hus t her e i s n o g u a r e n t e et h a t
A s i n t h e c a s e w i t h e p i d e r n r a l s t e m c e l l s, u se o f
they r,r,illmature to adult-type cells. Therefore, it is
feeder layer is advantageous for epithelial cell
necessaryto characterize the cells by appropriate
c u l t ur e .
methods.
E p i t h e l i a l c e l l s o f p r o s t a t e g l a n d h a ve b e e n
EPI THELI AL STEM CELLS s u c c e s s f u l l yg r o w n i n s u s p e n s i o n c u l t u r e s i n th e
The epit helial c ells ( e. g. epi d e r m i s l i n i n g o f g u t ) presenceof 3T3 feeder layer. The same method is
are constantly being shed from their outer surface. a l s o u s e d f o r c u l t u r i n g h u m a n b r e a s te p i t h e l i a l ce l l s
This c ellular los s is c om pens a t e db y a c o n t i n u o u s and colorectal carcinoma cells.
replacement process The replacement occurs in a
highly or ganiz ed and a r egula t e df a s h i o n . CHARACTERIZATION OF STEM C EL L S

I t is es t im at ed t hat in hum a n s t h e e n t i r e o u t e r lmmunological techniques are widely used for

lay er of s k in is s hed daily T h e e n t i r e e p i t h e l i a l the characterizationof different populations of stem
lining of t he m ous e gut is r e p l a c e d o n c e i n 3 - 4 c e l l s . T h e s e t e c h n i q u e s a r e m o s t l y ba se d o r l
day s . This pr oc es s of s heddi n g a n d r e p l a c e m e n t i m m u n o c y t o c h e m i s t r yu s i n g f l u o r e s c e n tmi cr o sco p y
c ont inues t hr oughout lif e. o r s t a i n i n g t e c h n i q u e i n v o l . , r i n gc o l o u r r e a cti o n s.

The epidermis of the skin has a prolit'erative T h e c e l l s o f t h e t i s s u e s p r o d u c e s p e ci fi c ce l l

c om par t m ent c ont aining s t em c e l l s a n d p o s t -
m it ot ic c ells , bes ides s om e t r a n s i t a m p l i f y i n g
populat ion of c ells . The t r an s i t a m p l i f y i n g c e l l s ,
produced from stgm cells with limited life span are
s hed f r om t he eoider m is .
surface and cytoplasmic proteins. The cell surface
p r o t e i n s s u c h a s i n t e g r i n sa n d t h e m e m be r s o f C D
( c l u s t e r o f d i f f e r e n t i a t i o n ) a n t i g e n s ( e g . C D .,o ,
C D 3 1 ',C D 4 4 ) c a n b e u s e d a s m a r k e r s o f e p i th e l i a l
cell types. Further, the cytoplasmic proteins of
 Chaot er36 : P R IM AR Y A N D C EL LL I N E S
CULTURE 449

epithelialcells(of cytokeratin
family)arealsouseful Applications of tissue
for their identification. specific stem cells

Stem cells, isolatedfrom different tissuesof

APPLICATIONS OF CULTURED
humansand animals,and culturedin vitro are less
S T E M CE LLS
toitpotentthan EScells. They usuallydifferentiate
Embroynic stem cells in tissue repair into a single cell type and are referredto as
The culturestemcellscan be usedfor the repair unipotent. However,stem cells from bone marrow
of tissueswith functional impairmentthat may and brain are capable of forming differentcell
occur due to damage or ageing. The cultured typesthoughto a lesserextentwhen comparedto
em br y onic s t em c e l l s c a n b e ma n i p u l a te dto E Scel l s.In mousel acki ngbone marrow w , hen the
produce cultures characteristicof a particular cul tured neuronal cel l s are pl aced,they devel op
tissue.Thus,thereexistsa possibility
of treating the i nto bl ood cel l s.
followingdiseases. Tissuespecificculturestemcellsare usedfor the
wit h p a n c re a tiicn s u l i np ro d u c i n gc e l l s. fol l ow i ngpurposes.
r Diabet es
r P ar k ins on' sdi s e a s ew i th c u l tu re d d o p a m i ne- . In surgi calrepai rand ti ssuegrafti ng.
pr oduc ingneu ro n s .
. In genetherapy.
E m br y onic s te m c e l l s a re u s e fu l fo r th e
productionof definedtransgenic animals.lt is also Some rnoredetailscln EScells, with particular
possibleto modifyEScell genomeby genetargeting referenceto stem cel l engi neeri ng, are gi ven i n
using in vlfro transformation and selection. Chapter40.

Biotechnology [29]
 n t he l. r bor at or iess, m all s c al e c u l t u r e s o f c e l l s r n maintenance of cells in culture. A good
I
l f las k s ( us ually 1- 5 lit r e v olu m e ) a r e d o n e f o r u n d e r s t a n d i n go f t h e s e p a r a m e t e r s ,l i s t e d b e l o w r s
? es t ablis hingt he c ell lines . Suc h c e l l c u l t u r e s a r e a l s o n e c e s s a r yf o r s c a l e - u p
* us ef ul f or s t udy ing t he m o r p h o l o g y , g r o w t h , . Cell quantitation.
m et abolis m et c . Lar ge- s c alec u l t u r e s a r e r e q u i r e d
e Equipmentand medium.
f or s em i- indus t r ial( 100- , 1, 000| c a p a c i t y ) a n d l a r g e -
scale industrial (5,000-20,000 | capacity) use of . pH and buffer systems.
cells for production of wide range of biologically . Oxygen
important compounds (e g. enzynres, antibodies, . Crowth kinetics.
hormones, interferons, plasminogen activator,
o Typesof culture processes.
i nt er leuk i ns ) .
. Other practical considerations.
The terms fermenter and bioreactor are in
common use while dealing with the industrial use
CELL QUANTITATION
of cells. A fermenter usually refers to the
c ont ainm ent s y s t em f or t h e c u l t i v a t i o n o f T h e t o t a l n u m b e r o f c e l l s i n a c u l t u r e ca n b e
pr ok ar y ot icc ells ( bac t er ia,f ung i ) ,w h i l e a b i o r e a c t o r m e a s u r e d b y c o u n t i n g i n a h a e m o c y t o me te r l t i s
gr ows t he euk ar y ot ic c ells ( m a m m a l r a n ,i n s e c t ) . h o w e v e r , n o t p o s s i b l e t o i d e n t i f y t h e v ia b l e a n d
Scale-up refers to the process of developing the non-viable cells by this method
culture systems in stages from a laboratory to the C e l l v i a b i l i t y : T h e v i a b i l i t y o f c e l l s ca n b e
industry. Scale-up although tedious, labour detected by use of dyes e g. tryphan blue. The
int ens iv e and ex pens iv e, is r e q u i r e d f o r t h e p r i n c i p l e i s b a s e d o n t h e f a c t t h a t t h e d ye i s
pr oduc t r on of c om m er c ially im p o r t a n t p r o d u c t s . p e r m e a b l e t o d e a d c e l l s w h i l e t h e v i a b l e ce l l s d o
For a bet t er under s t andingo f s c a l e - L r p c, e r t a i n not take up dye
bas ic and f undam ent al c onc e p t s o f c e l l c u l t u r e lndirect measurements for cell viability : The
s hould be c lear v i a b i l i t y o f c e l l s c a n b e m e a s u r e d b y th e i r
metabolic activity Some of the most commonly
used parametersare listed
. Clucose utilization
. Oxygen consumption.

There are several parameters that need to be r P y r u v a t ep r o d u c t i o n .

consideredfor appropriategrowth, p r o l i f e r a t i o na n d . Carbon dioxide formation.

450
Chapt er37 : A N IN 4 A LC EL LC U L T U R E -C E NE R A C
L ON S ID E R A TION
ASN D S C A LE -U P 451

In recentyears,many laboratories hal'e started The commonly used buffer of Ihe in vitro culture
measuringthe activity of lactate dehydrogenasecarbon dioxide-bicarbonatesystem(2-5% CO, with
( LDH) t o det e c tc e l l v i a b i l i ty .D e a d c e l l s re l ease 1 0 - 2 5 m M N a H C O 3 ) i s c o m p a r a b l e t o t h e b l o o d
LDH and therefore,this enzyme can be used to buffer. The presenceof phosphatesin the medium
quant it at iv elm
y e a s u reth e l o s so f c e l l v i a b i l i ty improves the buffering capacity. Some laboratories
u s e H E P E Si n s t e a do f b i c a r b o n a t ef o r m o r e e f f i c i e n t
E O UI P M E NT A N D ME D IU M buffering.

T he v ar iou sa s p e c tso f e q u i p me nat n d m e d i um As glucose is utilized by the cells, pyruvic acid

usedin culturelaboratorvaredescribedin Chaoters a n d l a c t i c a c i d a r e p r o d r - r c e dw h i c h c a n a l t e r t h e
33 and 34. pH. lf fructose and galactose (instead of glucose)
are used, the acid formation is less, but the cell
Culturevessels: The materialsmadeup of glass growth is reduced.
or stainlesssteel are commonly used for cell
cultures.Borosilicate glass(e.g.Pyrex)is preferrecl
as it can better withstand autoclaving for OXYGEN
s us pens ion
c ul tu re sw, h e re i nc e l l a tta c h m e ntot the Oxygen has to be continuously supplied to the
surfacehas to be discouraged; the culturevessels medi umthroughout the l i fe of the cul ture.Thi shas
ar e us uallyt r e a te dw i th s i l i c o n e(s i l i c o n i z a ti on). to be done w i thout causi ngdamageto the cel l s.
Oxygencan be suppl i edto the cul turesi n one of
Mediumand nutrients: Appropriateselectionof
the fol l ow i ngw ays.
t he m edium i s d o n e b a s e d o n th e n u tri ti onal
requirements,and the purpose for which the Surface aeration : In closed system static
culturedcells are required.Eagle'sbasalmedium cul tures,the headspace i s usedfor the suppl yof
and minimal essential medium are the most oxygen.For i nstance, i n a.l l i trefl askw i th 100 ml
commonlyused.The mediamay be supplementecl medi um 900 ml of the spacecontai ni ngai r has
wit h s er um . about O.27 g of 02. Thi s O, i s capabl e of
supporti ng108 cel l sfor about450 hours
Additionalfeedingof certain nutrientsis often
r equir ed as t h e y a re q u i c k l y u ti l i z e d a n d get Sparging: The processof bubbling gas through
ex haus t ed. T he s ei n c l u d eg l u c o s e g , l u ta m i n eand the culture is referredto as sparging.This is an
cystine. efficientmeansof O, supply,but may oftendamage
the cells due to effectsof the bubble on the cell
For suspensioncultures, media lacking calcium
membranesurfaces U se of hi gher ai r bubbl es
and magnesium are used since their abserrce
mi ni mi zesthe damagi ngeffect
minimizesthe surfaceattachment.
Membranedi ffusi on: A dequatedi ffusi onof
Non-nutrient medium supplements: Certain
oxygen into the culture can be obtainedthrough
non-nutrientcomooundsare often added to the
si l i conetubi ngw hi ch i s hi ghl ypermeablto e gases
m ediumf or imp ro v e m e not f c e l l c u l tu re sSo di um
This approachhowever,is inconvenient,besides
c ar box y m et h yc le l l u l o s ea d d i ti o nto me d i u mh el ps
the hi gh costof si l i conetubi ng.
t o m inim iz em e c h a n i c adl a ma g eth a t ma y o ccur
due to forcedaerationor the forcesgeneratedby Medium perfusion: The medium is perfused
s t ir r edim pell e r.P o l y g l y c o (tra
l d en a me Pl u r oni c through an oxygenationchamberbefore it enters
F - 68)in t he m e d i u mre d u c e fo s a mi n gi n s ti rre dand the culturesystem.This methodensuresgood O,
aeratedcultures. saturati on.Medi um oerfusi oni s i n fact used rn
glassbead systemand microcarriersystems.
pH A ND B U F F ER S YS T EMS
The idealpH for animalcell culturesis around GR OW TH K IN E TIC S
7. 4. A pH be l o w 6 .8 i n h i b i tsc e l l g ro w th . The The standardpatternof growthof culturedcells
factorsthat can alterpH includethe stabiiityof the followsa lag phase,ar.rexponential(log)phaseand
medium, type of buffer and its buffering capacity, a stationaryphase.For more details,referChapter
concentration of glucoseand headspace. 35 and Fig. 35.5.
 452 B IOTE CHNO LO CY

proliferationof cells,besidesprolongingtheir life.

The other tulture processes describedbelow are
the modifiedbatch cultures.

Fed batch culture

Thereis a gradualadditionof freshmediumso

I
'6
that the cell proliferationis much higherthan the
batch culture.Thus, in the fed batch culturethere
is an increasein the volume of culture.
o
Semi-continuous batch culture
o
A portionof the culturemediumis intermittently
replacedwith an equal volume of fresh medium.
The growth patternof the cells is fluctuating,with
a rapid increase in" cell density after each
replacement of the medium.

perfusion culture
ol differentcultureprccesses. Continuous
Fig. 37.1: Comparison
'':
Thereis a continuousadditionof the mediumto
the cultureand a withdrawalof an equalvolumeof
Gro w th o f c e l l s u s u a l l ymeansan i ncreasetn used bell-freemedium.The continuousperfusion
cell numbers.However,increasein cell massmay processmay close or open for circulationof the
o c c u r w i th o u tre o l i c a ti o n . medi um.
The following terms are in common use to Continuous-flow cultute
growth of culturedcells.
represent
In the continuous-flowculture, a homeostatic
Specific growth rate : The rate of cell growth condi ti onw i th no change i n the cel l num ber s,
p e r u n i t a mo u n to f b i o m a s s . nutrientsand metabolitesis attained.This is made
i Doubling time : The time required for a possible by a balancebetweenthe additionof the
populationof cellsto double in numberor mass. medium and withdrawal of medium along with
a cells.This is mostlysuitablefor suspensioncultures.
Degreeof multiplication(numberof doublings):
T h e n u mb e r o f ti me s a g i ven i nocul um has OTHER PRACTICAL CONSIDERATIONS
reolicated. Besidesthe parameters describedabove,there
I
{ are severalother practicalconsiderations for in
TYPES OF CULTUBE PROCESSES vitro cultureand scale-up.Someimportantonesare
The differentculture processes and the growth given below.
patternsof cells (represented
by cell density)are Culture surfacearea i The availablesurfaceis
depictedin Fig 37.1. They are brieflydescribeo. important for the cells.to grow. In general, the
culture processes are plannedin such a way that
Batch culture the surfacearea is not a limiting factor.
l n th i ste c h n i q u ew, h e n th e cel l sare i nocul ated Inoculationdensityof cells : As such,there is
i n to a fi x e dv o l u meo f th e me di um,they uti l i zethe no set rule for the densityof inoculation.However,
nutrientsand grow,and simultaneously accumulate inoculationwith high densityis preferredfor better
metabolites. As the nutrientsget exhausted,toxic growth.
waste products accumulate and the cell
Growth phase of cells : Cells in the late
multiplicationceases. Further, the cell densitydrops
exponential (log) phase are most suitable for
due to deathof the cells.
inoculation.The cellsat the stationaryphaseshould
Batch culture is a standardtechnique.Several be avoidedsince they have either prolongedlag
modifications have been made to increase phaseor no growth at all.
Chaot er37 : A N IMA LC E L LC U L T U R E-C EN E R A C AS
L ON S ID E R A TION N D S C A LE -U P 453

Stirring rate of culture : The stirringratesof Sidearm

dif f er ent c ult u re l i n e s a re d e v e l o p e d i n tne Sidearm for
labor at or ieslt. i s u s u a l l yi n th e ra n g eo f 1 0 0 - 500 additionof
rpm for most of the cultures. mediumand cells

Temperatureof the medium : lt is advisableto

warm the medium to 37oC beforeadding to tne
c ult ur e.

t t t
t
a a at
Scale-up involves the development of culture ta
' at a
'
systems in stages from (small scale) laboratory to ta
a a ' a
'
(large scale) industry. The methodology adopted to
t .----l t
increa se the s c ale of a c ult ur e depends on t h e .
pendulum
pro lifera tion of c ells and is br oadly div ided i n t o
two categories.

1. Sca le-u p in s us pens ion.

2. Sca le-u p in m onolay er .
Fig. 37.2 : Diagrammatic representation of a
stirrer flask.

Scale-upin suspension is the preferredmethod Physical palameters

as it is s im pl e r.Sc a l e -u po f s u s p e n s i o n c u l ture o Configuration
of the bioreactor.
pr im ar ilyinv ol v e sa n i n c re a s ien th e v o l u meo f the
. Supplyof power.
c ult ur e.S m all s c a l eg e n e ra l l yme a n sth e c u l ture
capacitylessthan 2 litresvolume (or sometimes5 o S ti rri ngof the medi um.
Iitres).
Ghemical parameters
Stirred suspension cultures . Medi umand nutri ents.
I t is us uallyn e c e s s a ryto ma i n ta i nc e l l s tra insrn . Oxygen.
stirred suspensioncultures,by agitation(or stirring)
of the medium.The stirringof the culturemedium o pH and buffersystems.
is achieved by a fiagnet encased in a glass . Removalof wasteproducts.
pendulumor b y a l a rg es u rfa c ea re ap a d d l e .The
Some of the relevantaspectsof the factorsin
s t ir r ingis us ua l l yd o n e a t a s p e e do f 3 0 -1 0 0 rpm
scal e-uphave al readybeen descri bedunder cel l
This is sufficientto preventsedimentation of cells
cul ture-general (S eep. 450).
consi derati ons
without creatingshearforcesthat would damage
c ells . The most commonly usedtechniquesfor stirred
suspension cultures are briefly described
Statie suspension cultures hereunder.
S om e c ells c a n g ro w i n s u s p e n s i o n c u l tures,
wit hout s t ir r in go r a g i ta ti o no f th e me d i u m ,and S TIR R E R C U LTU R E
form monolayercells. However,staticsuspension A diagrammaticrepresentation of a stirrerflask
c ult ur esar e un s u i ta b l fo
e r s c a l e -u p . is shown in Fig 37.2. The sizeof the stirrerflaskis
in the range of 2-10 litres. lt is fitted with a
FACTORS IN SCALING.UP magnetized rotating pendulum, and two side
For appropriatescale-up, the physical and arms- one for the addi ti onof cel l sand medi um,
chemicalrequirements of cellshaveto be satisfied. and the other for the supplyof COr.
454 BIOTECHNOLOGY

5%co2

u*
"3 *-Filter

Cell
Magne.tized suspensron
rotating
pendulum bath
31'c Medium
Cell

lce water lce water

bath bath
PRODUCTRECEIVER BIOREACTOR MEDIUMRESERVOIR

Fig. 37.3 : Suspension culture in a biostat (chemostat).

The stirrerculturevesselis autoclaved(at 15 Ib/ medium flow can be regulatedby a peristaltic

i n 2 fo r 1 5 mi n u te s ),a n d i s t hen set up as i n pump.B y thi s techni que,i t i s possi bl e
to keept he
Fig. 37.2. The flask is seededwith the culture. cultureconditionsconstantratherthan to produce
l argenumberof cel l s.
Then medium along with an antifoamagent is
added.The flaskis connectedto CO, and stirredat The continuousflow cultures are useful for
a speedof 60 rpm.The flaskis incubatedfor about monitoringmetabolicchangesin relationto cell
2 h o u rs . densitv. However, these cultures are more
susceptible to contantination.
The contents of the small stirrer flask are
transferredto a largeflask and the entire set up is
A IR .LIFT FE R ME N TE R C U LTU R E
restarted.lncubationat 37oC is carried out for
4-7 days.The growth of the cells is monitored The major limitationof scale-upin suspension
daily,and the cellsarecounted.Thereis a tendency cultureis inadequate mixingand gasexchange.For
of the cellsto enterapoptosis,if the concentration small cultures,stirring'of the medium is easy,but
exceeds1 x 106 cells/ml. the problem is with largecultures.The designof
fermentershouldbe suchthat maximummovement
C ON T IN U O U S F L O W C U L TU R E of l i qui d i s achi evedw i th mi ni mum shear t o
damagethe cel l s.
In a continuousflow culture,it is possibleto
keep the cells at a desiredand set concentration, A diagrammaticrepresentationof an air-lift
and maintain.This is carriedout bv a biostator fermenteris depictedin Fig. 37.4. A 5% CO, in air
chemostat (Fig 37.3). is pumpedthroughthe bottom of the fermenter.The
bubblesformed move up to agitateand aeratethe
Continuousflow cultureconsistsof growingthe
cul ture.Thesebubbl escarrya fl ow of l i qu idalong
cells at the mid-logphase,removalof a measured
with them and releaseat the top which goesto the
vqlume of cells, and replacementby an equal
bottom for recycling.
volume of medium. The equipment, specially
designed for this purpose has the facility for It is possibleto continuouslysupplyOr to the
removalof the cells and additionof medium.The culture in this technique.AirJift fermenter culture
flow rate of the medium addition can be technique is suitable for fragile animal as well as
determinedfrom the growthrateof the culture.The plant cells.Thisfermenteris extensively usedin the
 AS
C ON S ID E R A TION
Chapt er37 : A N IMA LC EL LC U L T U R E-C EN E R A L N D S C A LE -U P 455

Gas out Perfusedsuspension culture : This also hastwo

compartments.The cells are kept in a low-voiume
compartmentat high concentration,while the
medium is perfusedin adjacentcompartment. The
productcan be collectedin a third compartment.

Cell
suspensron
in medium
Cells
The monolayer culture are anchbrage-
Bafile dependent. Therefore, for the scale-up of
Air bubble monolayer cultures, it is necessaryto increase the
surface area of the substrate in proportion to the
number of cells and volume of the medium.

As already stated, suspension cultures are

Gas sparging
preferred as they are simple. The advantagesand
disadvantagesof monolayer cultures are listed.

Advantages

. Change of medium and washing of cells easy.

Fig. 37.4 : Diagrammaticrepresentationof airlift
r lt is easy to perfuse immobilized monolayer
, , , , , .fe rfh e n te n r rl r.r,r,
cells.

biotechnologyindustryfor culture capacitiesupto

20,000 litres.

NASA bioreactor
NASA (National Aeronautics SPace
Administration,USA) constructed a bioreactorto
grow the cellsat zero gravityby slowly rotatingthe
chamber (Fig. 37.5). The cells remain stationary
and form three-dimensional aggregatesand this
enhancesthe product formation. In the NASA
Rotatingculture
bioreactor,thereis almostno shearforce;hencethe chamber
cells are not damaged.
As the culture chamberstopsits rotations,the Suspendedcell
sedimentand the medium can be
cell aggregates aggregates
replaced.

O T HE R S Y S T E M S F OR SU SP EN SION
CULT URE
Rotatingchambers: The mixingand aerationof
the culture medium can be achievedby 2 or 3
TI
Fresn- Spent
rotatingchambers.The chambersare so designed medium medium
t hatt he c ell s usp e n s i oann d m i x i n ga re h i g h i n o ne input and productoutpul
chamber rvhile the product and spent medium
,remainin the other chamber.Thesechambersare
separatedby semipermeable membrane.
 456 B IOTE CHNO LO CY

MULTISURFACE CULTURE
The most commonl y used mu lt isur f ace
Roller propagatorof monolayer is Nunclon cell factory
bottle (in short Nunc cell factory). lt is composed of
Cells rectangularpetri dish-likeunits with huge surface
area (1,000-25,000 cm2). The units are inter-
cap
connectedat two adjacentcornersby verticaltubes
(Fig, 37.V. The medium can flow between the
Medium comoartments from one end.
The cell factory is almost like a conventional
petridishor a flaskwith multiplayerunits.
The main limitationof cell factory is that it is
Rollers very difficult to monitor the growth of cells. The
majoradvantage however,is its simpleoperationto
Fig. 37.6 : Roller bottle culture. producel argenumber' ofcel l s.

MULTIARRAY DISKS,
. T h e c e l l p ro d u c t fo rma ti o n (pharmaceuti cal l ySPIRALS AND TUBES
importantcompoundse.g interferon,antibodies) The surface area for growth of monolayer
i s mu c h h i g h e r. cul turescan be i ncreasedby usi ngdi sks,spir alsor
o The samesetup and apparatus can be repeatedly tubes. They are however, not in common use as
usedwith differentmedia and cells. thei r commerci i
al moortance
i s l i mi ted.

Disadvantages MICROCARRIER CULTURE

. Tediousand costly. Monolayers can be grown on small spherical

carriers or microbeads (80-300 pm diameter)
. Requiremore space.
referredto as microcarriers.
The microcarriers
are
. Crowth of cellscannotbe monitoredeffectively. made up of any one the followingmaterials(trade
r Difficultto measurecontrolparameters (Or. pH, namesgiven in brackets).
CO, etc.) . Plastic(acrobeads,
bioplas).
Forscale-upof monolayercultures,a wide range . Class (bioglass,
ventreglas).
of tissueculturesand systemhavebeendeveloped. . Gelatin(ventregel, cytodex-3).
A selectedfew of them are brieflydescribed. . Collagen(biospex,biospheres)
. Cellulose(DE-52/53).
ROLLEB BOTTLE CULTURE r DEAEDextran(cytodexl, dormacell).
A round bottleor tube is rolled around its axis
(b y ro l l e rsa) s th e m e d i u ma l o ngw i th the cel l sruns
aroundinsideof the bottle(Fig.37.6).As the cells
are adhesive,they attachto inner surfaceof the
bottle and grow forminga monolayer.
Roller bottle culture has certain advantages.
. The medium is gentlyand constantlyagitated.
. The surfacearea is high for cell growth.
e Collectionof the supernatant
medium is easy.
Thereare limitationsin roller culture.
I Monitoringof ceils is very difficult. Fig. 37.7 : A diagrammatic representation of
Nunc cel! factorv.
a In v e s tm e ni st ra th e rh i e n .
C hapt er37 : A N IMA LC E L LC U L T U R E-C EN ER A C
L ON S ID E R A TION
ASN D S C A LE -U P 457

- oo o o
5
Water iackel Water
jacket

Glassbeads Glass
OO beads
--+

Mediumand t
I
inoculationinlet
Mediumand
inoculationinlet
(A) Fixed-bed reactor (B) Fluidized-bed reactor

Fig. 37.8 : Fixed and fluidized-bed teactors.

The microbeadsprovide maximum surfacearea PERFUSED MONOLAYER CULTURE

for monolayer cultures.This actually dependson The growth surface areas of the monolayer
the size and densityof the beads.The cells can cultures can be perfused to facilitate medium
grow well on the smoothsurfaceat the solid-liquid replacement and improved product formation and
interface.However, microcarriersneed efficient
lecovery. The perfusion can be carried out
s t ir r ing wit hout g ri n d i n g th e b e a d s . T h e m a i n with pumps, oxygenatorand other controllers.
advantage with microcarrier cultureis that it can be Perfusionof fixed and fluid-bedreactoris brieflv
treated as a suspensionculture for all practical described.
purposes.
Microcarrierscan be cultured in stirrer flasr Fixed.bed reactors
(SeeFig. 37.A or in continuoussuspension(See The fixed-bedreactor has a bed of glassbeads
Fig. 37.3). In fact, the'suppliersof microcarriers (Fig. 37.5A). The medium is perfusedupwards
orovidethe technicalliteratureand other relevant throughthe bed.The cellsaregrownon the surfaces
informationfor settingup a microcarrierculture. of the beads.The oroductscan be collectedfrom
the top along with the spentmedium.
Factors affecting microcarrier culture
r Compositionand coatingof beads(gelatinand lnsteadof glassbeads,porousceramic rnatrix
with microchannelscan also be used in fixed-bed
collagen beads are preferredas they can be
reactors.
solubilisedby proteases).
o Higherstirringspeedis usuallyrequi:'ed. Fluidized-bed reactors
r Class beads are used when the microcarriers ln a fluidized-bed react6r, the beads are
need to be recycled. suspendedin a stream of medium (Fig. 37.8A.
Thesebeadsare porousin nature;and are madeup
Analysis of microcarrier culture of ceramicsor a mixtureof ceramicsmixed with
The cell countingtechniquesare difficultto be naturalproductssuch as collagen.They are of low
usedfor microcarriercultures.The growthratecan densityand float in the medium.The flow rate of
be detectedby analyzingDNA or protein. the perfusedmediumis equalto the sedimentation
458 B IOTE C HNO LO CY

Medium

co2---+
Samplestakenout for
Numberof cells
Monitoring by Productsformed
probes Leftovernutrients
Volume Contaminationcheck
pH {-
vv2
^n
Water jacket
O2
Glucose
Bioreactor
Ternperature
Turbidity

rateof the beads.The cellscan grow as monolayers specialiTed productsformed(e.g.immunoglobulins

on the outer surfacesand inside of the porous by hybri domacel l s).
beads.
The cell proliferationand rate of biomass
Other perfused monolayel cultures formation can also be determinedby estimating
DNA, proteinand ATP.
Membrane perfusion, hollow-fiber perfusion,
matrix perfusion and microencapsulationare The different parametersfor monitoring of a
among the other techniques for perfusion of bioreactorfor suspensionculture are depicted in
mo n o l a y ecr u l tu re s . Fig 37.9.

MONITORING OF MONOLAYER
C U LTU R E S
It is ratherdifficult to monitor monolavercell
culturesfor scale-up.This is due to the fact that in
most of the techniquesemployedfor monolayer
Mcnitoringof the progressof cell growth and cultures,the cells cannot be observeddirectly to
the culturesystemsare very importantin scale-up. monitorthe progressof the culture.
In recent years, nuclear magnetic resonance
MON IT O R IN G OF
(NMR)techniqueis usedto assaythe contentsof
SUSPENSION CULTURES
NMR spectragenerated
culture.The characteristic
The progressof suspensioncultures can be by specificmetabolitesenablesthe identification
monitored in situ by measuringglucose, 02, CO? and quantitationof metabolites,
besidesdetecting
pH or metabolitesproduced (lactate,ammonia)or the progressof cell growth.
 I s the celis are removed from the living (in vivo)
Ae nviron men t and s ubjec t ed t o ex per im en t a l
manipulations in the culture systems(in vitro), Ihetr
viab ility assu mess ignif ic anc e.Viabilit y of t he c e l l s
represents the capability of their existencel
survival and development.
Man y e xp eri m ent sar e c ar r ied out wit h c ells i n
the cu lture ra ther t han us ing t he anim al m ode l s .
Th is is p artic ular ly s o wit h r egar d t o th e
determination of safety and cytotoxicity of several
comp ou nd s (ph ar m ac eut ic alsc,os m ot ic s ,ant ic an c e r
drugs, food additives). ln vitro testing Ior
cytotoxicity and safety evaluation is in fact
cost-effective, bresides reducing the use of
animals. Studies on cytotoxicity broadly involve
the metabolic alterations of the cells, including
the death of cells as a result of toxic effects
of the compounds. For instance, in case of anti-
cancer drugs, one may look for death of
ce lls, wh ile f or c os m et ic s t he m et abo l i c
alterations and allergic responses may be more
rmoortant.
LIMITATION OF V'TBO
CYTOTOXICITY "V
STUDIES
To xicity of a giv en c om pound is a c om pl e x
process as it occurs in vivo. This may result in
d irect da mag e to c ells , alt er at ionsin phy s iologi c a l
a nd b ioche mica l f unc t ions , inf lam m at or y c hang e s
and other systemic effects, not only at the site of
a pp lica tion b ut als o at t he ot her s it es .Som e of t h e
important in vitro limitations are briefly described
with special reference to systemic responses,
metabolism and pharmacokinetics.
Tissue and systemic responses
The cultured cells reoresent the cells in
i s o l a t i o n ,a n d n o t a n i n t e g r a l p a r t o f a t i s s u eo r a n
organ. As already stated, the nature of the in vitro
effect is measured by cell survival or by an
altered metabolic effect. On the other hand,
the in vivo reactions are complicated that may
lead to tissue responses (fibrosis, inflammatory
reaction) or systemic responses (pyrexia,
vascular dilation). Therefore, the in vitro
cytotoxic responses have to be considered
carefully.
In recent years/ some attempts are made to
create in vitro organotypic cultures by assembly of
different cell types. Even with this approach, it is
not possible to observe all the tissue, and systemic
responses.
Metabolism
The whole body metabolism is complex and
well integrated. Some of the compounds that are
toxic in vitro are detoxified by the liver. On the
other hand, certain non-toxic or less toxtc
compounds may be converted to more toxic ones
in the liver. For these reasons, it is necessarythat
the in vitro cells are exposed to the same type of
compound that is formed in liver after
detoxification
460 B IOTE CHNO LO CY

Pharmacokinetics Dye exclusionassay: The principleof this assay

is basedon the factthatviablecellsareimpermeable
When the body is exposedto a drug, there
to severaldyes such as naphthalene black,trypan
occurstissuepenetration,
metabolism, clearance
and
blue,eosinY, nigrosingreenand erythrocinB. The
excretion, constituting an organized pharma-
of mi xi ngth e cells in
techni quebasi cal l yconsi sts
cokinetics.It is not possibleto stimulatethese
suspension with the dye and examiningthem under
parameters in the isolatedcells. However,use o{
the microscopy.The stainedcells and the total
multicellular
tumorspheroids hascertainadvantages
number of cells are counted. The percentageof
with regardto understandingof drug penetration.
unstained cells representsthe viahle cells.
Dye exclusionassayis convenientand suitable
to suspensi oncul turesthan to monol ayer s.
This is
due to the fact that as the dead cellsdetatchfrom
the monolayersthey are lost from the assay.
Despite the limitation stated above, there are The major limitation of this assay is that
several assays developed in the laboratory for reproductivelydead cells do not take up the
measuring the cell viability and cytotoxicity. They dye, and will be counted as though they are
are broadly categorized into the following types. vi abl e.
. Cytotoxicity and viability assays. Dye uptakeassay: The viablecellscan take up
. Survival assays. the dye diacetyl fluoresceinand hydrolyseit to
o Metabolic assays. fluorescein.The latter is held up by the viable
cells, as it is impermeableto membrane.The
. Transformationassays.
viable cells therefore emit fluorescin green while
o Inflammation assays. the deadcel l sdo not.Thus,the vi abl ece llscan be
identified.
CYTOTOXICITY AND
VIABILITY ASSAYS Labeled chromium uptake assay : Labeled
chromi um(s1C r)bi ndsto the i ntracel l ular
pr ot er ns
A majority of the cytotoxicity and viability assays through basi c ami no aci ds. W hen t he cell
are based on the measurement of membrane membraneis damaged,the labeledproteinsleak
integrity, cellular respiration, radioisotope out of the cell, and the degree of leakage is
incorporation,colorimetricassaysand luminescence- proportionalto the amount of damage.Labeled
based tests. slCr uptakemethodis used in the immunological
studies to determine the cytotoxic activity of
Based on membrane integrity T-lymphocytes againsttargetcells.
The most common measurements of cell Enzymereleaseassays: The membraneintegrity
viability are based on membrane integrity. The of cells can also be assessedby estimatingthe
dam age t o m em br ane m ay o c c u r d u e t o c e l l enzymes released. Lactate dehydrogenase (LDhI)
disaggregation, cell separation or freezing and has been the most widelv used enzyme for this
thawing. Membrane integrity can be determined by purpose.
uptake of dyes to which viable cells are
im per m eable ( e, g. napht halen e b l a c k , t r y p a n b l u e , Based on cellular respiration
erythrosin) or release of dyes normally taken up Respirationof the cells measuredby oxygen
and retained by viable cells (e.g. neutral reo, utilizationor carbon dioxide oroductioncan De
diacetvl fluorescein). usedto assess cel l vi abi l i ty.Thi si s usuallydoneby
The other assays for membrane integrity are usingWarburgmanometer.
r eleas eof labeled c hr om ium ( 5 1 C r ) ,e n z y m e s a n d
Based on radioisotope incorporation
us e of f luor es c ent pr obe s . C e l l viability
measurements,based on membrane integrity are By usingradiolabeled substrates
or metabolites,
immediate that can be detected within a few hours. the radiolabel in the oroducts formed can oe
However, these measurementscannot predict the detected.-Thismethodis particularlyusefulfor the
ult im at e s ur v iv al of c ells . cytotoxicityassaysof drugs.Someof the important
Chaoter38 : CELLVIABILITYAND CYTOTOXICITY 46t

radioisotope incorporation methods are briefly o Changesin the morphology.

8 rve n. o Detection of phosphatidyl serine in the
Labeled nucleotides : Incorporation of (3H) membraneby using annexin V conjugatedto
thymidin e in to DNA, and ( 3H) ur idine int o RNA a r e (FITC)or biotin.
fluoresceinisothiocyanate
widely used for the measurementof drug toxicity. o D N A l adderi ng.
labeled phosphate : The cells are prelabeled
with 32P.When the damage occurs to cells, they SURVIVAL ASSAYS
releaselabeled phosphatewhich can be measured. The testsdescribedabove for measurement of
The efficacy of drugs can be evaluated by this cell viability and cytotoxicityare short-term,and
approacn. they identifythe dead/livecellsat the time of assay.
Many times,when the cellsaresubjectedto toxicity
Based on colorimetlic assays (i.e. exposedto drugs, irradiated),the effectsare
not immediate,but may be observedafter several
The recent developments in the colorimetric
hours or sometimes even days. The assays
assays by uslng sophisticated microplate readers
based on the survivalof cells (i.e. retentionof
are fru itfully u t iliz ed f or quant it at ion of c ells . A
regenerative capacityor reproductiveintegrity)are
good correlation between the cell number and
preferred.
colorimetric assayare observed.Some highlights of
this approach are given below.
Glonogenic assay
. Protein content can be estimated bv methvlene
In clonogenicassay,the survivalof the cells is
blue, amido black, sulforhodamine.In the Lowry
measuredhy plating efficiency(i.e. the percentageof
method for protein estimation, Folin-Ciocalteau
cellsseededat subculture that give riseto colonies).
reagent is used.
The plating efficiencymeasuresthe proliferative
o DNA can be quantitated b y s ta i n i n g wi th capacityfor severalcell generations. Clonogenic
fluorescence dyes e.g. 2 -d i a mi n o d i no- assaybroadlyconsistsof the followinBstages.
ph en yli n do ne
1. Treatment of the cel l s w i th varyi ng
. Lysosomal and Golgi body activity by using concentrations
of experimentalagentfor about24
neutral red. hours.
. Enzyme activity assayse.g. hexosaminidase, 2. Trypsinization
followedby seedingof cellsat
mitochondrialsuccinatedehydrogenase. low density.
3. Incubati onof the cel l sfor 1-3 w eeks.
Based on luminescence test
4. S tai ni ngand counti ngof the col oni es.
T hev iabilit yo f c e l l l c a n b e m e a s u rewd i th g o od
A survivalcurve (semilogplot) representing the
sensitivity by estimating ATP levels by
survival fraction of the cells against drug
lum ines c encbea s e dte s t.T h e p ri n c i p l ei s b a s e don
concentration is depicted in Fig 38.1. The
the following reaction.
inhibitory concentration (lC) refers to the drug
A T P+ D -L u c i fe ri n + 0 2 concentrationrequiredto inhibit the viability of
II Luciferase cells. Thus, lCso and lCgo represent the
concentrationsof a compound that respectively
J
inhibit 50% and 90o/oof colony formation.
AMP + ZPi + CO2+ Light
As is evidentfrom the graph,the curve has a
A good sensitivityof this test is reportedfor the kneew herei nl C rol i es,w hi l e l C nofal l si n the l i near
cells in the rangeof 20 to 2 x 107 cells/ml. range. Therefore,the differenceswill be more
si gni fi canti n the l i nearrange.
Based on apoptosis
The clonogenicassayis influencedby several
M os t of t h e a n ti c a n c e rd ru g s k i l l c e l l s by factors,the importantones are listed.
apoptosis which can be measured for the
assessmentof cytotoxicity. Apoptosis can be o Concentration of the toxic agent.
detectedby the following ways. o Durationof exposure.
462 B IOTE C HNO LO CY

o Thesetestare carriedout afterexposureof the cells
I 0 .1
o populationdoublings).
$ n n{
U)
Concentrationof compound ----J
Fig, 38.1 : Survival curve in clonogenic assay.
. C e l l d e n s i tyd u ri n ge x p o s u re.
. C e l l d e n s i tyd u ri n gc l o n i n g .
METABOLIC ASSAYS
The metabolic assays are based on the
measurements of metabolicresponsesof the cells.
to cytotoxicdrugs(eitherimmediatelyor after2-3
The most commonly used metabolic
measurements are DNA, RNA or proteinsynthesis
(by estimatingtheir concentration),besidesthe
assay of certain dehydrogenase enzymes.Some
detailson the estimationof DNA and proteinare
al readygi ven (S eep. 493).
limitationsof metabolicassays: The estimation
of the total contentof DNA proteinmay or may not
be i ndi cati veof i ncreasei n cel l numbe r .This is
becausetheseassayscannotdiscriminatebetween
the proliferativeand metabolic activity of cells.
Some workers, therefoie prefer to confirm the
metabolicmeasurements by colonogenicsurvival
a5say.

. Sizeof colony. TRANSFORMATION ASSAYS

MTT.based cytotoricity assay The followingare the commonlyusedassaysfor

measurement of in vitro transformation.
T h e te tra z o l i u ms a l t 3 , (4 .5-di methyl -thi azol -
2-yl)-2, 5-diphenyl tetrazolium bromide) is . Evidenceof mutagenesis.
commonlyknown as MTT. lt is dye, and is widely . Anchorageindependence.
used in cytotoxicityassays.
. Reduceddensitylimitationof cell proliferation.
The growingcells in the log phaseare exposed
to cytotoxicdrug. The drug is then removedand Mutagenesis
the cells are allowed to proliferate for 2-3 Mutagenesiscan be assayedby sisferchromatid
p o p u l a ti o nd o u b l i n gti me s (P DTs).
The numberof exchange (SCD. SCE basically involves the
surviving cells can be detected by MTT dye reciprocalexchangeof DNA segmentsbetween
reduction. The concentrationof MTT-formazan sisterchromatidsat identicalloci in the S-phase
formedcan be determinedspectrophotometrically.of cell cycle. Sister chromatid exchanges
MTT-basedcytotoxicityassayis carried out in are more sensitive to mutagenesis than
the following stages. chromosomalbreaks.For this reason,SCEsare
preferred in mutagenesis research and
1 . In c u b a ti o n o I mo n o l a yer cul tures w i th
plates.
in microtitration transformationassay.
varyingdrugconcentrations
2. Removalof drug and feeding of plates to The S C E techni que basi cal l y i nvolves t he
achieve2-3 PDTs. incorporation of radioactive nucleotides into
replicating DNA and detection of SCEs by
3. Treatmentof plateswith MTT, and removal fl uorescence pl us Gi emsa(FP C )techni q ue.
of medium and MTT.
4. Measurement of MTT-formazan in an ELISA INFLAMMATION ASSAYS
plate reader.
Inflammationassaysare requiredfor testingthe
When the absorbance of testwellskontrolwells various forms of aller:gyinduced by cosmotics,
of the microplate is plotted against the pharmaceuti caland s other xenobi ot ics These
concentrationof the cytotoxic drug, a sigmoid assaysare at the earlystagesof developmentin the
curve is obtained. cul turecel l s.
 ell transformation due to changes in the
/-
\- e en etic ma ter ial, and c ell c loning inv olv ing
t he rod uction of a populat ion s ingle c ell ar e
p
de scribe d in th is c haPt er .
Transformation broadly refers to the change in
phenotype of a cell due to a new genetic material'
As regaris the cultured cells, transformationinvolves
soontaneous or induced permanent phenotypic
DNA,
alterationsas a result of heritable changes in
and consequentlygene expression'
Transformationof cells may occur due to any
one o f the fo llowin g c aus est hat ult im at ely r es ult i n
a cha ng ed ge ne tic m at er ial'
o Spontaneous
. lnfe ctio n with tr ans f or m ingv ir us '
r From gene transfection'
. Expo su reto ch em ic al c ar c inogens '
. Expo su reto ion iz ing r adiat ions '
of trinsformed cells
Gharacteristics
The general characterso{ transformed cells are
given in Table 39.1. They are grouped as genetic,
structural, growth and neoplastic, and listed'
control and malignancy.Theseaspectsare briefly
descri bed.
GE N E TIC IN S TA B ILITY
, cel l l i nesi n cul tureare proneto
In generalthe
A maj ori tyof normalfi ni te cel l
geneti ci nstabi l i ty.
l i nesare usual l ygeneti cal lstabl
y ew hi l e cel l l i nes
from other species(e.8. mouse) are genetically
unstable,and can get easily transformed.The
conti nuouscel l l i nes deri vedfrom tumorsof al l
speciesare unstable.
i n the
The normal l yoccurri nggeneti cvari ati ons
following causes.
cultured cells are due to the
1. High rateof spontaneous mutationsin the ln
vitro conditions,possiblydue to high rate of cell
prol i ferati on.
2 The continuedpresence of mutantcellsin the
cul ture,as they are not normal l yel i mi nated'
IMMOB TA LIZA TION
The acquisition of an infinite life spanby a cell
is referredto as immortalization.
Most of the normalcells(fromdifferentspecies)
have a finite life span of 20-100 generations. But
some cells from mouse,most of the tumor cells
have infinite life span, as they go on producing
conti nuouscel l l i nes.
Control of finite life span of cells

Transformation is associated with genetic The finite life spanof culturedcells is regulated
immortalization, aberrant growth by about 1O senescence genes.Thesedominantly
instabitity,

463
464 B IOTE CHNO LO CY

productof this gene(T antigen)bindsto senescence

genessuch as R b and ps3.Thi s bi ndi ngr est r ict s
surveillance genes.The result
activityof senescence
is an increasedgenomic instabilityand activity,
Genetic characters
leading to further mutations favouring
Aneuoloid i mmortal i zati on.
Heteroploid
Highspontaneousmutation
rate Forthe processof immortalization, the cellsare
i nfectedw i th retrovi ruses
contai ni ngi mm or t alizing
Overexpressed
oncogenes
genebeforetheyentersenescence. By this way,the
Mutated
or deleted
suppressorgenes
life span of the cells can be extendedby 20-30
Structural characters populationdoublings.Thereafter, the cellsceaseto
proliferate, and entera crisisphasethatmay lastfor
Altered
cytoskeleton
severalmonths.At the end of the crisis phase,a
Changed extracellular
matrix
small portion of cells can grow and eventually
Modifiedexoressionof celladhesion
molecules becomeimmortalized*
Disruptedcellpolarity
lmmortalizationof human fibroblasts : The
Growth characters human f ibroblasts are most successfu llv
lmmortalizedcells immortalizedby the viral genenamelySV4OLT. The
Lossof contactinhibition processof fibroblastimmortalizationis complex
Anchorage independent and indirectwith a very low probabilityi.e. about
I rn -l^u'
a cel l s.
Density of growthreduced
limitation
Growthfactorindependent
lmmortalization of cells by
Lowserumreouirement telomerase-induction
Shorterpopulationdoublingtime
The most importantcauseof finite life spanof
Neoplasticcharacters cel l s (i .e. senescence)i s due to t elom er r c
Tumorigenic shortening,followed by cell death (apoptosis).
Invasive lf the cellsare transfected with telomerasegene
protease
Increased secretion htrt, the life spanof the cells can be extended.And
a smallproportionof thesecellsbecomeimmortal.

actinggenessynthesize productswhich inhibitthe ABERRANT GBOWTH CONTROL

cell cycle progression.
The transformedcellsand the cellsfrom tumors,
It is strongly believed that immortalization grow n i n cul ture show many aberrat ionswit h
occursdue to inactivationof someof the cell cycle respect to growth and its control. The growth
regulatorygenese.g. Rb, p53genes. of thesecell are listedin Table39.1,
characteristics
and someof them brieflydescribedhereunder.
lmmortalization of cells by viral genes
Severalviral genescan be usedto immortalize Anchorage independence
cells.Someof thesegenesare listedbelow.
Thereoccur severalchangeson the cell surfaces
SV4OLT of transformedcells. These include alterations
H PV 1 6 E6 /E 7 in the cell surfaceglycoproteinsand integrlns,
and loss of fibronectin.Some of the transformeo
hTRT
cells may totally lack cell adhesion molecules
Ad 5 E la (CAMs).
EBV.
The modificationson the surfaceof transformeo
Among the above viral genes,SV4OLTis most cel l s l eadsto a decreasei n cel l-cel l , and cell-
commonly used to induce immortalization. The substrate adhesion.The net resultis that there is a
 Chapt er39 : CE L LT R AN SF OR M AT ION
AN D C E LLC LON IN C 465

reducedrequirement for attachmentand spreading Forthe malignanttransformation of cells,several

of the cells to proliferate.This phenomenonis steps may be required. The following two
referredto as anchorageindependence. approachesare in use to understandmalignant-
associated propertiesof culturedcells.
Anchorage independent cells grow in a
disorganized fashion. These cells may be 1. The cel l s can be cul turedfrom mal i gnant
comparablewith the tumor cellsdetachedfrom the tumorsand characterized.
nativetissuewhich can grow in foreigntissuesi.e.
formation of metastases. 2. V i ral genesor chemi calcarci nogens can be
usedto transformthe untransformed cells.
Gontact inhibition
fhe transformedcellsare characterizedby loss
of contact inhibition. This can be observedby the
morphologicalchanges in the disorientedand
disorganizedmonolayer cells. This results in a In the traditionalculture techniques,the cells
reduceddensitylimitationof growth,consequently are heterogenous
in nature.lsolationof pure cell
leadingto higher saturationdensitycomparedto
strainsis often requiredfor variouspurposes.CeIl
normalcells (ReferChapter35 and Fig. 35.6).
cloning broadly involvesthe processesconnected
with the production of a population of cells
Low serum requilement
derived from a single cell. Cloning of continuous
ln general,transformed cellsor tumor cellshave cel l l i nes i s much easi ercomparedto that of the
lower serum dependencethan the normal cells. pri marycul tures,and fi ni tecel l l i nes.
This is mostlydue to the secrectionof autocrine
There are certai n l i mi tati onsfor cl oni ng of
growth factorsby the transformedcells (Note : The
culture cells derived from normal tissues.These
normalcellsin culturearedependenton the serum
cells survivefor a limited numberof generations,
for the supply of growth factors).Some of the
and therefore cloning may not result in any
growth factorsproducedby tumor cells are given.
si gni fi cantnumberof cel l s. On the other hand,
r Colony stimulatingfactor (CSF). cl oni ng of conti nuouscel l l i nes due to thei r
o Transforminggrowth factor (TCFa). transformed status is much easier. Thus, the
transformedcells have higher cloning efficiency
o lnt er leuk ins
1, 2 a n d 3 . comparedto normalcells.
. Vasoactiveintestinalpeptide(VlP).
Cloningmay be carriedout by two approaches-
. Castrinreleasingpeptide. monol ayerand suspensi on cul tures.
It may be noted that many normal cells .l
. Monolayerculture : Petridishes,multiwell
(fibroblasts,endothelialcells) also produce auto- plates or flasks can be used for cloning by
crinefactorsduringactivestageof cell proliferating. monolayerculture.lt is relativelyeasyto remove
Hence, thesefactorswill not be of much use to the individualcoloniesof cells from the surfaces
serveas markersof cell transformation. where they are attached.

T UM O RI G E NI C IT Y 2. Suspension culture : Cloningcan be carried

out i n suspensi on by seedi ngcel l s i nto vi scous
Cell transformationis a complex processthat solutions(Methocel)or gels(agar).As the daughter
often resultsin the formationof neoplasticcells. cells are formed in suspension, they remainintact
The cell linesobtainedfrom malignanttumorsare and form coloniesin susoension.
already transformed.Such cells may undergo
furthertransformation in the in vitro culturedue to
D ILU TION C LON IN G
. Increasedgrowth rate.
D i l uti on cl oni ng i s the most commonl yused
o lm m or t aliz at i o n .
techni quefor cl oni ng of monol ayercel l s, and
o Reducedanchoragedependence. involvesthe following stages(Fig 39.1).

Biotechhology
[30]
466 B IOTE CHNO LO CY

STIMULATION OF PLATING
___t
t---------------
w.r.r.7fifi.m.t
EFFICIENCY

Exponentially(log phase) Plating efficiency representsthe percentageof

growingcells in culture
I efficiencyare said to be identical,if each colony is
I Trypsinization
J
Singlecell suspension
diluted(1G-100cells/ml)
cells seeded at subculture that gives rise to
colonies. The plating efficiency and cloning
derivedfrom a singlecell.
The plating efficiency is around 107" for
continuouscell lines, while for primary cultures
and fi ni tecel l l i nes,i t i s qui te l ow -0.5 t o 57oor
sometimeseven zero. Severalattemptsare madeto
i mprove the pl ati ng effi ci ency i n th e cult ur e
laboratories. Theseapproachesare basedon the
assumptionthat the cells at low densitiesrequire
more nutrientsand/or growth factors.
The stimulationof platingefficiencywith regard
to culture factors, conditioned medium and feeder
layersis brieflydescribed

Multiwelldish Petridish Plasticbottle Culture factors

Medium : A mediumrich in variousnutrientsis
bettersuitedfor improvingthe platingefficiency.

Serum: Fetalbovineserumis betterthan horse

or calf serum,if the additionof serumis required.
Trypsinization Cloningrings Inadiation

COLONIESISOLATED
JJ Addition of metabolites: Supplementingthe
medi um w i th i ntermedi ary metabo lit es ( e. 9.
pyruvate, o-ketoglutarate) and nucleosides
stimulatesplatingefficiency.

Fig. 39.1 : Dilution cell cloning technique for

Addition of hormones : Dexamethasone, a
synthetic analogue of hydrocortisone,improves
platingefficiencye.g. fibroblasts,melanomacells,
chi ck myobl asts.Insul i n al so sti mul a t esplat ing
1. Trypsinizationof cells (at log phase) to efficiencyof severalcell types.
producesinglecell suspensions. Carbon dioxide : CO, significantlyinfluences
.l
2 . D i l u ti o no f th e c e l l sto about 0-100 cel l s/ platingefficiency.
Mostof the cellsrequire5"h CO2
ml. while for some cells, a lower CO, concentration
(around2% CO2) is bettere.g. humanfibroblasts.
3. Seed the cells in multiwell dishes, petri HEPESis used to protect the medium from pH
dishesor plasticbottles. fluctuations.
4. Incubate under appropriateconditionsfor
Pretreatmentof substrate: The platespretreated
1-3 weeks.
with fibronectinor polylysineshow an improved
5 . l s o l a tei n d i v i d u acl o l o ni es. platingefficiency.

The clones can be isolateddirectly from the
multiwelldishes(by trypsinization),
by cloningring
techniquefrom petridishes,and by irradiatingthe
plasticbottle.
Use of purified trypsin : Some workers prefer
to use purified trypsin insteadof crude trypsin
for trypsinizationso that the plating efficiencyis
better.
 ChA P t CT
39 : CE LLT R AN SF OR M AT ION
A N D C ELLC LON IN C 467

Gonditioned medium
A medium that has already been used for the
growth of other cells contains certain metabolites,
growth factors and other products that stimulate CellsIn suspensron Cellsin monolayer
culture culture
growth. Such a medium, referredto as conditioned
me diu m, whe n a dded t o c ell c loning m edium II I
improves plating efficiency. + +
Counting Trypsinization
Feeder layers (---- andcounting
I
A layer growth-arrestedliving cells, referred to ---r
as feeder layer, promotes plating efficiency. This rs
because the feeder cells provide nutrients, growth
factorsand matrix constituentsthat support survival,
growth and proliferation of cells.

S USPENSION CLO NI NG
Stockof cells
Certa in ce lls, par t ic ular ly t he t r ans f or m ed I
fibroblasts and hematopoietic stem cells. can oe I
mo re co nven ien tlyc loned in s us pens ionr at hert han +
monotayers. Seiialdilutions

Suspe nsionclon ing c an be c ar r ied out by us ing

agar or Methocel, which can hold the cells of a
given colony together, and prevent mixing of
colon ies,
Th e techn iqu e of c loning in agar s us pens ion,r s
carried out in the following stages(Fig 39.2).
1. Ce lls fro m sus pens ionc ult ur e or m onolay er s
can be used . Th e m onolay er c ells hav e t o
t rypsin ize d wh ile t he s us pens ion c ells c an be
directly u se d.
2. Co un t the cells and dilut e s er ially s o t hat
10- 20 0 cells/ml are f inally pr es ent .
3. Freshly p repar ed agar m edium wit h
approp riate dilu tion is us ed.
4. The a ga r medium ' is inoc ulat ed wit h t he
dilute d cells.
5 . In cu ba tion for 1- 3 week s s iv en c lones r n
Clonesin susoension'
cu lture. (after1-3 weeks)
IS OL ATION OF CLO NES
After the cloning is complete, selection and Fig. 39,2 .' Suspension eloning technique in agar.
isolationof specific cell strainsis the next important
step. This is essentiallyrequired for the propagation
Micromanipulation
of ce lls.
lf the monolayer cells are cloned directly in the The genuinenessof a colony, that is the colony
multiwell plates, the colonies can be isolated by is derived from a single cell can be done by
trypsinization of the individual wells. However, micromanipulation. This can be achieved by
w.hen the cloning is carried out in petri dishes,the monitoring the colony formation at the early stages
colonies can be separated from the medium by of cell cloning. Micromanipulation, if properly
plac ing sta inle ssste el or c er am ic r ings ar ound t he done, is believed to be the conclusive method for
colonie s. d e t e r m i n i n gt h e g e n u i n e c l o n a l i t y o f a c l o n e .
 cells. The recent developmentsin the
'f- he c ell c ult ur es a r e w i d e i y u s e d i n t h e with isolated
fl laboratoriesr.t,orldoverfor various purposes' /n
v it r o s t udies wit h is ola t e d c e l l s a r e u s e l u l f o r
under s t anding of m any c e l l f u n c t i o n s s u c h a s
t r ar . r s c r ipt ion, t r ans lati o n , c e l l proliferation,
r es pir at ion and giy c oly s i s . T h u s f o r t he study of
biology and nr at r y iunc t i o n s , t h e c e l ls grown In
c onv ent ional and m on o l a y e r c r - i l t u r e s m a y b e a p p r o a c h e si n t h i s d i r e c t i o n
aclequate. However, 1or the study of integrated 1 . O r g a n c u l t u r e s : T h e w h o l e o r g a n s o r sm a l l
c ellular f unc t ions or or g a n f u t . t c t i o n s i,s o l a t e dc e l l s fragmentsof the organs that retain the special and
will be not be of m uc h u s e , a s e x p l a i n e d b e l o w i n t r i n s i c p r o p e r t i e sa r e u s e d i n c u l tu r e

Cet lular Ent er ac t ions in $rgan functions 2 H i s t o t y p i c c u l t u r e s : T h e c e l l l i n e s g r o w n tn

to high densitv represent
There occurs interaction amons various cells.in i[:;*f:;i,t;:l.t"t'-
v iv o, r es ult ingin a c as c a d eo f e v e n t s .T h e s ec e l l u l a r
int er ac t ions( m os t ly due t o h o r m o n a l s t i m u l a t i o n ) 3 . o r g a n o t y p i c c u l t u r e s : I n t h i s ca se ' th e ce l l s
are very important for the expression of their from different Iineages are put together in the
f unc t ions , as indic at ed b y t h e f o l l o w i n g e x a m p l e s d e s i r e d r a t i o a n d s p a t i a l r e l a t i o n sh i p sto cr e a te a
c o m p o n e n t o f a n o r g a n i n t h e l a bo r a to r y'
. Hor m onal s t im ulat iono f f i b r o b l a s t si s r e s p o n s i b l e
for the releaseof surfactantby the lung alveolar ORGAN CULTUBES
c ells '
T h e u s e o f o r g a n c u l t u r e s (o r g a n s o r th e i r
. Androgen binding to stromal cells stimulates representative fragments) with reference to
prostate epithelium. structural integrity, nutrient and gas exchange,
growth and differentiation, along with the
Besides hormones, nutritional factors and
advantages and limitations is briefly described'
xenobiotics also exert stimulatory effects on the
c ells t o f unc t ion in a c o o r d i n a t e d f a s h i o n '
Structural integritY
oRGANOTYPIG MoDELS As already stated, the isolated cells are
w h i l e i n t h e o r g a n c u l tu r e ' th e ce l l s a r e
As ex plained abov e , t h e c e l l u l a r i n t e r a c t i o n s i n d i v i d u a l ,
as a single unit The cell to cell
that occur in the in vivo system are not possible integrated

468
 r 40 : OR G A NAN D H IST OT Y PIC
C U L TU R E S
A,N D TIS S U EE N C IN E E R IN C

association, and interactionsfound in the native . Semisolidgel of agar.

tissuesor organsare retainedto a largeextent.
. Clottedplasma.
As the structuralintegrityof the originaltissueis
preserved, . Microporousfilter.
the associated cellscan exchangesignals
t hr oughc ell adh e s i o no r c o mmu n i c a ti o n s . o Lenspaper.
o Stripof Perspexor Plexiglas.
Nutrient and gas exchange
Thereis no vascularsystemin the organculture. ln recent years,filter-well insertsare in use ro
fhis limits the nutrient supply and gas exchanges attainthe naturalgeometryof tissuesmore easily.
of the cells.Thishappensdespitethe adequatecare
taken in the laboratoryfor the rapid diffusionof Procedure for organ culture
nutrientsand gasesby placingthe organcultures The basictechniqueof organcultureconsistsof
at the interface between the liquid and the followingstages.
gaseousphases.As a consequence/ some degree
of necrosisat the central part of the organ may 1. Dissection and collectionof the organtissue.
occur. 2. Reducethe size of the tissue as desireo,
Some workers prefer to use high 02 preferablyto lessthan I mm in thickness.
concentration(sometimeseven purA Or) in the 3. Placetissueon a support(listedabove)at the
organcultures.Exposure of cellsto high O, content gas medium interface.
is associated with the risk of O, inducedtoxicity
e.g. nutrient metabolite exchange is severery 4. Incubatein a humid CO, incubator.
affected. 5. C hangethe medi um(M199or C MR L1066)
as frequentlyas desired
Growth and differentiation
6. The organ culture can be analysed by
ln general,the organ cultures do not grow histology,autoradiography and immunochemistry.
exceptsomeamountof proliferation that may occur
on the outer cell layers. Organ culture on stainless steel
support grid
Advantages of organ cultures
Smallfragmentsof tissuecan be culturedon a
. Providea directmeansof studyingthe behaviour filter laid on top of a stainless steelgrid (Fig.aL.l).
of an integrated tissuein the laboratory.
. Under s t andinogf b i o c h e mi c a la n d m o l e c u l ar Organ culture on filter.welt insens
functionsof an organ/tissue becomeseasy. Filter-wellinsertshavebecomevery popularfor
organcultures.This is mainly becausethe cellular
Limitations of organ cultures interaction, stratification
and polarizationare better
. Organculturescannotbe propagated, in these culture systems. Further, the recombinatron
hencefor
eachexperimentthereis a needfor a freshorgan of cells to form tissue- like densities,and access
{rom a donor. to medium and gas exchangeare better.

o V ar iat ions Thefour differenttypesof filterwellsfor growing

ar e h i g h a n d re p ro d u c i b i l i ty
is low.
tissuesin the form of cell layersare depictedrn
. Difficultto prepare,besidesbeing expensive. Fig. 40.2.
o Crowth of cell layer on top of firter
TE CHNI O UE S O F OR G A N C U L T U R E
(Fig. 40.24).
The most important requirementof organ or
r Crowth of cell layers on matrix (collagenor
tissuecultureis to placethem at sucha locationso
that optimalnutrientand gasexchanges matrigel)on top of filter (Fig.a0.281.
occur.This
is mostly achievedby keepingthe tissueat gas- . Cell layersgrown on the interactivecell layers
limited interfaceof the following supports. placed on the underSideof filter (Fig. 40.2e.
47|J B IOTE CHNO LO CY

Organculture

Fig, 40.1 : Organ culture on stainless steel support grid.

. Cell layer grown on the matrix with interactive epithelium, - stratified epidermis, intestinal
cell layer on the underside of the filter epithelium and renal (kidney) epithelium.
ffig. ao.2D).
HISTOTYPIC CULTURES
Filterwell-insertswith differentmaterials(ceramic,
Crowth and propagationof cell lines in fhree-
collagen, nitrocellulose)are now commercially
dimensional matrix to high cell density represent
availablefor use in culture laboratories.
histotypiccultures.The advantage with this culture
Filter-well inserts have been successfully used systemis that dispersedmonolayerculturescan be
to develop functionally integrated thyroid usedto regenerate tissue-likestructures.

Petridish or
multiplatewell

Cell layer
Collagen
Filter or Matrigel

(A) Monolayergrown on top of filter (B) Monolayergrown on matrix

on top ol filter

Petridish or -)
multiplatewell

Cell layer
Collagen
Filter or Matrigel
lnteractive
cell layer
(C) Interactive cell layer Medium (D) Interactivecell layer on the
on the underside of filter undersideof filter with matrixcoating
Fig. 40.2 : Diagrammatic representation of organ culture in filter-well insefts.
Chapt er40 : O R C A N AN D H IST OT Y PIC A,N D TIS S U EE N C IN E E R IN G
C ULTU R E S 471

The commonly usecitechniquesin histotypic three dimensionalstructureof MCTS allows the

cultures use gel and sponge hollow fibers and experimentalstudies related to drug therapy,
s pher oids . penetrationof drugs, resistanceto radiationetc.
Further,MCTShavealsobeenusedto studyseveral
Gel and sponge technique biologicalprocesses.
T he c ells (n o rm a l o r tu mo r) i n c u l tu re can o R e g u l a t i o n o f cell proliferation and
penetrate gels(collagen) or sponges(gelatin)which d ifferentiation.
providesa matrix for morphogenesis of primitive
o tmmune responses.
cells. This approach has been used for the
dev elopm enot f m a m m a rye p i th e l i u m,a n d s ome . C e l l d e a t h .
tubularand glandularstructures. r Cell invasion.
. Cene therapy.
Hollow fibers technique
In recentyears,perfusionchamberswith a bed The mai n advantage of three di mensi onalcel l
of plasticcapillaryfibershavebeendeveloped.The culture (in the form of TVICTS)is that they provide
advantageof using hollow fibers in histotypic a well-defined geometry of cells planar or
c ult ur esis t ha t n u tri e n ta n d g a s e x c h a n g ei s m ore spherical which is directlyrelated to the structure
efficient.As the cells attachedto capillaryfibers and function. lt is now well acceptedthat the
grow, there occurs an increasein cell density MCTS behavelike the initial avascularstagesof
to form tissue-like structures.Many rvorkers solid tumors in vivo. However,bevond a critical
c laim t hat t h e b e h a v i o u ro f h i g h -d e n s i t ycel l s size (> 500 mm), most of the MCTS develop
formed on hollow fibers is comparableto their necrosis(deathof cells)at the centresurror-rnded by
in vivo behaviour.For instance,choriocarcinoma vi abl e cel l s. A di agrammati c
representati on
of
cells grown in hollow fiber culturesreleasemore MCTS in comparisonwith tumor is depicted in
c hor ionicgon a d o tro p h i th n a n i n a c o n v e n ti onal Fig. 40.3.
monolayer.
Technique of MCTS production
Hollow fiber culturetechniquesare regardedas
idealsystems for the industrialproductionof several Single-cell suspensionobtainedfrom trypsinized
biologic ally i mp o rta n t c o mp o u n d s . Wo rk i s monol ayer cel l s or di saggregatedtumor i s
pr ogr es s ingin th i s d i re c ti o n . i nocul atedi nto the medi um i n magneti csti rrer
flasksor roller tubes.As the incubationis carried
T HRE E DI ME N S IO N AL C U L T U R E S out for about 3-5 days, aggregatesof cells
representing spheroidsare formed. lt is observed
Spheroids in histotypic culture that spheroid formation is more efficient unoer
Spheroidsrepresentthe clusfersof cellsusually static conditionson stationaryand non-adhesive
formed by the reassociationof dissociatedcultured surfaces. For this reason, agar/agdrose-coated
cells.lt is knownfor someyearsthatthe dissociated cul turedi shesto w hi ch cel l s do not adhereare
embryoniccells reassemble to form a specialized frequentlyusedto initiatespheroidformation.
structure.
Once the spireroidsare formed, they are
Thebasicprincipleof usingspheroids in histotypic transferi'ed to 24 well platesfor analysis.Spheroid
cultureis that the cells in heterotypic or homotypic growth is quantifiedby measuringtheir diameters
aggregates are capableof sortingout themselves into regularly.This can be done by usinga microscope
groupsto form tissue-like architecture. eyepiecemicrometeror an imageanalysisscanner.
The major drawback of spheroids is the Good growthof spheroidsis observedwhen grown
e f n u tri ents i n w el l s.
lim it at ionin t h e d i ffu s i o na n de x c h a n g o
ano gases. Transfectant mosaic spheroids : lt is now
possibleto producespheroidsfrom cellsthat have
Multicellulal tumor spheroids (MCTSI been transfectedwith different genes. Mosaic
Multicellulartumor spheroidsprovidean in vitro spheroidsare formed by mixing transfectedand
model for studieson tumor cells.The
proliferating non-transfected spheroids in the desired proportion.
472 BIOTECHNOLOCY

.)o o o
d,?..o-q
n
v
(J^" (J
()n
oo -ov^(
iz"::$
NEst

Capillary

Fig. 40.3 : A diagrammatic representation of a tumor in comparison with a multicellular tumor spheroid.

MCTS co-cultures : MCTS can be produced . To evaluateradiationeffectson targettissues.

from heterogenous cells also, forming MCTS co- . Forthe development of tissuesand tissuemodels.
cultures. This is comparable to heteroiogous
spheroids(in short heterospheroids) consistingof
ORGANOTYPIC CULTURES
tumorcellsin combinationwith hostcells.Someof
the MCTSco-culturesare listed. Organotypic culture basically involves the
. MC T Sa n d i mmu n ec e l l s . combination of cells from differentlineagesin a
determinedratio to create a componentof an
r MCTSand fibroblasts. organ.With the advancesin the organotypic culture
MCTSand endothelialcells. techniques,it is now possibleto developcertain
"
ti ssuesor ti ssuemodel s.
Heterospheroids with heterotypic cell interaction
serve as good models for studyingseveralin vivo . Skin equivalentshave been created by co-
processes e.g. inflammation.MCTSco-culturesare cLrlturingdermiswith epidermiswith interviewing
v e ry u s e fu l i n ti s s u e mo del l i ng and ti ssue l ayers
of col l agen.
engineering, the detailsof which are given later. . Models for prostateand breast.

Applications of spheroids or MCTS . Modelsfor control of growth and differentiation

of l ung.
Spheroidshave a wide rangeof applications.
Someof the importantones are listed.
r Serve as models for a vascular tumor growth.
o For the study of gene expressionin a three
d i me n s i o n aclo n fi g u ra ti oonf cel l s. Tissueengineering(TE)refersto the application
o To determine the effect cytotoxic drugs, of the principles of engineering to cell culture for
antibodies,radionucleotides usedfor therapeutic the construction of functional anatomical units
purposes. (tissues/organs).The ultimatepurposeof TE is to
supply various body parts for the repair or
o To study certain disease processes e.g.
replacementof damagedtissuesor organs.Tissue
rh e u ma to i d
a rth ri ti s . engineeringmay be regardedas the backbone'of
r Forthe developmento{ genetherapiesfor several reconstructivesurgery. lt is possible to supply
diseases e.g. cancer. al mostal l surgi cali mpl ants(ski n,bl oo d vessels,
 Chapt er40 : O R C AN AN D H IS T O T Y P|C
CU L TU R E S
A,N D IIS S U EE N GTN E E R IN C 473

Iigaments,heart valves, joint surfaces, nerves) Thereare certain disadvantages

associatedwith
throughthe developments in tissueengineering. autologouscells.
Thereare two schoolsof thoughtwhile dealing o lt is not always possibleto obtain sufficient
w it h t is s ueengi n e e ri nte
g chniques. biopsymaterialfrom the patient.
1. Some workersbelieve that the living cells . Diseasestate and age of the patient will be
possess an innate potential of biological l i mi ti ngfactors.
regeneration. This impliesthat when suitablecells
are allowed to gro\A,on an appropriatesupport Allogeneic cell sources
matrix,the cellsproliferate, and ultirnatelyresultin
lf the cells are taken from a person other than
a n or ganiz eda n d fu n c ti o n a lti s s u e .T h i s ti s s ue
the patient,the sourceis saidto be allogeneic. The
resemblesthe original tissue in structure and
advantages of allogeneiccell sourceare listed.
f unc t ion. T his a p p ro a c h i s v e ry s i m p l e , a nd
ec onom ic al, alt h o u g hth e s u c c e s si s l i mi te d . . Obtainedin good quantityfrom a healthydonor.
2. A c c or din gth e s e c o n d s c h o o l o f th o u g ht, " C el l scan be cul turedi n a l argescal e.
there are severalcontrol processes to produce a .
Cost-effective with consistentquality.
n ew andf unc t io n atil s s u eT h u s ,ti s s u ere g e n e rati on
in vivo or tissue production in vitro are very . Availableas and when requiredby a patient.
complex. Therefore,tissue engineeringis not a The major problem of allogeneiccell sourcers
simple regenerationof cells, and it requiresa
the immunological complications that may
comprehensive approach with a thorough ultimately lead
to graft rejection.The immune
u nder s t anding o f c e l l u l a r c o n fi g u ra ti o ns, p a ci al responses
however,are variabledependingon the
arranSement and control process. type of cel l s used.For i nstance,endothel i alcel l s
Tissueengineeringis a complicatedprocess. are more i mmunogeni cw hi l e fi brobl astsand
Some fundamentaland basic aspectsof TE with smoothmuscl ecel l s are l ess i mmunogeni c. The
specialreference to the followingaspects arebriefly age of the donor is anotherimportantfactor that
described. contri butes to i mmunol ogi calcompl i cati ons. Thus,
. Cell sourcesand culture cel l s from adul t donors are hi ghl y i mmunogeni c
w hi l e fetal or neonatalcel l s el i ci t l i ttl e or no
o Cell orientation
rmmuneresponse.
. Cell supportmaterials
o Designand engineeringof tissues. Xenogeneic cell sources

C E LL S O URC E S A N D C U L T U R E When the cells are taken from different species

(e.9.pig sourcefor humans)the sourceis saidto be
Adequate quantities of cells are required xenogeneic.This approachis not in common use
for tissue engineering.There are three types of due to i mmul ogi calcompl i cati ons.
cell sources-autologous,
allogeneicand xenogeneic.
C U LTU R E OF C E LLS
Autologous cell sources
The methods adapted for culturing of cells
The cell sourceis said to be autologouswhen requiredfor tissueengineeringdependon the type
the patient's own cells are used in If. This is a
and functionsof cells. For most of the cells, tne
straightforwardapproach.A pieceof desiredtissue
conventionalmonolayerculturesservethe purpose.
is takenby biopsy.lt may be enzymaticaliydigesied The major drawbackof monolayerculturesis that
or explantcultured,and the cellsare grown to the cells may lose tlreir morphology,functionsand
r equir ednum be r. proliferativecapacityafterseveralgenerations.
The main advantages
of autologouscells in TE
Someworkerspreferthreedimensionalcultures
are :
for the cells to be used in tissueengineering.
For
. A v oidanc eof i mmu n ec o mp l i c a ti o n s detai l s on these techni ques,S ee p. 471. The
o Reductionin the possibletransferof inherent nutrient and gaseousexchangesare the limiting
i nfections. factorsin three dimensionalcultures.
474 B IOTE CHNO LO CY

Genetic alterations of cultured cells endothelialgrowth factor (VECF),oligosaccharide

for use in TE fragmentsof hyaluronan,fibronectin,collagen.
Gene therapy can be successfully employed in There are certain practical difficulties in
tissue engineering. This can be achieved by maintainingeffectivechemical gradientsfor the
transferring the desiredgenesto cells in culture. cel l s i n three di mensi onal cul ture s. This is
The new genes may increasethe productionof particularlythe limiting factor when the cells
an existing protein or may synthesizea new becomedense.
protein.Some successhas been achievedin this
d i re c ti o n . Mechanical cues
r Genetically altered fibroblasts can produce The responseof the cellsto mechanicalsignals
transferrin,
clottingfactorVlll and clottingfactor is complexand this may resultin any one or more
tx. of the following.
r Mo d i fi e de n d o th e l i acl e l l scan synthesi ze
ti ssue . C hangesi n the cel l al i gnment.
plasminogenactivator.
o Deformationof cytoskeleton.
. C e n e ti c a l l y e n g i n e e re d kerati nocytes can
produce transglutaminase-l (This enzyme is . Alteredmatrixformation.
lacking in patients suffering from a dermal .
Synthesisof regulatorymolecules(e.g growth
disorder, lamellar icthyosis). The altered
factors,hormones).
keratinocytes proved successful when
transplantedin animal (rat) models of this There are mainly three mechanical cues
disease. governi ngcel l popul ati ons.

1. Tensionalforces.
CELL ORIEI{TATION
forces.
2. Compressional
The orientationof cells with regardto specific
shapeand spatialarrangement is influencedby the 3. Shearforces.
followingenvironmental factors.
or contactguidance.
1. Substrate CELL SUPPORT MATERIALS
2 . C h e m i c a gl ra d i e n ts . The supportmaterialsof cells largelydetermine
the nature of adherentcells or cell types, and
3 . Me c h a n i c acl u e s .
consequently tissueengineering.Thereare a large
numberof supportmaterialswhich may be broadly
Substrate guidance
categorizedas follows.
The topographical features of the substrate
. Traditionalabiotic materials.
determinethe contact guidance.These features
may be in the form of ridges,alignedfibersetc. lt . Bioprosthesis
materials.
is possible to use differential attachment to
substratesas a means of producing different r Syntheticmaterial.
a l i g n m e n t o f c e l l s . l n re cent years, syntheti c . N aturalpol ymers.
polymer substratecollagenfibrils and fibronectin
are used as bioresorbabletemplatesfor tissue . S emi -natural
materi al s.
e n Sr n e e nn 8 .
Traditional abiotic materials
Ghemical gradients
The traditionalabioticsupportmaterialsinclude
Developmentof chemicalgradientsis required plastics,ceramics and metals. These materials
for cellular orientation and for the stimulation of cannot be resorbed or become biologically
cellular functions. Certain growth factors integratedinto the tissues. it is preferable
Therefore,
and extracellularmacromoleculesare capable to avoid the traditional materials in tissue
of creating chemical gradients e.g. vascular enSrneenn8.
Chapt er40 : O R C A N A N D H IS T O T Y PIC A,N D TIS S U EE N C IN E E R IN C
CU LTU R E S 475

Bioprosthesis materials promote intimate molecular packing. The so formed

The natural materials modified to become
biologically inert representbioprosthesismaterials.
They are formed by extensivechemical cross
link ingof n a tu ra til s s u e sF. o r i n s ta n c eth, e natural
c ollagen- b a s ceodn n e c ti vti e s s u e(e .9 .p o rc i neheart
values) can be stabilized by treatment with
glutaraldehyde.The product formed is non-
im m unoge n ith c a t re m a i n su n c h a n g e a d t the si teof
transplantation for severalyears.However,growth
of somecells or even connectivetissuecan occur
on bioprosthesismaterials. The design and
fabricationof thesematerialsis done in sucha way
that their functions are not affected by the
s ur r oundi n h
g o s tti s s u e s .
molecules can effectively serve as support
materials.
Semi.natural materials
Semi-natural materials are derived from the
n a t u r a l m a c r o m o l e c u l a rp o l y m e r s o r w h o l e t i s s u e s .
They are the modified materials to achieve
aggregation or stabilization. Some examples of
semi-natural materials are listed below.
. Chemically cross-linked hyaluronan, stabilized
bv benzvl esterification.
. C o l l a g e n c r o s s - l i n k e dw i t h a g e n t ss u c h a s t a n n i c
acid or carbodiimide.

Synthetic materials
DESIGN AND ENGINEERING
A wide range of synthetic bioresorbable
OF TISSUES
polymersare availableas supportmaterials.The
most commonly used polymers in tissue The following surgical criteria are taken into
engineeringare poly (glycolic'acid)(PCA), poly c o n s i d e r a t i o nw h i l e d e a l i n g w i t h t i s s u ee n g i n e e r i n g .
( lac t ic ac id ) (P L A),a n d c o p o l y me rPL GA (pol y
. Rapid restorationof the desired function.
( lac t ic - c o-g l y c o l iacc i d ). T h e c o mp o s i ti onand
dimensions of thesepolymerscan be so adjustedto . E a s eo f f i x i n g t h e t i s s u e .
make them stable ln vlvo, besidessupportingin
r Minimal patient discomfort.
vivo cell growth.
Thereare certainadvantages in usingsynthetic For designing tissue engineering, the source of
donor cells is very critical (See cell sources
BOrymers.
p . a 7 3 ) . U s e o f p a t i e n t so w n c e l l s ( a u t o l o g o u sc e l l s )
. Productionis easyand relativelycheap.
is favouredto avoid immunological complications.
o Compositionof polymersis reproducibleeven in A l l o g e n e i c c e l l s a r e a l s o u s e d , p a r t i c u l a r l y w h e n
largescaleproduction. the TE construct is designedfor temporary repair. It
There are however, some disadvantagesalso. is observed that when the cells are cultured and/or
preserved(i.e. cryopreservation),the antigenicity of
o Com pati b i l i ty
w i th c e l l si s n o t a s g o o da s natural
allogeneic cells is reduced.
poly m er s .
. On degradation,they may form some products Another important criteria in TE is the support
whic h c a u s eu n d e s i ra b lcee l l u l a re ffe c ts. material, its degradation products, cell adhesion
characteristicsand mechanical cues.
Natural polymers The design and tissue engineering with respect
The mostwidely usednaturalpolymermaterials to skin, urothelium and peripheral nerve are briefly
are collagen-chondroitin sulfateaggregates. These described hereunder.
m at er ialsa re c o m m e rc i a l l a
y v a i l a b l ew i th varyi ng
compositionunder the trade name Integra. The Tissue engineered skin
other naturalpolymersfor cell supportare usually
lt was first demonstrated in 1975 Ihat human
obtainedby their aggregation in cultureas it occurs
keratinocytes could be grown in the laboratory in
in vivo e.g. collagengels,fibrin glue,Matrigeland
a form suitable for grafting. Many improvements
somepolysaccharides. Amongthe polysaccharides,
have been made since then. lt is now possible to
chitosanand hyaluronanare usedas hydratedgels.
grow epithelial cells to produce a continuous sheet
T he natu rapl o l y m e rsma i n l ya c t o n th e p ri nci pl e which progresses to form cornified layers. The
of intermolecuiar interaction within the polymersto major difficulty with TE skin is the dermal layer
476 BIOTECHNOLOCY

Proximal --------f Directionof I Distal

srump regeneration slump

Additions
Conduit
material
Regenerating
nerve

Fig, 40.4 : A diagrammatic representation of the basic design for peripheral nerve implant.

sw eatgl ands shaoedinto a bladderand musclecellswerecoated

p o s s e s s i nbBl o o d c a p i l l a ri e snerves,
,
and other accessory organs. on the outersurface.The lumenalsurface(i.e.inner
surface)coatedwith pre-culturedurothelialcells.
Some developmentshave occurred in recent The bladder constructedin this way functioned
yearsto produceimplantableskinsubstitutes which al mostl i ke a normalone, and w as mai n t ainedf or
n ra y b e re g a rd e d a s ti s s ue engi neeri ngski n about one year.
constructs.
Tissue engineered peripheral
lntegra'" : This is a bioartificial material nerve implants
composedof collagen-glycosaminoglycan. Integra'"
Peripheral nerveinjury is a commonoccurrence
is not a true TE construct.lt is mainlyusedto carry
of trauma and tumor resection surgery,often
the seededcells.
leadingto irreversiblemuscleatrophy.Therefore,
Dermagraft'" : This is composed of poly the repair of injured peripheralnervesassumes
(g l y c o l i ca c i d ) p o l y m e rme s hseededw i th human si gni fi cance.
dermalfibroblastsfrom neonatalforeskins. A diagrammaticrepresentationof the basic
designof a peripheralnerveimplantis depictedin
Apligraf'" : This has human dermal fibroblasts
Fig 40.a.
s e e d e d i n to c o l l a g e n g e l . A l ayer of human
keratinocytes is then placedon the upper surface. The regenerationof the injured nerve occurs
from the proximalstumpto rejoin at distalstump.
The tissue constructsdescribedabove have The regenerationis guided by three types of
limitedshelf-life(about5 days).However,they can substances.
integrateinto the surroundingnormal tissueand Conductmaterial: This is the outer layerand is
form a good skin cover. Further,there is no the primarysourceof guidance.Conductmaterial
e v i d e n c eo f i mmu n o l o g i c aclo mpl i cati onsw i th TE is composed of collagen-glycosaminoglycans,
constructs. PLGA(polylactic-co-glycolic and
acid).hyaluronan
materials.
fibronectin.All theseare bioresorbable
Tissue engineered urothelium
Fillingmaterial: This supportsthe neuralcells
It i s n o w p o s s i b l e
to c u l tu r eurothel i alcel l sand
for regeneration, besidesguiding the processof
b l a d d e rs m o o thm u s c l ec e l l s .Thi s rai sesthe hope
regenerati on. Fi l l i ng materi alcontai n scollagen,
that the constructionof TE urotheliumis possible.
fibrin, fibronectinand agarose.
In fact, some successhas been reported in the
developmentof a functionalbladderin dogs. For - Additives: Additivesincludea largenumberof
this purpose,poly (glycolicacid)polymerbasewas growth factors; neurotrophic factors (in different
Chapt er40 : O R C A N AN D H IS T O T Y PIC A,N D TIS S U EE N C IN E E R IN C
C U LTU R E S
forms, combinations, ratios) e.g. fibroblast growth
{actor (FCF), nerve growth factor (NCF). Additions
of $chwann cells or transfectedfibroblastspromote
nerve generatronprocess.
TISSUE MO DELLI NG
Researchis in progressto create tissuemodels in
the form of artificial organs. Some of the recent
de ve lop men t on ex per im ent alt is s ue m odellin g a r e
b riefly o utlin ed.
Artificial liver
Hepatocytes, cultured as spheroids or
hepatocytes and fibroblasts cultured as hetero-
sph ero ids ca n . be us ed. They ar e held in t h e
artificial support systems such as porous gelatin
sponges, agarose or collagen. Addition of
exogenous molecules is useful for the long - term
culture of liver cells. Sonre progress has been
reported in creating artificial liver as is evident from
the hepatocytes three-dimensional structure and
meta bo lic fun c t ions .
pancreas
Artificial
Sph ero ids o f ins ulin s ec r et ing c ells hav e b e e n
d evelo pe dfro m m ous e ins ulinom a bet a c ells . S o m e
workers could implant fetal islet-like cell clusters
un de r th e kidn ey s of m ic e, alt hough t he f unc t i o n s
were not encouraging due to limitation of oxygen
sup ply.
Other tissue models
Pituita ry gland : M ult ic ellular s pher oids co u l d
be created to study ceftain hormonal release e.g.
lute inizing ho r m one ( LH) , f ollowing s t im ulat io n b y
lute inizing ho r m one r eleas ing hor m one ( LH R H ) .
Some success has also been achieved to create
spheroids for the production of melatonin.
Thyroid gland : Thyroid cell spheroids can be
used for the study of cell adhesion, motility, and
thyroid follicle biogenes is .
Brain cell cultures : Three dimensional brain
cell cultures have been used for the study of neural
mye lina tion and dem y elinat ion, neu r o n a l
regeneration,and neurotoxiclty of lead. Aggregated
brain cells are also used for the study of Alzheimer's
d ise asean d Par k ins on' sdis eas e.
Heart cell cultures : Aggregatedheart cells have
been used for the studv of cardiac development
and physiology.
477
The cells of the body, when grown in culture
g e n e r a l l y m a i n t a i n t h e i r o r i g i n a l c h a r a c t e r .T h u s ,
liver cells behave like liver cells, and keratinocytes
as keratinocytes.This is possiblesince each type of
specialized cell has a memory of its developmental
h istory.
Stem cells are undifferentiated cells that can
divide continuously (indefinitely) to produce
daughter cells. The daughter cells undergo
d i f f e r e n t i a t i o ni n t o p a r t i c u l a r c e l l t y p e s .
Embryonic stem (ES) cells are an extraordinary
class of stem cells that can proliferate indefinitely
in culture, as they possessvery high developmental
p o t e n t i a l .T h e c u l t u r e d E S c e l l s w h e n p u t b a c k i n t o
the animal can develop into different cell types and
t i s s u e s .T h i s d e p e n d s o n t h e s i t e a t w h i c h t h e y a r e
i n t r o d u c e d . F o r i n s t a n c e .E S c e l l s i n l i v e r a d o o t t h e
c h a r a c t e ra n d b e h a v i o u r o f n o r m a l l i v e r c e l l s .
ES CELL CULTURES TO PRODUCE
DIFFERENTIATED CELLS
The ES cells obtained from an early mouse
embryo can be cultured indefinitely. The cultures
may be continued as monolayersor allowed to form
aggregatescalled embryoid bodies. The cells of the
embryoid bodies can specialize and differentiate
under suitable conditions (by using various
combinations of signal proteins) into different cells
Gig. a0.5). For instance,when treated with certain
growth factors, ES cells can form astrocytes and
oligodendrocytes.These are the main types of glial
cells of the central nervous system.
Cultured ES cells, appropriately treated with
growth factors, rvhen injected into t'he brain can
serveas progenitorsof glial cells. Thus, it is possible
to correct a mouse dificient in myelin-forming
oligodendrocytes. The grafted cells are capable
of forming myelin sheathsaround axons lacking
them.
HUMAN EMBRYONIC STEM
CELL RESEARCH
It was in 1998, researchersfor the first time
reported the estabrlishmentof human embryonic
stem cell lines.These cells, existing indefinitely in
culture, are capable of giving rise to any human
cell type.
 5
{
o
Fibroblast
growth factor,
Epidermal
growthfactor(1, 2)
Platelet-derived
growth factor
Oligodendrocyte

Retinoicacid

Earlyembryo
(blastocyte)

Retinoicacid,insulin,
thyroidhormone

CulturedES cells

Cells of inner mass Macrophagecolonystimulating

factor,interleukin(1 and 3)
O

Retinoicacid ---l
m
o
T
Z
--
a\
various cells. o
Chapter40 : ORCAN AND HISTOTYPIC
CULTURES,
AND TISSUEENCINEERINC 479

Applications of human ES cells contaminationis identifiedto be due to a non-

The applicationsof humanembryonicstemcell human mol ecul e namel y N -gl ycol yl neurami ni c
lt
research Someof them are listed acid. is suggested
are extraordinarv. that for humanEScell cultures,
productsof animaloriginof any kind shouldnot be
below
used.An idealalternativeis to usehumanembryo-
o Corrections with lossof normalcells- derivedconnectivetissuecells as the feederlayer
of disorders
diabetes, Alzheimer's disease, Parkinson's i n E Scel l cul tures.
disease.
Thiscan be achievedby appropriate cell
therapy. Ethical issues of ES cell research
o.,Engineering of varioustissues.
and replacement
Many people object to growing of human
o Forthe discoveryof new drugs,and testingsafety embryosin the laboratories, even if it is limitedto
of drugs. the early stagesof development.Some countries
r To studythe developmentof humans. have,in fact,bannedthe useof Covernmentfunos
for humanembryo research.However,someprivate
Limitations of human ES cells companiesare conductinfinancing researchon
humanembryoni ccel l s.
There are several practical and technical
difficultiesassociated with EScell culturesand their There are many serious ethical, legal and
utility.lt is recently(2004)foundthatcontamination pol i ti cali ssues surroundi ng
humanE Scel l research.
with medium (the most commonly used being Thi s i s i n addi ti on to the enormoustechni cal
anim al- de ri v e dm e d i a ) c re a te d i mmu nol ogi cal difficulties.lf may takesomemoreyearsbeforethe
complications when such cells are utilized appropriateapplicationsof ES cell researchto
f or t her a p e u ti c p u rp o s e s i n h u m a n s. Thi s human welfarebecomesa reality.
J he dependenc e of m an on a n i m a l s s u c h a s
I c at t le, s heep, poult r y , pig a n d f i s h f o r v a r i o u s
purposes(miik, meat, eggs,wool etc.) is well known.
lntprovement in the genetic characteristics of
l iv es t oc kand ot her dom es t icanim a l s( e . g . ,h i g h m i l k
yield, weight gain, et c . ) , in t he e a r l y d a y s , w a s
carried out by selective breeding methods
T his t ec hnique pr im ar ily inv olv e s a c o m b i n a t i o n
of mating and selection of animals with improved
genetic triats. Although selective breeding is very
tim e c ons um ing and c os t ly , i t w a s t h e o n l y
m et hod av ailable,t ill s om e y ea r s a g o , t o e n h a n c e
the genetic characteristicsof animals. For larger
a nim als wit h long ges t at ion p e r i o d , i t m i g h t
take severaldecadesto createa desiredcharacterby
conventional breeding. With the advent of modern
biotechnology, it is now possible to carry out
manipulationsat the genetic level to get the desired
characteristics in animals. Transgenesisrefers to the
phenomenon of introduction of exogeneous DNA
into the genome to create and maintain a stahle
heritable character. The foreign DNA that is
introduced is called transgene. And the animal
whose genome is altered by adding one or more
transgenesis said to be transgenic.The transgenes
behave like other genes present in the animals'
genome and are passedon to the offsprings.Thus,
transgenic animals are genetically engineered or
genetically modified organisms (GMOs) with a new
heritable character. lt was in 1980s, the genetic
m anipulat ion of anim als by int r o d u c i n g g e n e s i n t o
fertilized eggs became a reality.
480
IMPORTANCE OF TRANSGENIC
ANIMALS.GENEBAL
Transgenesishas now become a powerful tool
for studyingthe gene expressidnand developmental
p r o c e s s e s i n h i g h e r o r g a n i s m s , b e s id e s th e
i m p r o v e m e n t i n t h e i r g e n e t i c c h a r a cte r i sti cs.
Transgenic animals serve as good models for
u n d e r s t a n d i n gt h e h u m a n d i s e a s e s .F u r t h er ,se ve r a l
proteins produced by transgenic animals are
i m p o r t a n t f o r m e d i c a l a n d p h a r m a ce u ti ca l
a p p l i c a t i o n s .T h u s , t h e t r a n s g e n i cf a r m a n im a l s a r e
a part of the lucrative world-wide biotechnology
industry, with geat benefits to mankind.
Transgenesis is important for improving the
quality and quantity of milk, meat, eggs and
wool producfion, bresides creating drug resistant
a ni m a l s .
Milk as the medium of
protein production
M i l k i s t h e s e c r e t i o n o f m a m m a r y g l a n d s th a t
can be collected frequently without causing any
h a r m t o t h e a n i m a l . T h u s , m i l k f r o m t h e t r a n sg e n i c
animals can serve as a good and authenticated
source of human proteins for a wide range of
applications.Another advantagewith milk is that it
c o n t a i n s o n l y a f e w p r o t e i n s ( c a s e i n , l a c t al b u m i n ,
immunoglobuline etc.) in the native state,therefore
isoiation and purification of a new protein from
milk is easy.
 Chapt er4 1 : T R A N S C E N IC
A N IMA L S 481

Gommonly used animals for RETROVIRAL VECTOR METHOD

transgenesis
The transferof small pieces(B kb) of DNA can
The first animalsused for transgenesis was a be effectivelycarried out by retroviruses.This
mouse. The 'Super Mouse', was created by method, however,is unsuitable for transferof larger
insertinga rat gene for growth hormoneinto the genes.Further,even for smallgenes,there is a risk
mousegenome.The offspringwas much largerthan of losingsomeregulatorysequences. Aboveall, the
the parents.SuperMouseaftracteda lot of public biggest drawback is the risk of retroviral
attention, since it was a product of genetic contaminationin the products(particularly in foods
manipulationratherthanthe normalrouteof sexual for human consumption), obtained from transgenic
reoroduction. animals.Because of theselimitations,the retroviral
vectormethodis not in regularusefor transgenesis.
Mousecontinuesto be an animal of choice for
most transgenic experimenfs.The other animals MIC R OIN JE C TION ME TH OD
usedfor transgenesis include rat, rabbit,pig, cow,
goat, sheepand fish. The introductionof DNA by microinjection
methodinvolvesthe following sleps(Fig.41.1).

l. The young virgin female mice (4-5 weeks

age) are subj ectedto superovul ati on. Thi s i s
achi evedby admi ni strati on of fol l i cl e-sti mul ati ng
hormone (pregnantmare' s serum),fol l ow ed by
As alreadystated,mouseis the animalof choice (2
days l ater)humanchori oni cgonadotropi nThe
f or t r ans g e n iecx p e ri me n tsB.e i n ga s m a l lani mal ,i t
superovulated mouseproduces30-35 eggs(instead
can be easily handled,and mouseis regardedas
of normal5-10 eggs).
researcher-friendly by biotechnologists. Another
improtantreasonof choosingmice for transgenic 2. The abovefemalemiceare matedwith mares
experimentsis it produces more eggs (normal and sacrificedon the followingday. The fertilized
mouse5-10 eggs,superovulated mousecan yield eggsare removedfrom the fallopiantubes.
up t o 40 e g g s u ) n l i k eth e l a rg ed o m e s ti ca n i mal s.In
the last two decades,hundredsof differentgenes 3. B y mi cromani pul ati onusi ng a mi cro-
have been introduced into the various mouse injection needleand a holdingpipette,the DNA is
strains. And as such, the methodology of injected into the male pronucleus of the fertilized
transgenesisis well developed with laboratory egg. Adequate care must be taken to ensurethat
m ous e. w hi l e the el asti cnucl earmembranei s punctured ,
the needl edoesnot touchthe nucl eol iA . di ssecti on
Transgenicmice have significantlycontributed microscopecan be used for identifyingthe male
to the understanding of molecularbiology,genetics, pronucl eus (l argeri n si ze)and for mi croi nj ecti on.
im m unol o g ya n d c a n c e r,b e s i d e sc re a ti ngani mal
modelsfor severalhuman geneticdiseases. 4. The eggs with the transgenesare kept
overnightin an incubatorto developto a 2-cell
There are three mefhods for introducing a stage. These eggs are then implanted
foreign gene (transgene)into mice, and in fact the microsurgically into a foster mother i.e., pseudo-
s am em eth o d sa re a p p l i c a b l eto o th e r a ni mal sas mouse pregnantfemal e mouse w hi ch has been
well. mated the previousmight with vasectimized(or
infertile)male.The fostermothercan deliverpups
1. Retroviral vector method.
after 3 weeksof implantation.
2. Microinjection method.
The presenceof transgenein the pups can be
3. Embryonic stem cell method. identifiedby polymerase chainreactionor Southern
blot hybridization.
lt may be noted here that the term transfection
is usedfor introducinga foreigngeneinto animals. The mouse carrying the foreign gene is the
This is quite comparable to transformation that is transgenic founderfromwhich puretransgenic lines
carriedout in lower organisms(ReferChapter6). can be established.

Biotechnology
[31]
482 B IOTE CHNO LO CY

Limitations of microinjection method

Besidesthe low efficiency (discussedabove),
there are some other disadvantagesin this
technique.The foreign DNA randomly integrates
into the host genome. Sometimes,even many
Mouse piecesof DNA get incorporatedat a single site.
Further,transgenesmay not be expressedat all or
sometimesunderexpressed, or evenoverexpressed.
A l l these D rocessesw i l l di sturb th e nor m al
Female Male physi ol ogyof the transgeni cani mal .In addit ion,
pronucreus pronucteus mi croi nj ecti onproceduresare ti me consum ing,
costl y and l abour i ntensi ve.D espi te all t hese
Fertilizedegg
l i mi tati ons,thi s techni quei s routi nelyused f or
produci ngtransgeniani c mal s.
j Transgene
E MB R Y ON IC S TE M C E LL ME TH OD

Holding Cellsfrom the inner cell massof the blastocyst

pipette Microinjection stageof a developingmouseembryocan proliferate
pipette
in cell culture.Thesecells,referredto as pluripotent
embryonic stem (ES) cells, are capable of
I differentiating into other types of cells (including
I germ line cells) when transferredto another
blastocystembryo.
The embryonicstem cell technologybasically
involves the introduction of a foreign DNA into ES
cell (Fig.41.2). Embryonicstemcellsin culturecan
Pseudopregnant female
be subjectedto genetic manipulationswithout
mousewith implantedeggs changingtheir plutipotency.Foreign DNA can be
introduced into ES cells by electroporation or
microinjection.The desiredgeneticallyengineered
cellswith transgene can be identifiedby a selection

@@@@
procedureusi ng a markerB ene or P CR analysis
(describedbelow). The transfectedcells can be
cul tured, i ntroduced (by mi croi nj ect ion)int o
Transgenic blastocystand then implantedinto foster mother
(i.e.,pseudopregnant femalemouse).By this way,
transgenicfoundermice are produced.Transgenic
Fig. 41.1 : DNA microinjection method to produce l i nes can be establ i shedby sui tab le br eeding
transoenic mice. strategies of the foundermice.

Selection of transgene containing cells

The microinjectionmethod involves several
stepsand none of them is 100% efficientfor any
animal to developinto transgenicanimal. In case
of moLrse,it was found that about 65% of tne
fertilized eggs survive microinjectionprocedure,
about 25o/oof the implanted eggs develop into
pups,and only 25ohof them are transgenic. Thus,
if one startswith I000 fertilizedeggs,only 30 to 50
transgenic pupsmay be producedi.e.,3-5% of the
i n o c u l a te de g g sd e v e l o pi n to transgeniani
c mal s.
Severalstrategieshave been developedfor the
sel ecti on of transgenecontai ni ng cells. The
importantones are brieflydescribed.
Selection by use of marker gene coding for
thymidinekinase: lt is worthwhileto know the role
of thymi di neki naseto understand i ts u t ilit y as a
marker gene. There are two pathwaysfor the
synthesisof deoxyribonucleotides (dATB dCTR
dCTPand dTTP),the basicunitsof DNA structure.
 AN IM AL S
Chaot er41 : TR A N S G E N IC 483

One is the salvage pathway that recycles the

degraded nitrogenous bases formed from DNA.
The other alternate pathway is an endogenous
lnnercell synthetic pathway from different precursors
mass (glycine, aspartate, glutamine, methyl tetra-
hydrofolate etc.).
Donorblastocyst
The enzynSethymidine kinase (TK) is involved
J in the salvage pathway. T K phosphorvlates

ES cells
Et thymidine to produce thymidine monophosphate
(dTMP) which is finally converted to thymidine
I
triphosphate (dTTP).
-Transoene
ia The gene that encodes the enzyme thymidine
, kinase can be used as a marker to determine
JTransfection
@ @ @_ES whether the transgene has been inserted. This is
cel with
l'a;;s';;; illustrated in Fig. 41.3. The mammalian cells are
@O@-O
capable of synthesizirrgdTTP by salvage pathway
I Selectionandculture and endogenous synthetic pathway. The cells
I to enrichtransfected
EScells lackingTK gene cannot produce dTTP lf such cells
J
are cultured in a HAT mediurn containing
(o(o@
v/-a\v/-^\\
hypoxanthine(H), aminopterine(A) and fhymidine
\Y-\Y-l (T), they cannot grow and therefore die. This is
ES cells with transgene
b e c a u s et h y m i d i n e c a n n o t b e u t i l i z e d i n t h e s a l v a g e
into
I Microinlection pathway due to lack the enzyme thymidine kinase.
blastocYst Further, aminopterine blocks the endogenous
J
pathway (by inhibiting the enzyme dihydrofolate
reductase,required for one carbon metabolism).
If a transgene is joined to a TK gene, inserted
into a mammalian cell (TK-)and then the cells can
g r o w i n H A T m e d i u m . T h i s i s p o s s i b l eo n l y i f t h e
T K g e n e i s i n c o r p o r a t e di n t o t h e m a m m a l i a n c e l l s .
Recipientblastocyst
A n d l o g i c a l l y ,t h e c e l l s t h a t s u r v i v e i n H A T m e d i u m
carry the transgene. In this fashion, thymidine
kinase can be effectively used as a marker gene.
There are other enzymes that serve as markers
for identifying transgeneinsertion. These include
dihydrofolate reductase (resistantto methotrexate)
and neomycin phosphotransferase (resistant to
antibiotic C41B) and PCR analysis for selecting
Fostermother transgene containing cells. The last one is a more

+
I direct and recent method, and is successfullvuseo
f o r d e t e c t i n g t r a n s g e n ec o n t a i n i n g c e l l s .

ffi,\ffinffi-\
q.44V t}
Promoter sequence to

ggs 4zL* H 4,<Vo IJ facilitate transgenesis

I n t h e e a r l y e x p e r i m e n t s o n t r a n s g e n e s i s ,t h e
Transgenic mouse metallethionein (MMT) gene promoter was
founder
used. MMT gene encodes for a metal binding
protein that is involved in metal homeostasis.The
Fig. 41.2 : Embryonic stem cell (ES) nethod to
foreign gene (i.e., a rat growth hormone gene) can
produce transgenic mice.
be linked to a promoter sequence of MMT. By
484 B IOTECHNO LO CY

(A) Normal mammalian cell (B) Mammalian cell lacking TK gene (C) Mammalian cell (TK-)
with transgene and TK gene

Capableof synthesizing Cannotsynthesize Transgene

dTTP by salvageand dTTP when grown
in HAT medium. Can svnthesizedTTP
endogenoussynthetic
Cellsdie in HAT medium.Cellscan grow.
pathways.Cellscan grow

Fig.41.3: Thymidine kinase (TK) marker gene for selecting transgene containing cells (X-represents the genes
for synthesizing dTTP by endogenous synthetic pathway).

doing so, the promoterswitcheson the growth
hormonegene when the MMT is activatedby a
me ta l i n th e e n v i ro n m e n(e
t .g.,cadmi um).Thus,
the metal inducer(Cd)can stimulatethe promoter
(MMT promoter)to facilitatethe transgene (growth
hormonegene)to express.Therefore,additionof
cadmiumtriggersthe growth hormoneproduction.
By inserting a transgene (foreign gene) into a
c hr om os om e, a new f unc t io n i s i n t r o d u c e d w h i l e
producing transgenic animals (described above).
On the other hand, in a processreferredto as gene
knockout an existing function can be blocked hy
destroying a specific gene. The target gene
disruption can be carried out by incorporatinga
DNA sequence,usually a selectablemarker gene
(smg)into the coding region.This is depictedin
Fig.41.4. The chromosome carryingthe targetgene
(with 4 exons)with flankingsequences is subjected
to homologous recombination with a Vector
carryinga selectable markergene.The homologous
recombi nati onresul ts i n gene kno ckout i. e. ,
disruptionof the targetgene.
ln the gene knockout, the loss-of-function
occurs in transgenicanimals.This is in contrastto
gain of functionthat takesplace by introducinga
foreigngene.

Chromosome

t
Homologous
Target gene t
Homologous
reglon regron
J + I

Recombination
J

with geneknockedout
Chromosome
Fig. 41.4 : Gene knockout by homologous recombination
 41 : TRANSCENI CANI M ALS
Ch AOICT 485

Applications of gene knockout production of therapeutic agents. Adequate care,

Disruption of the target genes (gene knockout) rs
impo rtan t fo r un der s t andingt he dev elopm ent an d
p h ysiolo gical cons equenc es in an or ganis m
Furth er,the bio ch em ic al and pat hologic al bas is o f
se ve ral h uma n dis eas es c an be appr opr iat e l y
understood by inactivating specific genes. About
200 knockout mice have been so far created rcr
serve as animal models for the study of a large
n u mbe r o f h uma n abnor m alit iesand dis or der s .I n
fa ct, kn ocko ut mous e whic h lac k genesf or a s ing l e
organ or organ system can be produced (For more
d eta ils, See p. 48 6) .
In th e con ve nt ional t r ans genes is ,s m all s iz e d
transgenes(< 20 kb) are transferredthrough vectors.
T his involves a r is k of los ing s om e inr por t a n t
sequ en ce s in cfu ding t he r egulat or y ones Fur t he r ,
several genes exist as complexes and are too large
to be handled by conventional vectors. Yeast
artificial chromosomes(YACs)can carry large-sized
genes (size range 100-1 ,000 kb), and are effectively
u sed in tran sg en es is .
By using microinjection technique or transfection
of ES cells with YACs, transgenic mice have been
produced. Selectedexamples are cited below.
1 . YACs are used to carry human B-globin gene
comp lex (5 ge ne s wit h 250 k b s iz e) and pr oduc e
t r an sg en icmice.
2 . YACs a re e m ploy ed f or pr oduc ing hum a n
antib od ies in tr ans genic m ic e. Sy nt hes is o f
antibodies is very complex process.YACs carrying
varying sizes of DNA sequences (800-1 ,000 kb
YACs) have been employed to generate different
antib od ies.
however, must be exercised before extrapolating
d a t a o f t r a n s g e n i cm i c e t o h u m a n s
Mouse models for several human diseases
( c a n c e r s ,m u s c u l a r d y s t r o p h y ,a r t h r i t i s ,A l z h e i m e r 's
disease, hypertension, allergy, coronary heart
disease, endocrine diseases, neurodegenerative
disorders etc ) have been developed. A selected
few of thern are briefly discussed.
THE HUMAN MOUSE
The transgenic mice with human immune
system were produced, and they are commonly
referred to as human mice (Fig. 41 .5). For this
purpose/ mice with severe combined immuno-
deficiency (SCID-acondition characterizedby total
l a c k o f i m m u n e s y s t e mc e l l s ) w e r e c h o s e n . H u m a n
thymus tissue from an aborted fetus was
t r a n s p l a n t e du n d e r t h e c a p s u l e m e m b r a n e o f t h e
kidney of the mouse Human lymph node was
placed under the opposite kidney. After about a
w e e k , i m m a t u r e i m m u n e c e l l s ( T - l y m p h o c y t e sf)r o m
a human fetus were injected into the mouse tail
vein. These lymphocytes enter the thymus tissue
under the kidney and mature to T-lymphocytes.The
so produced T-lymphocytes enter the circulation
a n d i n t h e l y m p h n o d e ( p r e s e n tu n d e r t h e s e c o n d
kidney), they multiply to form a full-pledged
functional immune system. lt takes about two
weeks after the transplant for the mice to display
Ihe human immune system (characteristics of both
T-lymphocytesand B-lymphocytes).
The human mouse, being a close animal model
for human immune system is a boon for
i m m u n o l o g i s t s ,p a r t i c u l a r l y w o r k i n g o n A I D S . T h i s
is because the various immunological aspects of
AIDS including the possible development of an
AIDS vaccine can be explored by using human
mouse (Note : There is no good animal model for
A I D S r e s e a r c he x c e p t c h i m p a n z e e )

T H E A L Z H E I M E R 'S MOUSE
Alzheimer's disease affects about 1% of the
population between 60-65 years old, and about
Mou se , a ltho ugh not c los e t o hum ans in i t s 30"/" of the populatiCrnover B0 years old This
biology, has been and continues to be the most disease is characterized by progressive loss of
exp loite d an im al m odel in t r ans gene s i s memory and personality changes (decline in
experiments. The Common feature between man t h i n k i n g , j u d g e m e n t e t c . ) . T h e p o s t m o r t e ma n a l y s i s
an d mo use is tha t bot h ar e m am m als . Tr ans ge n t c o f b r a i n s o f A l z h e i m e r 's p a t i e n t s r e v e a l e d p l a q u e s
mice are extensively used as animal models for of dead nerve cells entangled in a protein called
understandine human diseases and for the amyloid. This protein w'as purified and its amino
486 B IOTE CHNO LO CY

acid sequence determined. Later, amyloid

precursor protein (APH and its gene sequence
were also identified.Alzheimer'sdiseaseis more
prevalentin some fanrilies,clearly indicatinga
geneticbasisfor this disease.
Transgenicmice were developedby introducing
amyloid precursorgene into fertilizedegg cells of
mi ce.The synthesiof s humanamyl oi dp r ot einand
i ts accumul ati onas typi cai pl aquesi n t he m ice
brain were observed.The Alzheimer'smouse is
A mousewith SCID very usefulin understanding the pathologicalbasis
of the disease.Certainmutationsin APPgene,and
the involvementof someother genesare believed
to be responsible for Alzheimer'sdisease.

TH E ON C OMOU S E
The animal model for cancer is the oncomouse
(onco refersto cancer).Firstdevelopedfor breast
cancer,the oncomouseis usefulfor r-inderstanding
of cancer and evol vi ne modal i ti esf or cancer
therapy.
fhe oncogenec-myc in associationw'ith mouse
mammarytumor (MMT) virus was found to be
responsible for breastcancerin animals.Transgenic
mice were producedby introducingchimericDNA
consistingof c-myc gene and sectionsMMT virus
i nto ferti l i zed mouse egg cel l s. B reastcancer
develooedin adult femalemice and the trait was
passedon to the offspring.
Oncomouse (the mouse that is genetically
alteredand is susceptiblefor cancer)was patented
by U.S. Patentoffice in 1988. In fact, it was the
lmmaturehuman
immunecells very first animal to be patented.

A mousewith SCID THE PBOSTATE MOUSE

The prostategland, surroundingthe urethraof
t males is responsible for secretingsemenfluid. In
the oider men, particularlyabove60 yearsof age,
prostate gland gets elarged and may become
cancerous.The oncogenefor prcstatecancerwas
i denti fi ed(i nt-2). A chi meri c D N A b y joining
'.ri int-2 with viral promoter was prepared and
Human(T- and B-)
introduced into fertilized mouse eggs. In the
iransgenicmice so developeci,enlargernentof
prostategiand was observed.Thereprostatemice
w ere al sopatentedi n 1991.

THE KNOCKOUT MOUSE

Fig. 2.5 : The human mouse (SCID-Severe
The basi cpri nci pl esunderl yi nggene knockout
havebeendescribed(Seep.484). Severalknockout
 A N IMA L S
Chaot er41 : T R A N S C E N IC 447

mice have been developed. lt is not an
exaggerationthat'the knockout mice have become
as common as a test tube in the laboratory for a
biotechnologist. lt must be noted that not all the
knockout mice are immediately useful for human
health and welfare. A selectedfew of the knockout
mice are described.
SGID mouse
Se ve reco m bined im m unodef ic ienc y( SCID ) i s a
condition with a total lack of immune system.SCID
mice were developed by eliminating a single gene
and the resultant mice lost the ability to produce
B-lymphocytes and T-lymphocytes. The human
mouse was later developed from the SCID nrouse
(Se e p . 48 5) .
Knockout mouse for allergy
The receptorsiteson certainbody cells for lgE
antibodies are believed to be responsible for
triggeringallergy reactions.Knockoutmice were
developedfor allergyby removingthegeneencading
for receptor protein. The result is that antibodies
cannotbind to cellsdue to lackof receotors and the
mice are unaffectedby allergic reactions.lt is
expectedthat somebreakthrough may occur in the
near future to benefitthe millions of sufferersof
spreadthroughoutthe world.
allergicreactions,
Knockout mouse with memory loss
The memoryprocesses
in brain are believedto
be carried out by a specialized area called
hi ppocampus.B y a gene knockout techni que,
researchers have developed mice that lack
hippocampus.These knockout mice lacked the
ability to remember.
Knockout mouse with
retinitis pigmentosa
By inactivatingthe mouserhodopsingene,rods
cells of the retinal deterioratein the transgenic
mice. This retinal degenerationis comparableto
human retinitis pigrnentosa. The rhodopsin
knockout mouse is useful for understanding the
retinal degeneration, besidesevolvingtherapeutic
strategies.
TR A N S GE N IC MIC E FOR
H U MA N D IS E A S E S
Transgenic mice are important for the
devel opmentof therapeuti cdrugs and possi bl e
gerretherapies,besidesthe understanding of the
humandi sease. Many transgenimic cew i th human
diseaseequivalentshave been developedand a
selectedfew of them are given in Table41.1.

Knockout mouse for transplantation

By using a suicide gene, the liver cells were
destroyedin a mousethat alreadylacks immune
s y s t emS. am p l eh u m a nl i v e rc e l l sw e retra n s pl anted In generaltransgenesi
, i ns l argeani mal si s more
in t his m ou s e . En c o u ra g e b d y l a c k o f i mmune difficult than with mice. Thereare severalfactors
s y s t emt,he h u ma n l i v e r c e l l sc a n d e v e l o p.In thi s for the lower efficiencyof transgenesis in large
fashion,organreplacementin animalsis possible. animals.Theseinclude less number of eggsthey

rnsgenic rnice with human diseaseequivalents(sclcctedexamples)

Human diseaseequivalent Genetic change in mice

anemia
Sickle-cell Insertion
of B-globin hemoglobin.
of sickle-cell
Lesch-Nyhan
syndrome of mousegeneencoding
Inactivation hypoxanthine-guanine
phosphoribosyltransferase
Kaposis
sarcoma oftllV tatgene
Introduction
of malignancy
Development fnsertion
of human rasandc-myc oncogenes
X-Linked
muscular
dysirophy Mutation
at a locuson X-chromosome
Hypertension* Introduction
of mouse
reninoene
*This wasdonein transqenic
rals
488
pr oduc e and t ec hnic al dif f i c u l t i e s i n h a n d l i n g ,
besidei long gestationalperiods to get the offspring
(lt takes about 2 years to produce a calf from a
f er t iliz ed egg) .
Some of the early experiments to produce
transgenic large animals were far from satisfactory.
For instance,transgenicsheepoverproducinggrowth
hormone grow leanerwith increasedfeed efficiency
But they are more susceptibleto infection, become
inf er t ileand t end t o die at y o u n g a g e . A l l t h i s m i g h t
be due to ineffective control of gene regulation.
Several improvementshave been made to produce
t r ans genicanim als wit h des i r a b l ec h a r a c t e r s .
Biot ec hnologis t sar e par t i c u l a r l y i n t e r e s t e d t o
im pr ov e t he qualit y of an i m a l s , w i t h i m p r o v e d
r es is t anc e t o dis eas es , be s i d e s e n ! r a n c i n g t h e r r
ability produce foods.'Building a better animal ,
being the moffo. Further,production of commercial
and phar m ac eut ic al c om p o u n d s b y t r a n s g e n i c
anim als is als o gaining im po r t a n c e in recent y e a r s . l i n k e d d i s u l f i d e b r i d g e s . F o r g o o d p ro d u cti o n o f
The protocol adopted for producing other
t r ans genicanim als is c om pa r a b l e w i t h t h a t a l r e a d y
described for transgenic mice, with certain
m odif ic at ions .
TRANSGENIC CATTLE
fhe mammary gland of the dairy cattle is an genes for the synthesis of cysteine. The two
ideal bioreacfor for producing several new proteins enzymes/ synthesized by the transgenes, are
(of pharmaceutical importance), besides improving
t he qualit y and quant it y of t h e e x i s t i n g o n e s . F o r in the intestine to produce cysteine. fhus, good
instance,a transgeniccow, with an over-expressecl
c as eint r ans gene,c an giv e m i l k w i t h h i g h e r c o n t e n t quality and quantity of wool.
of casein. lf lactase transgene is introduced and
ex pr es s edin t he m am m ar y g l a n d , m i l k f r e e f r o m
lac t os ewill be s ec r et ed.Suc h a m i l k w i l l b e a b o o n
f or ' lac t os e int oler ant peo p l e w h o e x p e r i e n c e
ind iges t ion and ot her c o m p l i c a t i o n s , a f t e r hemoglobin have been successfullydeveloped.This
c ons um ing nor m al m ilk and m i l k p r o d u c t s .
Some success has been achieved in creating
transgenic cattle with improved resistance to viral,
bacterial and parasitic diseases. However, this
is not an eas y job due t o t h e c o m p l e x i c i t y o f
genetic control to combat the disease-producing (weeks
organrsms.
Attempts have been made in recent years to
pr oduc e c at t le wit h inh e r i t e d i m m u n o l o g i c a l
protection by transgenesis.Introduction of genes
t hat c ode f or heav y and ligh t c h a i n s o f m o n o c l o n a l
ant ibodies has m et wit h s o m e s u c c e s s i n t h i s
dir ec t ion.
B IOTECHNO LO CY
l n v i v o i m m u n i z a t i o n o f a n a n i m a l al th o u g h n o t
yet fully successful,is ideal for disease protection.
l n v i v o i m m u n i z a t i o n p r i m a r i l y i n vo l ve s th e
insertion of a transgene for an antibody that
s p e c i f i c a l l yb i n d s t o a n a n t i g e n .
TRANSGENIC SHEEP AND GOATS
Transgenesisexperiments in sheep and goats
mostly involve the development of mammary
glands as bioreactorsfor the production of proteins
for pharmaceuticaluse. This is possible despite the
fact that quantity of milk produced by sheep and
goats is lessthan that of dairy cattle (cow, buffalo).
Some proteins produced by sheep and goats have
g,ood pharmaceutical ure (Table 41.2).
Transgenic sheep with
increased wool ptoduction
K e r a t i n i s t h e w o o l p r o t e i n w i t h h i g h l y cr o ss-
q u a l i t y w o o l , t h e a m i n o a c i d c y s te i n e ( o r i ts
precursor methionine) is required in large
quantities. However, cysteine supply to sheep is
always inadequate, since the microbes harboring
t h e r u m e n u t i l i z e i t a n d r e l e a s e i n th e fo r m o f
sulfide. This problem can be overcome by
p r o d u c i n g t r a n s g e n i c s h e e p c o n t a i n i n g b a cte r i a l
capable of trapping the hydrogen sulfide liberated
supply of cysteine to the sheep improves the
PIGS
TRANSGENIC
Transgenic pigs that can produce human
human hemoglobin can be separated from pig
h e m o g l o b i n b y s i m p l e a n a l y t i c a l te ch n i q u e s.
Hemoglobin, the oxygen carrying protein of RBC,
can be used as a substitute in blood transfusion
experiments.In fact, hemoglobin can be stored for
longer period (a few months) than whole blood
only). Further, there is no problem of
c o n t a m i n a t i o n ( l i k e H I V ) a s i s t h e c a se w i th w h o l e
blood. However, the free hemoglobin (naked
hemoglobin) cannot transport oxygen as effectively
a s t h e h e m o g l o b i n o f R B C . I n a d d i ti o n , n a ke d
hemoglobin is easily degraded and the breakdown
products cause damage to kidney. There also exists
a r i s k o f c o n t a m i n a t i o n b y p i g v i r u s e s a n d o th e r
 AN IM AL S
Chapt er41 : T R A N S C E N IC 489

Transgenlcanimals as
of therapeutlcally inportant proteins

lransgenic animal Protein product Biological importance

Cow Lactofenin Promotes

inteslinal
ironabsorotion
andhence
canbe usedto overcomeiron-deficiency
Possesses
anemias. activity
antibacterial
Cow lnterferon Provides
resistance
against
viralinfections
Sheep o,-Anlitrypsin Usedin thetreatment of emphysema
(promotes theexchange of gasesin lungs)
Goat Cysticfibrosis
transmembrane Forthetreatment ol patients
sufferingfrom
regulator(CFTR) (promotes
cysticfibrosis transport
ol ions)
Goat Tissueplasminogen thepatients
Usedin treating of myocardial
activator(tPA) (dissolves
infarction bloodclots)
Goat Antithrombin
lll Regulates
bloodclotting
Rabbits cr-Glucosidase Treatmentof Pompe's
disease(a genetic
disorder by blockin glycogen
characterized
degradation)
Mouse Urokinase Fordissolving
bloodclots
Mouse (antibodies)
lmmunoglobulins Administration immunitv
enhances
Pig Hemoglobin Bloodtransfusion
Goatandotheranlmals (?)
Vaccines To immunize various
against diseases

compoundsto causeallergicreactions.With these showedsome promisingresults.The day may not

limitations,the initial enthusiasmfor substituting be very far for utilizingtransgenic pigsas donorsof
blood transfusion with free hemoelobin has human organs(For more details,Seep. 492).
remainedshort-lived.
It is now advisednot to use ;rakedhemoglobin TR A N S GE N IC C H IC K E N S
for transfusion,when there is a heavyblood loss. The productionof transgenicchickens(or other
However,it can be usedduringmajor surgeries for birds)is rathercomplicated. This is mainlybecause
supplementing the"wholeblood transfusion. duringfertilizationin chickens,severalspermsenter
the ovum insteadof one. This is in contrastto
Pig in organ farms mammalswhereusuallyonly one spermentersthe
The humanorganssuchas heart,liver,pancreas, egg.The identificationof male pronucleithat will
kidney and lungs are in great demand for fusewith femalepronucleiis quitedifficult.Further,
transplantationsurgery.The shortage of these embryonicstem(ES)cells have not been identified
transplantableorgans can be overcome by i n chi cken.D espi teal l thesel i mi tati ons, transgeni c
developingthem in animals. Pig is a favourite chickens have been develooed.
animal for harvesting human organs. This is The blastodermcells (from an egg) can be
becausethe physiologyof pigs is closeto that of
removed from a donor chicken. They are
humans. Further,pigs do not carry any major (usuallyby lipofection
transfected with transgenes
infectiousdiseasestransmissible to humans.The
with liposomes). The so modifiedblastodermcells
us e of pig s i n o rg a n fa rmi n g i s s ti l l at the
are reintroducedinto the subgerminalspace of
experimentalstages.
irradiatedblastodermof freshlylaid eggs.Someof
In the preliminary experiments, organ the resultingchickensmay carry the transgene.
transplantation from transgenicpigs into primates Transgenic linesof chickenscan be established.
490 B IOTE CHNO LO CY

Transgenesisin chicken can be used to develop
low fat and cholesterol,and high protein containing
eggs. Transgenic chickens that are resistant to viral
and bacterial diseases have also been developed.
Some attempts have also been made to develop
pharmaceutical proteins in the eggs of transgenic
c h ic k ens .
TRANSG ENI C FI SH
Several transgenic fish (catfish, salmon, trout
etc.) have been developed with increase in their
growth and size. This was carried out by
introciucing growth hormone transgene (by
microinjection or electroporation). The fertilized
eggs with inserted transgene are incubated rn
temperature-regulatedholding tanks. (Note : The
fish egg development is external in contrast to the
mammalian embryogenesis).The efficiency of fish
(ransgenesisis as high as 70"/o.
It was found that the transgenicsalmon fish (with
growth hormone transgene)were 10 times heavier
than the normal ones, at the end of one year
use. In fact, any protein synthesizedin the human
b o d y c a n b e m a d e i n t h e t r a n s g e n i c a n i m a l s,
provided that the genes are correctly programmed.
T h e a d v a n t a g e w i t h t r a n s g e n i c a n i ma l s i s to
p r o d u c e s c a r c e h u m a n p r o t e i n s i n h u g e q u a n ti ti e s.
Thus, the animals serving as factories for
production of biologically important products are
referred ro as animal bioreactors ol, sometimes
pharm animals. Frankly speaking, transgenic
a n i m a l s a s b i o r e a c t o r s c a n b e c o nr m e r ci a l l v
exoloited for the benefit of mankind.
Once developed, animal bioreactors are cost-
effective for the production of large quantities of
h u m a n p r o t e i n s . R o u t i n e b r e e d i n g a n d h e a l th fu l
l i v i n g c o n d i t i o n s a r e e n o u g h t o m a i n t a i n tr a n sg e n i c
animals.
The importance of transgenic animals has
already been discussed under the respective
i n d i v i d u a l a n i m a l s . A s e l e c t e d l i s t o f th e
therapeutically important proteins produced by
animal bioreactors is given Table 4l .2.

Position effect is the phenomenon of different

Dolly, the first ever mammal clone was
Ievels of gene expression that is observed after
devel opedby W i l mut and C ampbel il n 1997. lt is
insertion of a new gene at different position in the
a sheep(femalelamb) with a mother and no father.
eukaryotic genome. This is commonly observed in
transgenic animals as well as plants. These The technique primarily involves nuclear
transgenic organisms show' variable levels and transfer and the phenomenon of totipotency. The
patterns of transgene expression. ln a majority of characterof a cell to develop to differentcells,
cases,position effectsare dependent on the site of tissues,organs,and finally an organismis referred
transgene integration. to as totipotency or pluropotency. Totipotency is
the basic characterof embryonic cells, As the
In general,the def'ectiveexpressionis due to the
embryodevel ops, to f inallygive
the cel l sspeci al i ze
insertionof transgeneinto a region of highly packed
the w hol e organi sm. A s such,the cel l sof an adult
chromatin. The transgene will be more active if -fotipotency
lack totipotency. was inducedinto the
inserted into an area of open chromatin.
adul t cel l sfor devel opi ngD ol l y.
The positional effects are overcome by a group
The cl oni ng of sheep for producing Dolly,
of DNA sequencescalled insulafors.The sequences
illustratedin Fig. 41.6, is briefly describedhere.
referred to as specialized chromatin structure (SCS)
The marnmarygland cellsfrom a donor ewe \ /ere
are known to perform the functions of insulators.lt
isolated.They n,ere subiectedto total nutrient
has been demonstrated that the expression of the
deprivation (starvation)for five days. By this
gene is appropriate if the transgene is flanked by
process, the mammarycel l sabandonth eir nor m al
i nsuIators.
growth cycle, enter a dormant stageand regarn
An ovum (eggcell)was taken
totipotencycharacter.
from anotherewe, and its nucleuswas removedto
form an enucleatedovum. The dormantmammary
Transgenesisis wonderfully utilized for glandcell and the enucleatedovum were fusedby
productionproteinsof pharmaceutical
and medical pulse electricity. l-he mammary cell outer
 AN IM AL S
Chapt er4 1 : T R A N S C E N IC 491

membranew as broken, al l ow i ng the ovurmto

envel opethe nucl eus.The fused cel l , as i t had
gainedtotipotency,can multiply and developinto
an embryo.Thi s embryow as then i mpl antedi nto
another ewe which servesas a surrogate/foster
mother.Five monthslater,Dolly was born.

rA
As reportedby Wilmut and Campbell,theyfused
277 ovum cel l s,achi eved13 pregnanci es,
and of
theseonly one pregnancyresultedin live birth of
\Y/ the offspring-Dolly.
Ovum with nucleus

I
l-* aO
a)
Some of the companies involved in transgenic
experimentshave startedcloning pet animals like
cats and dogs. Little Nicky was the first pet cat that
was cloned at a cost of $50,00 by an American
\-/ company (in Dec. 2004). More cloned cats and
Enucleatedovum dogs will be made available to interested parties
(who can afford) in due course.

Some people who own pet animals are

interested to continue the same oets which is
//-\\ p o s s i b l et h r o u g h c l o n i n g . T h e r e i s s o m e o p p o s i t i o n
((^) ) to this approach as the cloned animals are less
\_--l
\
\/ /
healthy, and have shorter life span, besidesthe high
Fusedcell cost factor.
(mammarycell nucleuswith ovum envelope)
II hnitro
] embryoculture

Fetal cells such as fibroblastsare totipotent.

Fetalcell cloning was successfully carriedout by
some workers to produce transgenic sheep(Polly),
transgenic bull calf (Gene) and other animals.
T'ransgenes (foreigngenes)can be insertedinto the
fetal cell genomesto producedesiredproductsby
transgenianic mal s.
As an example,the developmentof transgenic
calvesproducedby cloningwith fetalcellsis shown
Fostermother in Fig. 4I .7. Fibroblasts
were collectedfrom a fifty
five day old bovine fetus. Thesefibroblastswith
totiootentcharacterare cultivatedin a nutritious
medium.The desiredforeigngenes(transgenes) can
be introducedinto fibroblasts.The nucleuswith the
geneticallyaltered DNA (fibroblastnucleus) is
Dolly removedfrom the cell. lt is then insertedinto a
bovi neenucl eated ovum.Thi sovum cel l mul ti pl i es
Fig. 41.6 : The cloning of sheep for
developing Dolly.
to form embryos.Theyare implantedin a surrogate
(foster)
mothercow to givebirthto transgeniccalves.
 B IOTE C HNO LO G Y
492

The calves representclones, since they have

originatedfrom a singlecell. Theyaretransgenicas
thev carry the transgenes (foreign Senes)'The
productionof transgeniccalvesis a good example
of nuclear transfertechnologY.
Introduction of foreign genescan be effectively
carried out in fetal cells, and the so produced
transgenic animals can serve as good animal
bioreactors to synthesize several products of
pharmaceuticalimportance.The progressin this
directionhas been ratherslow due to ethicalancl
moral i ssues.

Organ transplantation (kidney, liver, heart etc')

in humans has now become one of the advanced
surgical practices to replace the defective, non-
functional or severaly damaged organs. The major
limitation of transplantationis the shortageof organ
d o n o r s . T h i s o f t e n r e s u l t si n l o n g w a i t i n g t i m e s a n d
many unnecessarydeaths of organ failure patients'
Xenotransplantation refers to the replacement of
failed human organs by the functional animal
organs.The major limitation of xenotransplantation
is the phenomenon of hyperacute organ rejection
due to host immune system.The organ rejectionsis
mainly due to the following two causes
. The antibodies raised against the foreign organ'
. Activaiion of host's complement system.

Transgenic calves produced bY
PIGS IN XENOTRANSPLANTATION?
Some workers are actively conducting research
to utilize organs of pigs in xenotransplantation'lt is
now identified that the major reasonfor rejection of
pig organs by primates is due to the presence of a
s p e c i a l g r o u p o f d i s a c c h a r i d e s( C a l - c r 1 ,3 - C a l ) i n
pigs, and not in Primates.
The enzyme responsible for the synthesis of
s p e c i f i c d i s a c c h a r i d e si n p i g s h a s b e e n i d e n ti fi e d ' l t
is u 1,3-galactosyltransferase, present in pigs and
not in primates. Scientists are optimistic that
knackout pigs lacking the gene encoding the
enzyme ct, 1,3-galactosyltransferase can be
developed in the next few years. Another approach
is to introduce genes in primates that can degrade
or modify Cal-c 1,3-Cal disaccharidegroups (of
pigs).This will reduce immunogenecity.
 AN IM AL S
Chaot er41 : T R AN SC EN IC
Besidesthe above, there are other strategiesto
avoid hyperactive organ rejection by the hosts rn
xe notransolantation.
. Exp ressio n of pig
ant ibodies agains t t he
disacch ar ides .
. Expre ssionof c om plem ent- inac t iv at ingp r o t e i n
on the ce ll s ur f ac es .
By the above approaches,it may be possible to
overcome immediate hyperactive rejection of
organs. The next problem is the delayed rejection
wh ich invol v es t he m ac r ophagesand nat ur a l k i l l e r
cells of the host.
Another concern of xenotransplantationis that
the endogenouspig retrovirusescould get activated
after organ transplantation.This may lead to new
genetic changes with unknown consequences.
Th e use of t r ans genic anim al s in
xenotransplantation is only at the laboratory
expe rimen tal s t ages , inv olv ing anim als . l t i s
doubtful whether this will become a reality in the
near future.
There is a vigorous debate concerning the ethics
of xenotransplantation and the maiority of general
public are against it.
Several human diseaseshave a second host in
the life cycle of the parasite.Transgenesiswill be
useful for disease control by interrupting the life
cycle. Attempts are being made in the recent years
in this direction, although the sucess has been
limited Transgenic organisms may soon serve as
environmental replacements to control several
diseases.
TRANSGEN I C SNAI LS
Schistosomiasisis a parasitic diseasescaused by
Schistosoma which develops in the snails. The
parasiteinfects humans by penetratingthrough skin
when contacted in water. It is estimatedthat about
10 0 million people wor ldwide ar e t he v ic t i m s o f
schistosomiasis.This disease is characterized by
chills, fever , int es t inai ulc er at ionsand diar r h e a .
By developing transgenicsnails, it is possible to
interrupt the life cvcle of Schistosoma. Some
workers have attempted to create transgenic snails
that will resist the invasion of the parasife. lf such
493
s n a i l s a r e r e l e a s e di n t o t h e e n v i r o n m e n t , t h e y w i l l
break the life cycle of schistosoma.
TRANSGENIC MOSQUITOES
Mosquitoes are responsiblefor the transmission
of malaria. Some workers have identified certain
critical genes in mosquitoes (Anopheles genus that
transmitsPlasmodiumspeciesmalarial parasite)that
are responsible for harbor and transmission of the
parasite. Theoretically, it is possible to alter these
genes and produce transgenic mosquito. lf such
mosquitoes are released into the environment in
large numbers, they will dilute the population of
native mosquitoes, and halt the transmissionof
malarial parasite.
Another approach to control malaria is to
understand vector incompetence i.e., the genetic
basis of the vector to sustaina oarasiteand how to
alter it so as to make the vector inhospitablefor the
oarasite. Some success has been achieved in
developing transgenic mosquitoes with vector
incompetence. lf such mosquitoesare releasedinto
the environment, the life cycle of the malarial
parasite will be interrupted.
TRANSGENIC TSETSE FLIES
A protozoal diseaseAfrican sleeping sicknessis
transmitted by tsetse fly. This disease affects the
nervous systemand is often accompanied by coma.
Transgenictsetseflies were developed with a novel
approach. Researchersfound a protein that can kill
disease-producing protozoa. They identified the
protein encoding gene and inserted it into the
bacteria of tsetse fly gut. These bacteria produce
antiprotozoal protein that kills tsetse flies. ln this
manner the diseasetransmissioncan be prevented.
TRANSGENIC BOLLWORMS
Bollworms are the caterpillarsthat damage the
cotton crop. Researcheshave developed transgenic
bollworms with a suicide gene When released into
the environment,the transgenicbollworms will mate
with wild bollworms and produce caterpillarsthat
die. The cotton crop can be savedfrom the damage.
TRANSGENIC MEDFLIES
Medflies (Mediterraneanfruit flies) destroy fruit
and coffee crops throughout the world. Attempts
are being made to develop transgenic medflies to
replace wild medflies and save the crops..
 GO

il
42 P l ant Ti ssueC ul ture-C eneral 497
43 P l ant Ti ssueC ul ture Medi a 517
44 P rotopl astC ul ture and S omati c
H ybri di zati on 523
45 P roducti onof H apl oi d P l ants 538
46 S omacl onalV ari ati ons 546

o$ 47 Clonal Propagation
(Mi croproP agati on) 552
48 C ermpl asnt C onservati onand
C ryopreservati on 565
49 C eneti c E ngi neeri ngof P l ants-
Methodol ogY 572
PTANT/ 50 A ppl i cati ons of P l ant
ic
Transformation,rTransgen
AGRICULTURAI- P l ants 596
51 Transgeni cP l antsas
BIOTECHNOLOGY Bioreactors 622
52 C rorvth P romoti ng B acteri a i n
P l ants 639
53 Mol ecul ar Marker-A i dedP l ant
B reedi ng 648

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495

p lant tissue culture broadly refersto the in vitro
I cultivation of plants, seeds and various parts
of th e p lan ts (o r gans ,em br y os ,t is s ues ,s ingle c e l l s ,
pro top lasts) The c ult iv at ion pr oc es s is inv ar ia b l y
ca rried o ut in a nut r ient c ult ur e m edium un o e r
asep tic co nd itions
Pla nt ce lls hav e c er t ain adv ant agesov er ani m a l
cells in cu lture s y s t em s Unlik e anim al c ells , hig h l y
mature and differentialed plant cells retain the
ability of totipotency i.e the ability of change to
meristematic state and differentiate into a whole
p lan t.
BENEFITS O F PLANT TI SSUE CULTUR E
Pla nt tissu e c ult ur e is one of t he m os t r ap i d l y
g rowin g are asof biot ec hnology bec aus eof it s h i g h
potential to develop improved crops and
o rna men tal p lant s . W it h t he adv anc esm ade in t h e
tissue cultu re tec hnology , it is now pos s ible t o
regeneratespeciesof any plant in the laboratory.To
achieve the target of creating a new plant or a plant
with desired characteristics,tissue culture is often
co up led with r ec om binant DNA t ec hnology . T h e
techn iqu es o f plant t is s ue c ult ur e hav e lar g e l y
helped in the green revolution by improving the
crop yield and quality.
Th e kn owle dge obt ained f r om plant t is s u e
cultu res h as co nt r ibut ed t o our under s t andin go f
meta bo lism, gr owt h, dif f er ent iat ion and
morp ho ge ne siso f plant c ells . Fur t her ,dev elopm e n t s
rn tissu e cultu r e hav e helped t o pr oduc e s ev e r a l
[32]
-::echnology
iilihliiuiifri,-l, ,
merist€m,nucellus)
Cell culture
Protoplast culture
pathogen-free plants, besides the synthesis of many
biologically important compounds, including
pharmaceuticals Because of the wide range of
applications, plant tissue culture attracts the
attention of molecular biologists, plant breeders
and industrialists.
BASIG STRUCTURE AND
G R O WT H O F A P L A N T
A n a d u l t p l a n t b a s i c a l l y c o n s i s t so f a s t e m a n d
a root, each with many branches (Fig. 42.1). Both
the stem and root are characterizedbv the oresence
of apical growth regions which are composed of
meristematic cells These cells are the primary
source for all the cell types of a plant.
T h e p l a n t g r o w t h a n d d e v e l o p m e n to c c u r i n t w o
differenl ways.
1. Determinate growth : This is characterized
by ceasation of growth as the plant parts attarn
c e r t a i n s i z e a n d s h a p e . e . g . , I e a v e s ,f l o w e r s , f r u i t s .
2. Indeterminate growth : This refers to the
continuous growth of roots and stems under
suitable conditions lt is possible due to the
presence of meristems (in stems and roots) which
c a n p r o l i f e r a t ec o n t i n u o u s l y .
A s t h e s e e d g e r m i n a t e sa n d s e e d l i n g e m e r g e s ,
t h e m e r i s t e m a t i cc e l l s o f t h e r o o t a p e x m u l t i p l y .
Above the root apex, the cells grow in length
w i t h o u t m u l t i p l i c a t i o n . S o m e o f t h e e l o n g a t e dc e l l s
of the outer layer develop into root hairs to absorb
497
498
Leaf primordium
Axillarybud
Fig. 42.1 : A diagrammatic view of a plant structure
water and nutrientsfrom the soil. As the plant
grows, root cells differentiate into phloem and
xylem. Phloem is responsiblefor the absorptionof
nutrientswhile xylem absorbswater.
The meristematic cells of the shootapex divide
leadingto the growth of stem. Someof the stem
cells differentiateand develop into leaf primordia,
and then leaves.Axillarybudspresentbetweenthe
leaf primordia and elongatedstem also possess
me ri s te msw h i c h c a n mu i t i pl y and gi ve ri se to
branchesand flowers.
A diagrammaticview of a plantand a flowerare
respectivelyCepictedin Fig. 42.1 and Fig. 42.2
C ON VE I{ T IO N AL PL A N T B R E E D IN G
A N D PL A N T T IS SU E C U LTU R E
Si n c e th e ti m e i m m e m ori al ,man has beerr
Fig. 42.2 : A diagrammatic representatian of important
of pl antsto
c l o s e l yi n v o l v e di n th e i m p rovement
meet his basic needs.The conventional methoos
B IOTECHNO LO G Y
employed tor crop improvement are very tedious
and long time processes (sometirnes decades).
Further,in the conventional breeding nrethods,it is
not possibleto introduce desired genes to generate
new charactersor products.
With the developments in plant tissueculture, it
is now possible to reduce the time for the creation
of new plants with desired characteristics,transfer
of new genes into plant cells and large scale
production of commercially important products.
TERMS USED IN TISSUE CULTURE
A selectedlist of the most commonly used terms
in tissueculture are briefly explained
Explani: An excised piece of differentiated
tissue or organ is regarded as an explant. The
explant may be taken from any part of the plant
body e.g., leaf, stem, root.
- Callus : The unorganized and undiffererrtiated
m a s s o f D l a n t c e l l s i s r e f e r r e d t o a s ca l l u s.
C e n e r a l l y , w h e n p l a n t c e l l s a r e c u l tu r e d i n a
s u i t a b l e m e d i u m , t h e y d i v i d e t o f o r m c a l l u s i .e ., a
rrrassof parenchymatouscells.
Dedifferentiaticn : The pherrornenon of mature
cells reverting to meristematic state to produce
callus is dedifferentiation.
Dedifferentiation is oossible since the non-
d i v i d i n g q u i e s c e n tc e l l s o f t h e e x p l a n t , wh e n g r o w n
Style
Anther
Calyx
Darts in a flower.
fhapt er 42 : P LA N TT ISS U E
C U L IU R E-C EN ER A L 499

i a suitableculiuremediumrevertto meristematic
=:ate
Callusculture
Redifferentiationr The abiiityof tl.recalluscells
into a plant organor a whole plant
:o differentiate Organculture
s regardedas redifferentiation. (embryo,seed,
Anthel ovary,
Totipotency: The abilityof an individualcell to meristem,nucellus)
:er elop int o a who l ep l a n ti s re fe rre d
to a s c e l l u l ar Ceilculture
:rtifrotency.The inherentcharacteristic featuresof
r ant c ells na n re l y d e d i ffe re n ti a ti o n a n d Protoplastculture
':ditferentiation are responsible for the
Fig. 42.3 : Major types of plant tissue cultures.
rlenomenon of totipotency.
The othertermsusedin planttissuecultureare
Organ culture
e r . plained places.
at appro p ri a te
Cultureof isolatedplant organsis referredto as
BF I E F HI S T O RY O F P L AN T T IS SU E organ culture. The organ used may be embryo,
CULT URE seed, root, endosperm, anther, ovary, ovule,
meri stem(shootti p) or nucel l us.
About 250 years ago (1756), Henri-Louis
The organ culture may be organizedor un-
D uham el du M o n c e a u ' d e mo n s tra te dc a l l u s
organi zed.
formationon the decorticated regionsof elm plants.
vlany botanistsregardthis work as the fcrwardfor Organized organ culture : When a well
.l organized structureof a plant (seed,embryo) is
th e dis c ov erof y pla n tti s s u ec u l tu reIn
. 8 5 3 ,T re c ul
p ublis hedpic t ur e so f c a l l u sfo rm a ti o ni n p l a n ts . used in culture, it is referredto as organized
culture.ln this type of culture,the characteristic
Cerman botanist Gottlieb Haberlandt (1902), individualorgan structureis maintainedand the
regardedas the father of plant tissue culture, first progenyformedis similarin structureas that of the
developedthe concepto{ in vitro cell culture.He ori gi nalorgan.
was the first to culture isolated and {ully
Unorganizedorgan culture : This involvesthe
plan t c e l l s i n a n u tri e n tme d i u m .
d i f f er ent iat ed
isolationof cells or tissuesof a part of the organ,
Dur ing 1934-1 9 4 0 , th re e s c i e n ti s tsn a mel y and their culture in vitro. Unorganizedculture
Cautheret, White and Nobecourtlargelycontributed resul tsi n the formati onof cal l us.The cal l uscan be
to the developments made in plant tissueculture. dispersed into aggregates of cellsand/orsinglecells
to gi ve a suspensi on cul ture.
Cood progress and rapiddevelopments occurred
a f t er 1940 in p l a n t ti s u e c u l tu re te c h n i q u es. Cell culture
Stewardand Reinert(1959)firstdiscovered sornatic
embryo productionin vitro.Maheswariand Cuha Thecul tureof i sol atedi ndi vi dualcel l s,obtai neo
(1964)from India 'werethe first to developanther from an expl antti ssueor cal l usi s regarded as cel l
culture and poller culture for the productionof cul ture. These cul tures are carri edout i n di spensi on
h aploidplant s . medium and are ret'erred to as cell suspension
cultures.
TYPES OF CULTURE
Protoplast culture
Thereare differenttypesof plant tissueculture (i .e.,cel l sdevoi dof cel l w al l s)
P l antprotopl asts
te c hniques m
, ain l y b a s e d o n th e e x p l a n t u s ed are also used in the laboratory
for culture.
ffig. a23).
BASIC TECHNIQUE OF PLANT
Gallus culture TIS S U E C U LTU R E
This involvesthe cultureof differentiated trssue The generalprocedureadoptedfor isolationand
from explantwhich dedifferentiates in vitroto form cultureof planttissuesis depictedin Fig.42.4 and
cailus . brieflydescribedin the next page.
502 B IOTECHNO LO CY

Suspension culture from callus Formoredetailedinformationon the application

of cel l cul tures,S eep. 506.
Sus pens ion c ult ur es c a n b e i n i t i a t e d b y
t r ans f er r ingf r iable c allus t o l i q u i d n u t r i e n t m e d i u m
Gell culture technique
ffig. a2.5). As the medium is liquid in nature, the
pieces of callus remain submerged. This creates The in vitro cell culture technique broadly
anaer obicc ondit ion and ult i m a t e l vt h e c e l l s m a v d i e . involvesthe followingaspects:
For this reason, suspension cultures have to be 1. l sol ati onof si ngl ecel l s.
agitaiedby a rotary shaker.Due to agitation,the cells
2. S uspensi on cul tures grow thandsubcult ur ing.
geis dispersed,besidestheir exposureto aeration.
cultures.
3. Typesof suspension
Applications of callus cultures of suspensi ocult
4. S ynchroni zati on n ur es.
Callus c ult ur es ar e s low - g r o w t h p l a n t c u l t u r e 5. Measurement of growth of cultures.
systemsin static medium. This enabies to cono'uct of vi abi l i tyof cu lt ur edcells.
6. Measurement
several studies related to many aspects of plants
(growth, differentiation and rnetabolism) as lisied of the abovestepsare briefly
The salientfeatures
below.
descri bed.

o Nut r it ional r equir em ent so f p l a n i s . ISOLATION OF SINGLE CELLS

. Cell ar r d or gan dilier ent i a l i o n .
The cells employedfor in vitro culturemay be
. Development of suspensioi.r and protoplast obtained from plant organs.and from cultured
c u it ur es . trssues.
. Som ac lonal v ar iat ions . From plant organs r Plant leaves with
r Cenetic transformations ho..nogenous population of cells are the ideal
r Production of secondarv rnetabolites arrd their sourcesfor cel l cul ture.S i ngl ecel l sca n be isolat ed
from leavesby mechanicalor enzymaticmethods.
r egulat ion.
Mechanicalmethod: Surface sterilized leavesare
cut i nto smal l pi eces(< 1 cm2), suspended in a
medi um and subj ected to gri ndi ng in a glass
homogeniser tube.Thehomogenate is filteredthrough
The first attempt to culture single cells (obtained filtersand thencentrifuged at a low speedto remove
the cellulardebris.The supernatant is removedand
from leaves of flowering plants) was made in as
early as 1902 by Haberlandt. Although he was dilutedto achievethe requiredcell density.
unsuccessfulto achieve cell division in vitro, his Enzymaticmethod : The enzyme macerozyme
work gave a stimulus to several researchers.In later (undersuitableosmoticpressure)catt releasethe
years, good successwas achieved not only for cell i ndi vi dualcel l sfrom the l eafti ssue s.
M acer ozym e
div is ion but als o t o r ais e c o m o l e t e p l a n t s f r o m degrades mi ddl e l amel l a and ce ll walls of
s ingle c ell c ult ur es . parenchymatous ti ssues.
From cul tured ti ssues: S i ngi ecelis can be
Applications of cell cultures isolatedfrom calluscultures(grownfi'omcut pieces
Cultured celis have a wide range of applications of surface sterilized plant parts). Repeated
in biologv. subcul turi ng of cal l uson agar medium im pr oves
.l the fri abi l i ty
of cal l usso that fi ne cell suspensions
. Eluc idat ion of t he p a t h w a y s o f c e l l u l a r
are obtained.
m et abolis m .
2. Serve as good targets for mutation and S U S P E N S ION C U LTU B E S -
selection of desirable mutants- GROWTH AND SUBCULTURE
3. Production of secondary metabolites of
The i sol atedcel l s are grow n i n suspension
commercial interest.
cul tui ' es.C el l suspensi onsare maint ainedby
4. Cood potential for crop improvement. routi nesubcul tr,rri ngi n a fresh med ium .For t his
 42 : PL A N TT IS SU E
ChA O I CT C U L T U R E-C .EN E R A L 503

pu rpo se , th e c ells ar e pic k ed up in t he e a r l y

stationary phase and transferred.
As th e cells ar e inc ubat ed in s us pe n s i o n
cu iture s, th e c ells div ide and enlar ge. T h e
in cu ba tion p er iod is dependent on :

o lnitia l cell dens it y

. Duration of lag phase .g
o Crowth rate of cells.
5
Among these, cell density is very crucial. The o
initia l cell de ns it y us ed in t he s ubc ult ur esis v e r y
critical, and largely depends on the tvpe of
su sp en sio n cu lt ur e being m aint ained. W it h l o w
in itial ce ll d en s it ies t, he lag phas eand log phas e so f
growth get prolonged. Whenever a new suspension
cr-rltureis started, it is necessaryto determine the
o ptical cell de ns it y in r elat iont he v olum e of c u l t u r e __ | fims -- )
med ium, so t hat m ax im um c ell gr owt h c an b e
a ch ieved . With low c ell dens it ies ,t he c ult ur e w i l l Fig. 42.6 : A graphic rcpresentation of different
no t g row weil, and r equir es additi o n a l phases of batch suspension culture.
su pp leme nta t ionof m et abolit est o t he m edium .

Th e no rmal inc ubat ion t im e f or t he s us pe n s i o n The batch cul tures can be mai nta!ned
cultures is in the range of 21-28 days. smal lamountsof the
y transferri ng
conti nuousl by
susoensi onmedi unr (w i th i nocul unr)to fresn
TYPES OF SUSPENSION CULTURES medi umat regul ari nterval s(2-3 days).
Th ere are m ainly t wo t y pes of s us pe n s i o n Batchculturesare qharacterized
by a constant
cu lture s- b atc h c ult ur es and c ont inuous c ultu r e s . cl.range in the pattern of cell growth and
metabol i sm. For thi s reason,thesecul turesare not
Batch cultures i deal l y sui ted for the studi esrel atedto cel l ul ar
A bat c h c u l tu re i s a c e l l s u s p e n s i o n c u l ture behaviou r.
grown in a fixed volume of nutrient culture
m edium .I n b a tc h c u l tu re ,c e l l d i v i s i o na n d cel l Gontinuous cultures
growth coupled with increasein bicmassoccur In conti nuouscul tures, there i s a regul ar
unt il one of t h e fa c to rsi n th e c u l tu i ' ee n v i rc nment addition of fresh nutrient medium and draining
( nut r ientO
, , s u p p l y )b e c o m e sl i m i ti n g .T h e cel l s
out the used mediuntso that the culturevolume is
exhibit the following five phasesof growth when normallyconstant.Theseculturesare carriedout in
the cell number in suspension culturesis plotted speciallydesignedculturevessels(bioreactors).
againstthe time of incubation(Fig.42.6).
C onti nuouscul tures are carri ed out under
1. Lag phase characterizedby preparationof defined and controlled conditions-cell density,
c ellst o div ide . nutri ents,Or, pH etc. The cel l s i n thesecul tures
2. Log phase(exponential phase)wherethe rate are mostlyat an exponentialphase(log phase)of
i s h i g h e s t.
of c ell m ult ip l i c a ti o n growth.
3. Linearphaserepresented by slownessin cell Continuousculturesareof two types-open and
div is ionand i n c re a s ei n c e l l s i z ee x p a n s i o n. cl csed.
4. Deceleration phase characterized by Open continuouscultures: In thesecultures,the
in c e l l d i v i s i o na n d c e l l e x p a n s i o n '
dec r eas e inflowof freshnrediumis balancedwith the ouiflow
5. Stationaryphase representeC by a constant of the vol umeof spentmedi umal ongw i th the cel l s.
num berof c e l l sa n d th e i r s i z e . The additionof fresh mediumand cultureharvest
504
are so adjusted that the cultures are maintained
indefinitely at a constant growth rate. At a steady
state, the rate of cells removed from the cultures
equals to the rate of formation of new cells.
Open continuous culture system is regarded as
chemostat if the cellular growth rate and density
ar e k ept c ons t ant by lim i t i n g a n u t r i e n t i n t h e
m edium ( gluc os e, nit r o g e n , p h o s p h o r u s ) . I n
c hem os t atc ult ur es ,ex c ept t h e l i m i t i n g n u t r i e n t , a l l
oiher nutrientsare kept at higher concentrations.As
a r es ult , any inc r eas e or d e c r e a s e i n t h e l i m i t i n g
nut r ient will c or r es pondin g l y i n c r e a s eo r d e c r e a s e
the growth rate of cells. are subjected to lorv temperature (around 4'C)
ln turbidostat open continLrous cultures, s h o c k s y n c h r o n i z a t i o n o c c u r s . C o l d tr e a tm e n t r n
addit ion of f r es h m edium i s d o n e w h e n e v e r t h e r e r s combination with nutrient starvation gives better
an inc r eas e in t ur bidit y s o t h a t t h e s u s p e n s i o n results.
c ult ur e s y s t em is m aint a i n e d a t a f i x e d o p t i c a l
dens it y .Thus , in t hes e c ul t u r e s y s t e m s ,t u r b i d i t y i s culture can be selected based on the size of the
preselected on the basis of biomass density rn aggregates.and by this approach, cell synchro-
c ult ur es , and t hey ar e m a i n t a i n e d b y i n t e r m i t t e n t n i z a t i o n c a n b e a c h i e v e d .
addit ion of m edium and w a s h o u t o f c e l l s .
Closed continuous cultures : In these cultures,
t he c ells ar e r et ained wh i l e t h e i n f l o w o f f r e s h
medium is balanced with the outflow of
c or r es pondings pent m edi u m . T h e c e l l s p r e s e n t i n
the outflowing medium are separated(mechanically)
and added back to the culture system.As a result,
t her e is a c ont inuous incr e a s e i n t h e b i o m a s s i n
c los ed c ont inuousc ult ur es.T h e s ec u l t u r e sa r e u s e f u l
for studiqsrelated to cytodifferentiation,and for the
production of certain secondary metabolites e.g,
poly s ac c har idesc, oum ar in s .
SYNCHRONIZATION OF
SUSPENSI O N CULTUR E S
I n t he nor m al c ir c um s t a n c e s t, h e c u l t u r e d p l a n t
c ells v ar y gr eat ly in s iz e, s h a p e ,c e l l c y c l e e t c . , a n d
are said Io be asynchronous. Due to variations in
t he c ells , t hey ar e no t s u i t a b l e f o r g e n e t t c ,
bioc hem ic al and phy s iolo g i c a l s t u d i e s . F o r t h e s e
r eas ons , s y nc hr oniz at io n o f cells assumes
s ignif ic anc e
Synchronizationof cultured cells broadly refers
to the organized existence of majority of cells in
the same cell cycle phase simultaneously.
A synchronous culture may be regarded as a
c ult ur e in whic h t he c ell c y c l e s o r s p e c i f i c p h a s eo f
cycles for n.rajority of cultured cells occur
s im ult aneous ly .
B IOTECHNO LO CY
Several methods are in use to bring out
s y n c h r o n i z a t i o no f s u s p e n s i o nc u l t u r e s. Th e y m a y
b e b r o a d l y d i v i d e d i n t o p h y s i c a l a n d ch e m i ca l
methods.
Physical methods
T h e e n v i r o n m e n t a l c u l t u r e g r o w th i n fl u e n ci n g
physical parameters (light, temperature) and the
p h y s i c a l p r o p e r t i e so f t h e c e l l ( s i z e )c a n b e ca r e fu l l y
monitored to achieve reasonably good degree of
s y n c h r c l n i z a t i o nA. c o u p l e o f t h e m ar e d e scr i b e d
Cold treatment : When the suspensioncultures
Selection by volume : The cells in suspension
Ghemical methods
- f h e c h e m i c a l m e t h o d s f o r s y n ch r o n i za ti o n o f
s u s o e n s i o n c u l t u r e s i n c l u d e t h e u se o f ch e m i ca l
inhibitors, and deprivation of an essential growth
factor (nutrient starvation). By this approach, the
cell cycle can be arrested at a particular stage, and
t h e n a l l o w e d t o o c c u r s i m u l t a n e cu sl y so th a t
s y n c h r o n i z a t i o ni s a c h i e v e d .
C h e m i c a l i n h i b i t i o n : I n h i b i to r s o f D N A
synthesis (5-aminouracil, hydroxyurea, 5-fluoro-
deoxypurine),when added to the cultures resultsin
t h e a c c u m u l a t i o n o f c e l l s a t C 1 p h a se . An d o n
removal of the inhibitor. svnchronization of cell
division occurs.
Colchicine is a strong inhibitor to arrest the
growth of cells at metaphase. lt inhibits spindle
formation during the metaphase stage of cell
d i v i s i o n . E x p o s u r et o c o l c h i c i n e m u st b e d o n e fo r a
s h o r t p e r i o d ( d u r i n g t h e e x p o n e n t i a lgr o w th p h a se ) ,
as long duration exposure may lead to mitoses.
Starvation: When an essentialnutrient or growth
p r o m o t i n g c o m p o u n d i s d e p r i v e d i n su sp e n si o n
cultures,this resultsin stationarygrow,thphase. On
s u p p l e m e n t a t i o no f t h e m i s s i n g n u t r i en t co m p o u n d ,
cell growth resumptionoccurs synchronously.Some
workers have reported that deprivation and
subsequent addition of growth hormone also
i n d u c e s s y n c h r o n i z a t i o no f c e l l c u l t u r e s.
Chaot er42 : P L AN TT IS SU E
C U L T U R E-C EN E R A L
M E A S URE ME N T O F G R O W T H OF
CULT URE S
It is necessaryto assessthe growth of cells
in cultures. The oarametersselected for the
measuringgrorvthof suspensionculturesinclude
c ell c ount ing ,p a c k e d c e l l v o l u m e a n d w ei ght Fluorescein diacetate method
Increase.
Gell counting
Althoughcell countingto assess culturegrowth
is reasonablyaccurate,it is tedious and time
c ons um ing.Th i s i s b e c a u s ec e l l s i n s u s p e nsi on
culture mostly exist as coloniesin varying sizes.
Thesecells have to be first disrupted(by treating
with pectinaseor chromic acid), separated,and
then counfed using a haemocytometer.
Packed cell volume
Packedcell volume (PCV)is expressed as ml of
pellet per ml of culture. Tc determine PCV, a
m eas ur ed v o l u me o f s u s p e n s i o nc u l tu re i s
centrifuged(usuallyat 2000 xg for 5 minutes)and
the volume of the pelletor packedcell volume is
recorded.
After centrifugation the supernatant can be
discarded,the pellet washed,dried overnightand
weighed.This gives cell dry weight.
Gell fresh weight
The wet cells are collectedon a preweighed
nylon fabric filter (supportedin funnel).They are
washed to remove the medium, drained under
vacuumand weighed.Thisgivesthe freshweightof
cells. However,Iarge sampleshave to be usedlor
accurateweights.
M E A S URE M EN T O F VIA BIL IT Y O F
CULT URE D C E L L S
The viabilityof cellsis the mostimportantfactor on the filter paper (Fig. a2.7A). This filter paper,
f or t he gr owt ho f c e l l s .Vi a b i l i tyo f c u l tu re dcel l s wetted by the exudatesfrom callus tissue (by
can be measr.rred by microscopicexaminationof di ffusi on)suppl i esthe nutri entsto the si ngl ecel l .
c ellsdir ec t lyo r a fte rs ta i n i n gth e m .
Phase contrast microscopy
The viablecellscan be detectedby the presence
of healthy nuclei. Phasecontrastmicroscopeis
usedfor this purpose.
505
Evan's blue staining
A dilute solutionof Evan'sblue (0.025%w/v)
dye sfains the dead or damaged cells while the
l i vi ng (vi abl e)cel l sremai nunstai ned.
W hen the cel l susoensi oni s i ncubatedw i th
fluoresceindiacetate(FDA)at a final concentration
of 0.01oh,it is cleavedby esterase enzymeof living
cells.As a result,the polar portion of fluorescein
which emits green fluoroscenceunder ultraviolet
(UV) light is released-The viable cells can he
detected by their.fluoroscence,since fluorescern
accumul ates i n the l i vi ngcel l sonl y.
A clone is a rnassof cells,all of them derived
throughmitosisfrom a singlecell. The cellsof the
clone are expectedto be identicalwith regardto
genotype and karyotype.However, changes in
thesecel l s may occur after cl oni ng.S i ngl ecel l s
separated from plant tissues under suitable
conditionscan form clones.
' The variousmethodsfor the isolationof single
cells have already been described(Seep. 502).
S i ngl e cel l s can be cul tured bv the fol l ow i ng
methods:
1. Filterpaperraft-nurse
tissuetechnique
2. Microchambertechnique
3. Microdropmethod
4. B ergman'pl
s ati ngtechni que.
Filter paper raft.nurse tissue technique
Smallpiecesof sterilefilterpapersare placedon
established callusculturesseveraldays beforethe
startof si ngl ecel l cul ture.S i ngl ecel l i s now pl aced
The cell divides and forms clonies on the filter
paper.Thesecoloniescan be isolatedand cultured.
Microchamber technique
A microscopicslide or a coverslipcan be used
to create a micro?hamber.Sometimes,a cavitv
506 B IOTECHNO LO CY

(A) Bergmann's plating technique

B e r g m a n n ( 1 9 6 0 ) d e v e l o p e d a t e ch n i q u e fo r
c l o n i n g o f s i n g l e c e l l s . N o w a d a y s , Be r g n ta n n 's
plating technique is the mosf wiclely used method
f o r c u l t u r e o f i s o l a t e d s i n g l e c e l l s . T h i s m e th o d i s
depicted in Fig. 42.8 and briefly describedhereunder.

The cell suspensionis filtered through a sieve to

o b t a i n s i n g l e c e l l s i n t h e f i l t r a t e . T h e fr e e ce l l s a r e
s u s p e n d e di n a l i q u i d m e d i u m , a t a de n si ty tw i ce
than the required density for cell plating. Now,
equal volumes of melted agar (30-35'C) and
Coverslips m e d i u m c o n t a i n i n g c e l l s a r e m i x e d. Th e a g a r
m e d i u m w i t h s i n g l e c e l l s i s p o u r e d a n d sp r e a d o u t
in a petridish so thai the cells are evenly distributed
oil on a thin layer (of agar after it solidifies). The
Microscope petridishes(culturedishes)are sealedwith a parafilm
Singlecell slide and incubated at 25'C in dark or diffused light. The
in a drop of
s i n g l e c e l l s d i v i d e a n d d e . v e l o pi n t o c l on e s.
medium
T h e v i a b i l i t y o f c e l l s i n s i n g l e c l o n e s ca n b e
measured by the same techniques that have been
, d e s c r i b e d {o r s u s p e n s i o nc u l t u r e s .
uupraKo!sn
lvlicrowell

Outer chamber

Singlecell
in a drop ol
medium
Fig. 42.7 : Techniques for the culture of single cells
(A) Filter paper raft-nurse tissue technique
(B) Microchanber technique (side u,iew),
(C) Microdrop method (Refer Fig. 42.8 for
Bergmann's plating technique).
s lide c an be dir ec t ly us ed. A d r o p o f t h e m e d i u m
c ont aining a s ingle c ell i s p l a c e d i n t h e
m ic r oc ham ber .A dr op of m i n e r a l o i l i s p l a c e d o n
either side of the culture drop which is covered
with a coverslip (Fig. 42.78). On incubation, slngle
cell colonies are forme<-r.
Microdrop method
For t he c ult ur e of s ing l e c e l l s b y m i c r o d r o p
m et hod, a s pec ially des igne d d i s h ( c u p r a k d i s h ) i s
useci.lt has a snrall outer chamber (to be filled u,ith
s t er ile dis t illed wat er ) and a l a r g e i n n e r c h a m b e r
with a number of rnicrowells (Fig. a2.7A. The cell
density of the medium is adjusted in such a way
that it contains one cell per droplet.
Plant tissue cultures are associated with a
wide range of applications--the most important
being the production of pharmaceutical, medicinal
and other industrially important compounds. ln
addition, tissue cultures are useful for several other
purposes listed below.
'!
. To study the respiration and metabolism of
Dlants.
2. For the evaluation of organ functions in
olants.
3. To study the various plant diseasesanci work
out methods for their elimination.
4 . S i n g l e c e l l c l o n e s a r e u s e f u l fo r g e n e ti c,
m o r p h o l o g i c a l a n d p a t h o l o g i c a l s t u d ie s.
5 . E m b r y o n i c c e l l s u s p e n s i o n sc a n b e u se d fo r
large scale clonal prapagation.
6 . S o m a t i c e m b r y o s f r o m c e l l s u sp e n si o n sca n
be stored for long term in germplasm banks.
7 . l n t h e p r o d u c t i o n o f v a r i a n t c l o n e s w i th n e w
characteristics, a phenomenon referred to as
somaclonal variations.
Chapt er42 : PL A N TT IS SU E
C U L T U R E-GE N E R A L 507

8. Productian of haploids (with a single set of

chromosomes)for improvingcrops.
9. N4utantcells can be selectedfrom cultures
and usedfor crop improvement.
l 0.l mmatureembryoscan be cul turedi n vi tro
to producehybrids,a processreferredto as embryo
rescue.

The chemi calcompoundsproducedby pl ants

are collectively referred to as phytochemicals.
Biotechnologists havespecialinterestin plantiissue
cul ture for the l arge scal e producti on of
commerci al liymportantcompoundsThesei ncl ude
pharmaceuticals, flavours, fragrances,cosmetics,
food additives,feedstocks and antimicrobials.
Most
Moltenagar
medium
of these products are secondary metabolites-
chemical compounds that do not participate in
metabolismof plants. Thus,secondarymetabolites
are not directly neededbv plantsas they do not
^ oo
u
o o o o. performany physiologicalfunction(as is the case
^--":"-^ (
ooo o oo^Q^7
{o o ou oo-i, w i th pri mary metabol i tessuch as ami no aci ds,
Singlecells nucl ei caci dsetc.).
in medium
Although the native plants are capable of
I
J producingthe secondary metabolites of commercial
interest,tissueculture systemsare preferred.The
advantages and limitationsare Iisted.
ooo
ooo o
Oo oo Major advantages
Culturedish
1. C ompounds can be produced under

J controlledconditionsas oer marketdemands.

2. Culture systems are independent of
environmentalfactors,seasonalvariations,pestand
microbialdiseasesand geographical constraints.

Culturedish sealedwith parafiim

3. Cell growth can be controlledto facilitate
improvedproductformation.
+
I 4. The qual i tyof the productw i l l be consi stent
as i t i s producedby a speci fi ccel l l i ne.
Cellcolony 5. Recoveryof the productwill be easv,
6. P l antcr,rl turesare parti cul arluseful
y i n case
Too view of culturedish
of plants which are difficult or expensiveto be
grow n i n the fi el ds.
7. Mutantcel l l i nescan be devel opedfor the
Fig. 42.8 : A dlagrammatic representation of
production of novel compounds of commercial
Bergmann's cell plating technique.
importance,which arenot normallyfoundin plants.
508
B. Biotransformation reactions (converting
specific substratesto valuable products) can be
c ar r ied out wit h c er t ain c ult u r e d c e l l s .
9. The production control is not at the mercy of
oolitical interference.
10. The pr oduc t ion t im e is l e s s a n d l a b o u r c o s t s
ar e m inim al.
Considering the advantages listed above,
about 25-30V" of medicines for human use, and
t he v ar ious c hem ic al m at e r i a l s f o r i n d u s t r i a l
purposes are obtained from plant tissue cultures.
I n gener al, t is s ue c ult ur e p r o d u c t i o n o f n a t u r a l
materials is cheaper compared to synthetic
production. However, there are certain limitations
associatedwith tissue cultures.
Limitations/disadvantages
1. ln general, in vitro production of secondary
metabolites is lower when comoared to intact
plant s .
2. Many a times, secondary metabolites are
formed in differentiated tissues/organs.In such a
case, culture cells which are non-differentiatedcan
produce little.
3. Cultured cells are genetically unstable and
may undergo mutation. The production of
secondary metabolite may be drastically reduceo,
as the culture ages.
4. Vigorous stirring is nec€ssary to prevent
aggregation of cultured cells. This may often
damage the cells.
5. Strict aseptic conditions have to be
m aint ained dur ing c ult ur e t ec h n i q u e . A n y i n f e c t i o n
to the culture adversely affects product formation.
Why do plants produce
secondary metabolites?
Based on the existing evidence, it is believed
that the production of some secondary metabolites
is link ed t o t he induc t io n o f m o r p h o l o g i c a l
d ifferentiation.
Cons ider t he f ollowing exa m p l e s
1 . Cardiac glycosidesare found in the leavesof
Digitalis.
2. Q uinine and quinidin e a r e p r e s e n t i n t h e i nstance,one kg of vi ncri sti neand vinblast ine
bark of Cinchona.
B IOTECHNO LO CY
3. Tropanealkaloids(e.9.atropine)are found in
the roots of Atropa.
thatasthe cellsundergomorphological
It appears
and maturationduring plant growth,
differentiation
some of the cells specialiseto producesecondary
metabolites.It is also observed that in vitro
productionof secondary metabolites is muchhigher
from differentiatedtissueswhen comparedto non-
or lessdifferentiated
differentiated tissues.
APPLICATIONS OF
SECONDARY METABOLITES
From the ti me i mmemori al ,man has been
dependenton the plani products, besidesthe
supplyof food from plants.Theseplant products,
mostly the secondary metabolites include
flavours,perfumes,
pharmaceuticals, agrochemicals,
insecticidesand raw materials for industries.
Chemically,the plant productsmay be alkaloids,
terpenoids,glycosides(steroids,
phenolics)etc.
A s and w hen avai l abl e,the nat ur al plant
productsare preferredto syntheticproducts,by
man. Accordingto a WHO survey,nearly70-8(P/o
of the world population depends on herbal drugs.
It is a fact that many chemicalswith complex
structuresthat cannot be chernicallysynthesized
can be convenientlyproducedin plants.
The productionof specialitychemicalsby plants
i s a mul ti bi l l i oni ndustry.The pl ant c ell cult ur es
providelaboratorymanagedsourcesfor the supply
of usefulplantproducts.Althoughhundredsof new
compoundsare identifiedeveryyear in plants,only
a few of them are of commercial importance.
Attemptsare madeto producethem in cell culture
svstems.
A selectedlist of plant productsobtainedfrom
pl ant cel l cul turesal ongw i th thei r app licat ions
is
given in Table42.1.
S hi koni nei s a dye produced by t he cells
Lithospermum erythrorhizonon a commercialscale.
The other productssuccessfully producedin plant
cell cultures include analgistics(codeine)anfi-
malarial(quinine),musclerelaxants(atropine),drugs
to control cardiovascular disorders (digoxin),
hypotensives (reserpine), perfumes (jasmine),
insecticides (pyrithrins), food sweeteners(stevios
ioe,r
and anticancer agents(vincristine).Sometimes,the
costof the pl antproductsi s uni magi nably high.For
respectively cost $ 3,500,00and $ 1,000,000!
Chaot er42 : P L A N T T ISS U E
C U L T U R E -C E NE R A L 509

PRODUCTION OF SECONDARY
Tleu 42.1 A selected llst of secondary METABOLITES
metabolltesobtained from plant cell cultures
along with their application(s) The process of in vitro culture of cells for the
l a r g e s c a l e p r o d u c t i o n o f s e c o n d a r y m e t a b o l i t e si s
Product Uses complex, and involvesthe following aspects
Shikonine Lilhospemun Dye, .l
Selection of cell lines for high yield of
erythrorhizon pharmaceutical
secondary metabolites.
Codeine, Papaversomniferun Analgistic
2 Largescale cultivation of plant cells
m0rpnrne
Quinine Cinchonaofticinalis Antimalarial 3. Medium composition and effect of nutrients.
Atropine Atropabelladcnna Muscle 4. Elicitor-induced production of secondary
relaxanl metabolites.
Digoxin n;-;t^t:^:^^^.^
urguauJ tat ra@ Cardiovascular
disorders 5. Effect of environmental factors.
Reserpine Rauwolfia seryentina Hypotensive 6 . B i o t r a n s f o r m a t i o nu s i n g p l a n t c e l l c u l t u r e s .
Diosgenin Dioscorea deltoidea Antitertility 7 . S e c o n d a r y m e t a b o l i t e r e l e a s ea n d a n a l y s i s .
Vanillin Vanilla sp Vanilla
Jasmrne Jasniumsp Perf
ume SELECTION OF CELL L'NES FOB H'GH
Vinblastine, Catharanthus Anlicancer YIELD OF SECONDARY METABOLITES
ajmalicine, r0seus
vincristine The very purpose of tissue culture is to produce
high amountsof seccndarymetabolites.However, in
Taxol Taxusbrevifolia Anlicancer
g e n e r a l , m a j o r i t y o f c a l l u s a n d s u s p e n s i o nc u l t u r e s
Baccharine Baccharis negapotanica Anticancer
produce less quantities of secondary metabolites.
Cesaline Caesalpinia gillisesii Anticancer
This is mainly due to the lack of fully differentiated
Fagaronine Fagara zanthoxyloides Anticaner
c e l l s i n t h e c u l t u r e s .S o m e s p e c i a l t e c h n i q u e sh a v e
Maytansine Maytenus bucchananii Anticancer been devisedto select cell lines that can oroduce
Harring tonine Cephalotaxus harringtoniaAntrcancer higher amounts of desired metabolites. These
ThalicarpineThalictrum dasycarpun Anticancer methods are ultimately useful for the separation of
Ellipticine, Ochrosia moorei Anticancer producer cells from the non-producer cells. fhe
3-deoxycolchine techniques commonly employed for cell line
Pyrith rins Ttdolttc araala Insecticide s e l e c t i o n a r e c e l l c l o n i n g , v i s u a l o r c h e m i c a l
Chrysanthemun a n a l y s i sa n d s e l e c t i o nf o r r e s i s t a n c e .
cincerariefoliun
Rotenoids Derriselliptica lnsecticide Gell cloning
Tephrosia sp
This is a simple procedure and involves the
Nicotine Nicotiana tabacum Insecticide growth of single cells (taken from a suspension
Nicotiana rustica c u l t u r e s )i n a s u i t a b l em e d i u m . E a c hc e l l p o p u l a t i o n
Saff ron Crocus sativus Foodcolour is then screened for the secondary metabolite
andllavouring f o r m a t i o n . A n d o n l y t h o s e c e l l s w i t h h i g h - y i e l d i n g
agenr a b i l i t y a r e s e l e c t e da n d m a i n t a i n e d 'b y s u b c l o n i n g .
Stevioside Steviarabaudiana Sweetener
Thaumatin Thaumatococcus danielli Sweetener Single cell cloning : There are certain practical
difficulties in the isolation and culture of single
v dPJ dt uil l Capsicum frutesus chiili
cells.
Rosamarinic Coleus blunei Spice,
acrd antioxidant Cell aggregatecloning : Compared to single cell
Anthraquinones Morindacitrifolia Laxative,
dye cloning, cell aggregate cloning is much easier,
Berberine a^^t;^;^^^^i^a
wuPuJ Jdyut rtua Antibacterial hence preferred by many workers.
Sarcoplasmine Daturastramoniun Treatment
of A schematic representation of cell aggregate
(hyoscine) nausea yielding
cloning for the selection of cells high
510 B IOTECHNO LO CY

quantities of secondary metabolites is given in

Fig. 42.9. A high yielding piant of the desired
metabolite is selected and its exolants are first
c ult ur ed on a s olid m edium . A f t e r e s t a b l i s h i n gt h e HighyielCing
plant
c allus c ir lt ur es ,high m et ab o l i t e p r o d u c i n g c a l l u s e s
ar e ident if ied, and t hey ar e g r o w n i n s u s p e n s i o n
cultures. Cell aggregatesfront these cultures are
gr own on s olid m edium . Th e f r e s h l yd e v e l o p e d c e l l
aggregates(calluses) are divided into two parts.
O ne half is gr own f ur t her , w h i l e t h e o t h e r h a l f i s
used for the quantitatirre unu;Ur,r of the desired J
m et abolit epr oduc ed. The ce l l l i n e s w i t h h i g h y i e l d
Explant
of secondary metabolitesare selected and used for
culture
s c ale- upin s us pens ionc ult u r e s .T h i s i s f o l l o w e d o y
large scale tissue culture in a bicreactor.

Visual or chemical analysis

A direct measurementof some of the secondary, Established

callusculture
metabolites produced by cell lines can be done
eit her by v is ual or c hem ic a l a n a l v s i s .

Vis ual ident if ic at ion of c e l l l i n e s p r o d u c i n g

Selectionof
coloured secondary metabolites (pigments e.g., meiabolite
B-carotene,shikonin) will help in the selectian oI producingcalluses
high- y ieldingc ells . This m e t h o d i s q u i t e s i m p l e a n d
non- des t r uc t iv e.The m ajo r l i m i t a t i o n i s t h a t t h e
des ir ed m et abolit e s hould b e c o l o u r e d .
Establish
Certain secondarymetabolitesemit fluorescence suspensionculture
under UV' I ight , and t he c o r r e s p o n d i n gc l o i r e s c a n
be ident if ied.

Som e wor k er s us e s i m p l e . s e n s i t i v e a n d
inex pens iv e c hem ic al an a l y t i c a l m e t h o d s f o r Growthof cell
quantitative estimation of desired metabolites. aggregares
Analy s is is c ar : r iedout in s o m e c o l o n i e s d e r i v e d
f r om s ingle c ell c ult ur es . Ra d i o i m m u n o a s s a yi s t h e
m os t c om m only us ed a n a l y t i c a l m e t h o d .
Microspectrophotometry and fluorescent antibody Subculture Subculturefor
t ec hnioues ar e als o in us e. for gro\4/th metaboliteanalysis
lines(highyielding)
Selection for resistance

Cer t ain c ells r es is t antt o t o x i c c o m p o u n d s m a y

Scale-up in
lead to the formation of mutant cells which can suspensionculture
overproduce a primary metabolite, and then a
secondary metabolite. Such mutants can be
selected and used to produce the desired
metabolite in large quantit!es. One exarnple is
J
To bioreactor
des c r ibed.

Cell lines selected for resistance of 5-methyl- Fig. 42.9 : A schematic representation of cell
tryptophan (analogueof tryptophan)produce strains
which can overproduce tryptophan. These
 C U L T U R-C
" : P LAN TT ISS U E
Chapt er42 E ENERAL
trypto ph an o ve rpr oduc ing s t r ains c an s y nt f r es iz e
.l
0-5 0 time s h igh er lev els of t he nat ur al aux t n
name ly in do le acet ic ac id ( Not e : The s ec ondar y
metab olite ind ole ac et ic ac id is der iv ed f r om t ne
primary meta bo lit e t r y pt ophan) .
LABGE SCAL E ( M ASSI CULTM TTO N
OF PLANT CELLS
In ord er to a ch iev e indus t r ial pr oduc t ion of t he
desired me tab olite , lar ge s c ale c ult iv at ion of plan t
ce lls is re qu ired . Plant c ells ( 20- 1 50 pm in
dia mete r) a re ge ner ally 10- 1 00 t im es lar ger t han
bacteria l or fun ga l c ell W hen c ult ur ec l,plant c elis
ex hib it cha ng es in v olunr es anc i t hus v ar iable
sha pe s a nd size s. Fur t her ,c ult ur ed c ells hav e low
growth rate and genetic instability.All these aspects
have to be con sid er edf or nr as sc ult iv at ion of c ells
T h e fc,llo wing fou r dif f er ent c ult ur e s y s t em s ar e
rrridely used
1. Fre e-cellsus pens ionc ult ur e
2 . lmino bilized c ell c ult ur e
3 lvvo -ph ases y s t em c ult ur e
4. Hairy root culture.
Free-cell suspension culture
Mass cu ltiva tion of piant c ells is m os t f r equent l y
carrie d o ut by ce ll s us pens ionc ult ur es .Car e s hould
be taken to achieve good growth rate of cells and
efficient formation of the desired secondary membranes : Tubular hollow fibres composed of
meta bo lite. Ma ny s pec ially des igned bior eac t or s c e l l u l o s e a c e t a t e s i l i c o n e p o l y c a r b o n a t e a n d
ar e in u se for free - c ells us pens ionc ult ur es .Som e o f o r g a n i z e d i n t o p a r a l l e l b u n d l e s a r e u s e d f o r
t hese are listed be low :
o Batch bioreactors
o Con tinu ou s b ior eac t or s
o lvlultistagebioreactors
Airlift bioreactors
"
. Stirred tank bioreactors
Two imporiant aspectshave to be consideredfor
good successof suspensioncultures
1. Ade qu ate a nd c or r t inuousox y gen s upply .
2 . Minima i g en er at ionof hy dr ody nam ics t r es se s
du e to ae ratio n a git at ion.
lmmobilized cell cultures
Plan t ce lls can be m ade im m obiie or im m ov able agitated) and perfused at a slow rate with an
and u se d in cu ltur e s y s t em sThe
. c ells ar e phy s ic all y a e r a t e d c u l t u r e m e d i u m
511
immobilized by entrapment. Besides individual
cells. it is also possible to immobilize aggregate
c e l l s o r e v e n c a l l u s e s .H o m o g e n o u ss u s p e n s i o n so f
c e i l s a i 'e m o s t s u i t a b l e f o r i m m o b i l i z a t i o n .
Surface immobilized plant cell (SIPC) technique
e f f i c i e n t l yr e i a i n st h e c e l l s a n d a l l o w s t h e m t o g r o w
at a higher rafe. Further, through immobilization,
there is better cell-to-cell contact, and the cells are
p r o t e c t e d f r o r n h i g h l i q u i d s h e a r s t r e s s e sA . ll this
helps in the maximal production the secondary
metabolite.
The common rrethods adopted for entraprnent
of cells are briefly described.
1 . E n t r a p m e n to f c e l l s i n g e l s : T h e c e l l s o r t h e
protoplastscan be entrapped in several gels e 9.,
'Ihe
al6irnate,agat agarose,carrageenin. gels may
be used either individualiy or in combination The
t e c h n i q u e s e m p l o y e d f o r t h e i n . r r n o b i l i z a t i o no f
plarrt cells are comparable to those used for
i r n n r o b i l i z a t i o n o f m i c r o o r s a n i s m so r o t h e r c e l l s
( R e f e rC h a p t e r 2 1 ) .
2. Entrapnrent of cells in nets or foams:
Polyurethanefoams or nets with various pore sizes
are used. The actively growing plant cells in
s u s p e n s i o nc a n b e i m m o b i l i z e d o n t h e s e f o a m s .
T h e c e l l s d i v i d e w i t h i n t h e c o m o a r t m e n t so 1 f o a n r
and form aggregates.
3 Entrapment of cells in hollow-fibre
i m m o b i l i z a t i o n o f c e l l s . l t i s p o s s i b l et o e n t r a p c e l l s
within and between the fibres.
Membrane enrrapment is mechai-ricailystable
However, ii is more expensive than gel or foam
immobilization.
Bioreactors for use of
immobilized cells
Fluidized bed or fixed bed bioreactors are
employed to use immobilized cells for large scale
cu!tivation.
I n t h e f l u i d i z e d - b e d r e a c t o r s ,t h e i m m o b i l i z e o
c e l l s a r e a g i t a t e db y a f l o w o f a i r o r b y p u m p i n g t h e
medium. In contrast, in the fixed-bed bioreacior,
the immobilized cells are held stationary (not
512 B IOTECHNO LO CY

for the production secondarynetabolite(s)

Plant culture species lmmobilization method Substrate Product

Catharanthus roseus Entrapment
in agarose Cathenamine Ajmalicine
Digitalislanata in alginate
Entrapment Digitoxin Digoxin
Capsicum
frutescens in polyurethane
Entrapment foam Sucrose woPD4rvil |

Catheranthus
roseus Entrapment
in alginate,
agarose, Sucrose Ajmalicine
carrageenin
Petuniahybrida Entrapment
in hollow
fibres Sucrose Phenolics
Morindacitrifolia Entrapment
in alginate Sucrose Anthraquinone
Solanum aviculare polyphenylene
Attachment beads Sucrose glycosides
Steroid
Glycinemax Entrapment fibre
in hollow Sucrose Phenolics

Biochemicals produced by commercial production of secondary metabolites.

using immobilized cells A selected list of the plarrtsemployed in hairy root
cultures and the secondarvmetabolitesoroduced is
A s e l e c te dIi s t o f th e i mmobi l i zedcel l s from
given in Table 42.3.
s e l e c te d p l a n ts a n d th e ir uti l i ty to produce
importantbiochemicalsis given in Table42.2
MED'UM COMPOS'TION AND
EFFECT OF NUTRIENTS
Two.phase system culture
The in vitro growth of the plant cells occurs in
P l a n tc e l l sc a n b e c u l ti v atedi n an aoueoustw o
a s u i t a b l e m e d i u m c o n t a i n i n g a l l th e r e q u i si te
phase system for the production of secondary
elements.The ingradientsof the medium effect the
m e ta b o l i te sIn. th i s te c h n ique,the cel l s are kept growth and metabolismof cells.
apart from the product by separationin the
bioreactor.This is advantageous sincethe product For optimal production of secondarymetabolites,
can be removed continuously.Certain polymers a t w o - m e d i u m a p p r o a c h i s d e s i r a b l e . Th e fi r st
(e.g., dextran and polyethyleneglycol for the medium is required for good growth of cells
separation of phenoliccompounds) are usedfor the (biomass growth) while the second medium,
separationof phases. referred to as production medium promotes
secondary metabolite formation. The effect of
Hairy root culture
Hairy root culturesare used for the production
of root-associatedmetabolites. In general, these
cultureshavehigh growthrateand geneticstability. secondarymetabolite(s)
For the productionof hairy root cultures,the
PIant species Secondary metabolite(s)
t te ri a(p
e x p l a n ma l l a n ti
t s s u e)i s i nocul ated
w i th the
cells of the pathogenicbacterium,Agrobacteriurn Nicotiana
tabacum Nicotine,
anatabine
rhizogenes.This organismcontainsroot-inducing Atropabelladonna Atropine
(Ri) plasmidthat causesgenetictransformation of Daturastranonium Hyoscyamine
p l a n t ti s s u e sw
, h i c h fi n a l ly resul tsi n hai ry root Lithospernumerythrorhizon Shikonin
cultures.Hairy roots produced by plant tissues
roseus
Catharanthus Ajmalicine,
serpentine
have metabolitefeaturessimilarto that of normar
roots. Cinchonaledgeriana Quinine
alkaloids
Menthavulgaris Monoterpenes
Hairyroot culturesare mostrecentorganculture
systems and are successfu laciniatum
Solanum Sleroidalkaloids
lly used for the
 Chaot er42 : PL A N TT IS SU E
C U L T U R E-C EN E R A L 513

nutrients(carbonand nitrogensources,phosphate, Increase in phosphateconcentrationin the

growthregulators,precursors/vitamins,metalions) medium may increase,decreaseor may not affect
on different soecies in relation to metabolite productformatione.g.
formationare variable,some of them are briefly
described. 1. Increasedphosphateconcentration increases
alkaloid (in Catharanthusroseus),anthraquinone
(in Morinda citrifolia)and diosgenin(in Dioscorea
Effect of carbon source
deltoided oroduction.
Carbohydratesinfluence the production of
phytochemicals. Someexamplesare given below. 2. Decreasedphosphatelevel in the medium
increasesthe formation of anthocvaninsand
1. Increasein sucroseconcentration(in the phenolics(in Catharanthusroseus),alkaloids(in
range 4-1 07o) increasesallkaloid production rn Peganum harmala) and solasodine (in Solanum
Catharanthus roseuscultures. lanciatum).
2. Sucrose is a better carbon source than 3. Phosphate concentration (increase or
fructoseor galactosefor diosgeninproductionby decrease)has no effect on protoberberine(an
Dioscorea deltoidea or Dalanites aegyptiaca alkaloid)productionby Berberissp.
cultures.

3. Low concentrationof sucroseincreases the Effect of plant growth regulators

pr oduc t ion of u b i q u i n o n e -1 0i n to b a c c o cel l
Plant growth regulators(auxins, cytokinins)
cultures.
influence growth, metabolism and differentiation
of cultured cells. (For more details on the nature
Effect of nitrogen soutce
and differenttypesof auxinsand cytokinins,Refer
T he s t and a rdc u l tu rem e d i a u s u a l l yc o n t ai na Chapter43).Thereare a largenumberof reportson
mixtureof nitrateand ammoniaas nitrogensource. the influence of growth regulators for the
Majority of plant cells can toleratehigh levelsof productionof secondarymetabolitesin cultured
ammonia.The culturedcellsutilizenitrogenfor the cells.A few examplesare given.
o f a m i n o a c i d s , p ro te i n s(i n c l udi ng
bios y nt hes is
1. Additionof auxins(indoleaceticacid, indole
enzymes) and nucleic acids. The nitrogen
pyruvic acid, naphthaleneacetic acid) enhanced
containing primary metabolites directly influence
the production of diosgenin in the cultures of
the secondary metabolites.
Balanitesaegyptiaca.
I n gener a l h, i g h a m m o n i u mi o n c o n c e n trati ons
2. A uxi nsmay i nhi bi tthe producti onof certai n
inhibit secondary metabolite formation while
secondary metabolites e.g.,naphthalene aceticacid
lowering of ammonium nitrogen increases.lt is
and indole acetic acid inhibitedthe svnthesisof
reported that addition of KNO, and NH.NO,
anthocyaninin carrotcultures.
inhibitedanthocyanin(by 9O%) and alkaloid (by
B0%) production. 3. Anotherauxin,2, 4-dichlorophenoxy acetate
(2, 4-D ) i nhi bi tsthe producti onof al kal oi dsi n the
Effect of phosphate culturesof tobacco,and shikoninformationin the
culturesof Lithospermumerythrorhizon.
Inorganic phosphate is essentialfor photo-
synthesisand respiration(glycolysis).
In addition, 4. Cytokinins promote the production o(
many secondarymetabolites are producedthrough secondarymetabolites in manytissueculturese.g.,
phosphorylated which subsequently ajmalicine in Catharanthus
intermediates, roseus;scopolin and
release the phosphate e.8., phenylpropanoids,scopoletinin tobacco;carotenein Riclnussp.
terpenes,terpenoids.In general,high phosphate
levelspromotecell growthand primarymetabolism 5. In some ti ssuecul tures,cytoki ni nsi nhi bi t
while low phosphateconeentrations are beneficial productformatione.9.,anthroquinones in Morinda
for secondaryproductformation.However,this is citrifolia; shikonin in Lithospermumerythroshizon;
not always correct. nicotinein tobacio.

Biotechnology
[33]
514
I n t he abov e ex am ples , a u x i n s a n d c y t o k i n i n s
ar e s epar at ely dis c us s ed . I n a c t u a l p r a c t i c e , a
c om binat ion of aux ins and c y t o k i n i n s i s u s e d t o
ac hiev e m ax im um pr od u c t i o n o f s e c o n d a r y
m et abolit esin c ult ur e s v s te m s .
Effect of precursors
The substrate molecules that are incorporated
into the secondary metabolites are referred to
as precursors. ln general, addition of precursors
to the medium enhances product formation,
alt hough t hey us ually inh i b i t t h e g r o w t h o f t h e
c ult ur e e. g. , alk aloid s y nt h e s i s i n D a t u r a c u l t u r e s
in inc r eas ed while gr owt h i s i n h i b i t e d b y t h e
addit ion of or nit hine, pf r en y l a l a n i n e ,t y r o s i n e a n d
sodiunr phenylpyruvate;precursorstryptamine and
s ec ologanin inc r eas e ajm a l i c i n e p r o d u c t i o n I n
C. roseus cultures.
EL'CTTOR.'NDUCED PRODUCT'ON OF
SECONDARY METABOL'TES
The production of secondarymetabolitesin plant
cultures is generally low and does not meet the
commercial demands. There are continuous efforts
to understandthe mechanism of product formation
at the molecular level, anci exploit for increased
production. The synthesisof majority of secondary
metabolites involves multistep reactions and many
enzymes. lt is possible to stimulate any step to
increase product formation.
Elicitors are the compounds of biological origin
which stimulate the production of secondary
metabolites, and the phenomenon of such
stimulation is referred to as elicitation.
Elic it or s pr oduc ed wit h i n t h e p l a n t c e l l s a r e
endogenous elicitors e g., pectin, pectic acid,
c ellulos e,ot her poly s ac c ha r i d e sWh
. en the elicitors
are produced by the microorganisn.rs,they are
referred to as exogenous elicitors e.g., chiiin,
c hit os an, gluc ans . All t he e l i c i t o r s o f b i o i o g i c a l
origin are biotic elicitors. The term abiotic elicitors
is used to represent the physical (cold, heat, UV
light , os m ot ic pr es s ur e) a n d c h e m i c a l a g e n t s
(ethylene, fungicides, antibiotics, salts of heavy
metals) that can also increase the pi'oduct
formation. However, the term abiotic stressis useci
f or abiot ic elic it or s , whi l e e l i c i t o r s e x c l u s i v e l y
r epr es entbiologic al c om po u n d s .
B IOTECHNO LO CY
Phytoalexins
Plants are capable of defending themselves
w h e n a t t a c k e d b y m i c r o o r g a n i s m s ,by p r o d u ci n g
antimicrobial compounds collgctively referred to as
p h y t o a l e x i n s . P h y t o a l e x i n s a r e t h e ch e m i ca l
weapons of defense against pathogenic
m i c r o o r g a n i s m s . S o m e o f t h e p h y to a l e xi n s th a t
induce the production of secondarymetabolitesare
r e g a r d e da s e l i c i i o r s . S o m e c h e m i c a ls ca n a l so a ct
a s e l i c i t o r s e . g . , a c t i n o m y c i n - D , s o d i u m sa l t o f
a r a c h i d o n i c a c i d , r i b o n u c l e a s e - A c, h i to sa n ,p o l y- L -
lysine, nigeran. These compounds are regarded as
chernically defined elicitors.
lnteractions for elicitor formation
E l i c i t o r s a r e c o m p o u n d s i r r v o l v e d i n p l a n t-
microbe interaction. Three different types of
interactions betv,reen plants and microorganisms
are known that lead to the formation of elicitors.
1. Direct release of elicitor by th e
microorganisms.
2 . M i c r o b i a l e n z y m e s t h a t c a n a ct a s e l i ci to r s.
e.g. endopolygalacturonicacid lyase from Erwinia
carotovara.
3 Release of phytoalexins by the action of
p l a n t e n z y m e s o n c e l l w a l l s o f m i cr o o r g a n i sm s
which in turn stimulate formation elicitors froin
plant cell walls e.g., chitosan {rom Fusarium cell
walls; a-1, 3-endoglucanasefrom Phytophthoracell
walls.
Methodology of elicitation
Selection of rnicroorganisms: A wide range of
microorganisms(viruses,bacteria, algae and fungi)
that need not be pathogens have been tried rn
c u l t u r e s f o r e l i c i t o r i n d u c e d p r o d u cti o n o f
secondary meiabolites. Based on the favourable
e l i c i t o r r e s p o n s e , a n i d e a l m i c r o o r g a n i sm i s
s e l e c t e d .T h e q u a n t i t y o f t h e m i c r o b i a l i n o cu l u m i s
important for the formation elicitor.
Co-culture : Piant cultures (frequently'
s u s o e n s i o n c u l t u r e s ) a r e i n o c u l a te d w i th th e
selected microorganism to form co-cultures. The
cultures are transferredto a fresh medium prior to
t h e i n o c u l a t i o n w i t h m i c r o o r g a r r i s m .Th i s h e l p s to
stimulate the secondary metabolism.
Chaot er42 : P L A N TT ISS U E
C U L T U R E -C E N E R A L 515

Co-cultu res of plant c ells wit h m ic r oor ganism s

may sometimes have inhibitory effect on the plant T*v 42.4 A selectedlist of elicltor-induced
c ells. In su ch a c as e, elic it or pr epar at ionsc an b e secondary metabolite production in
obtaf,red by culturing the selected microorganism plant cell cultures
on a tissu e c ult ur e m edium , f ollowed b y
Elicitor Secondary
homo ge nisatio n and aut oc lav ing of t he ent i r e
nricroorganism metabolite(s)
c ultu re. Th is p roc es s r eleas eselic it or s . I n c as e o f
hea t lab ile e licito r s ,t he c ult ur e hom ogenat ehas to Aspergillus niger Cinchona ledgeriana,
Anthraquinones
be filte r sterilized ( ins t eadof aut oc lav ing) Rubiatinctoria
Pythium Catharanthusroseus Ajmalicine,
In some co-cult ur e s y s t em s , dir ec t c ont ac t o i
aphanidernatun Strictosidine
pla nt ce lls a nd m ic r oor ganis nr sc an be pr ev ent e d
Catharanthine
by immob iliza tion ( ent r apm ent )of one of t hem . I n Anlnrlic cn Papaver somniferumSanguinarine
t he se cultu res. o lant m ic r obial int er ac t ion oc c u r s
by d iffusion of th e elic it or c om pounds t hr ough t h e Phytophthora Glycine max lsoflavonoids
megasperma Gluceollin
m e diu m.
Dendryphion sp Papaver somniferumSanguinarine
M echa nism o f ac t ion of et ic it or s Alternaria sp Phaseolus vulgaris Phaseollin
Elicitorsare found t o ac t iv at egenesand inc r ea s e Fusarium sp Apiumgraveolens Furanocoumarins
t he synth esis of m RNAs enc oding enz y m e s Phythium Daucus carota Anthocynins
r espclnsiblefor th e ult im at e bios y nt hes iss ec onda r y aphanidermatum
m eta bo lites Penicilliun Sanguinaria Benzophenan-
expansun canadensis thridine
There are some recent reports suggestingthe
invo lve men t o f elic it or m ediat ed c alc ium - base d Alkaloids
s ign al tran sd uct ion s y s t em s t hat pr om ot es t h e
product formation When the cells are pretreated
with a ca lciu m ch elat e ( EDTA)pr ior t o t he addit io n 1 . B l u e l i g h t e n h a n c e sa n t h o c y a n i n p r o d u c t i o n
of e licito r, the r e oc c ur s a dec r eas e in t h e in Haplopappus gracilis cell suspensions.
production of secondary metabolite
2 . Wh i t e l i g h t i n c r e a s e s t h e f o r m a t i o n o f
anthocyanin in the cultures of Catharanthusroseus,
Elicitor-in du ce d pr oduc t s in c ult ur es
Daucus carota and Helianthus tuberosus.
ln Table 42.4', a selected list of elicitor-induced
3 . Wh i t e o r b l u e l i g h t i n h i b i t s n a p h t h o q u i n o n e
secondary metabolitesproduced in culture systems
biosynthesis in callus cultures of Lithospermum
are 8 rve n.
erythrorhizon.

EFFECT OF ENVIBONMENTAL FACTORS

Effect of incubation temperature
The ph ysica l f ac t or s nam ely light , inc ubat io n
The growth of cultured cells is increased with
temp era ture , p H of t he m edium and aer at ion o f
rncrease In temperature up to an optrmal
c ultu res influ en c e t he pr oduc t ion of s ec onda r y
temperature (25-30"C). However, at least for the
m eta bo litesin cu lt ur es .
production some secondary metabolites lower
Effect of light temperature is advantageous. For instance, in C.
roseus cultures, indole alkaloicJ production is
Lig ht is ab so lut ely es s ent iai f or t he c ar bo n
i n c r e a s e d b y t w o f o l d w h e n i n c u b a t e d a t 1 6 'C
fixa tion (p ho tosy nt hes is )of f ield- gr own plants .
i n s t e a d o f 2 7 " C I n c r e a s e dt e m p e r a t u r e w a s a l s o
Since the ca rbo n f ix at ion is alm os t abs ent or v er y found to reduce the production of caffeine (by
lo w in pla nt tissue c ult ur es , light has no ef f ec t o n
C. sineneis\ and nicotine (by N tabacum).
the primary metabolism. How'ever, the light-
mediated enzymatic reactions indirectly influence
Effect of pH of the medium
the secondary metabolite formation. The quality of
ligh t is also imp or t ant . Som e ex am ples of ligh t - For good growth of cultures, the pH of the
stinrulated product formations are given medium is in the rangeof 5 to 6. There are reports
516 B IOTECHNO LO CY

indic at ing t hat pH of t he m e d i u m i n f l u e n c e s t h e

formation of secondary r:netabolites. e.9., Tnsu 42.5 SelcctedexanPlesof
production of anthocyanin by cultures of Daucus by plant cell cultures
biotransformatlons
carota was much less when incubated at pH 5.5
Plant cell culture Product
than at oH 4.5. This is attributed to the increaseo
degradation of anthocyanin at higher pH. lanata
Digitalis Digitoxin Digoxin
Papaver
sonniferum Codeinone Codeine
Aeration of cultures
Nrcotiana
tobacun Carvoxine Cavaxone
Continuous aeration is needed for good growth Daucus
carota Digitoxigenin Periplogenin
of cultures, and also for the efficient production of
pruriens
Mucuna L-Tyrosine L-Dihydroxy-
secondary metabolites. phenylalanine
(L-D0PA)
BTOTBANSFOBMATION USING
Menthasp (-)-Menthone (+)-Neomenthol
PLANT CELL CULTURES
Coffeaarabica Vanillin Vanillin-D-
fhe conversion of one chemical into another glucoside
(i.e., a substrate into a final product) by using
tuberosun Solavetivone
Solanum Hydroxylated
hiological systems (i.e. cell suspensions) as derivatives
biocatalysts is regarded as biotransformation or
Galiunnollugo 2-Succinyl
benzoate quinones
Anthra
bioconversion. The biocatalvst mav be free or
inr m obiliz ed, and t he pr oc e s so f b i o t r a n s f o r m a t i o n Datura
sp Hydroquinone Arbutin
m ay inv olv e one or more enzymes. Citrussp Valencene Nootkatone'
Biot r ans f or m at ion inv olv in g m i c r o o r g a n i s m s a n d Choisya
ternata Ellipticine 5-Formyl-
anim al c ells ar e des c r ibede l s e w h e r e( C h a p t e r2 2 ) . ellipticine
The biot ec hnologic al ap p l i c a t i o n o f p l a n t c e l l purpurea
Digitalis I Steviol Steviocide,
cultures in biotransformationreactions involves the Stviarebandiana Steviobiocide
conversion of some less imoortant substancesto
v aluable m edic inal or c o m m e r c i a l l y i m p o r t a n t
Sometimes,the products formed within the cells
products. In biotransformation, it is necessary to
a r e r e l e a s e di n t o t h e m e d i u m , m a k i n g th e i so l a ti o n
select such cell lines that possessthe enzymes for
and analysis easy. For the secondary metabolites
catalysing the desired reactions. Bioconversions
stored within the vacuoles of cells, two membranes
may involve many types of reactions e.9.,
(plasma membrane and tonoplast) have to be
hydroxylation, reduction, glycosylation.
d i s r u p t e d . P e r m e a b i l i z i n ga g e n t s s u c h a s d i m e th yl
A good example of biotransformationby plant sulfoxide (DMSO) can be used for the release of
cell cultures is the large scale production of products.
cardiovascular drug digoxin from digitoxin by
I n g e n e r a l , s e p a r a t i o n a n d p ur i fi ca ti o n o f
Digitali Ianata. Digoxin production is carried out
products from plant cell cultures are expensive,
by im m obiliz ed c ells of D . l a n a t a. i n a i r l i f t
therefore every effort is made to make them cost-
bioreactors. Cell cultures of Digitalis purpurea ol
effective. Two approaches are made in this
Stevia rebaudiana can convert steviol into
direction : '
s t ev iobioc ide and s t ev ioc id e w h i c h a r e 1 0 0 t i m e s
sweeter than cane sugar. 1. Production of secondary metabolite should
be as high as possible.
A selected list of biotransformationscarried our
in plant c ell c ult ur es is giv e n i n T a b l e 4 2 . 5 . 2. Formation of side product(s)which interfere
w i t h s e p a r a t i o nm u s t b e m a d e m i n i m a l .
SECONDARY METABOLITE Once a good quantity of the product is released
RELEASE AND ANALYS'S i n t o t h e m e d i u m , s e p a r a t i o n a n d p u r i fi ca ti o n
The methods employed for the separation and techniques (e.g. extraction) can be used for its
purification of secondary metabolites from cell recovery. These techniques largely depend on the
cultures are the same as that used for olants. nature of the secondary metabolite.
fI ult ur em e d i aa re l a rg e l yre s p o n s i b lfo
e r th e i n
\- vitro g rowt h and m or phogenes is of p l a n t
tissue s. Th e s uc c es s of t he plant t is s ue c u l t u r e
d ep en ds on the c hoic e of t he nut r ient m edium . l n
fact, th e ce lls of m os t plant c ells c an be gr ow n i n
cultu re me dia .
Basica lly,th e plant t is s ue c ult ur e m edia s h o u l d
co nta in th e sam e nut r ient sas r eouir ed bv t he w h o l e
p lan t. lt ma y be not ed t hat plant s in nat ur e c a n
synthesize their own food material However,
plants growing in vitro are mainly heterotrophic
i.e. they cannot synthesize their own food.
Gomp osition of m edia
Th e co mpos it ion of t he c ult ur e m edi a r s
p rimarily de pe ndent on t wo par am et er s .
.l
The p ar t ic ular s pec iesof t he plant .
2 . The tvp e of m at er ial us ed f or c ult ur e r . e
cells, tissu es,or gans , pr ot oplas t s .
Thu s, th e c om oos it ion of a m edium i s
fo rmula ted co ns ider ingt he s pec if ic r equir em en t so f
a given cultu r e s y s t em . The m edia us ed m ay b e
solid (solid medium) or liquid (liquid medium) n
na ture . The selec t ion of s olid or liouid m ediu m i s
dependent on the better responseof a culture
MAJOR TYP ES O F M EDI A
Th e co mpo s it ion of t he m os t c om m only u s e d
tissue culutre media is given in Table 43.1, and
briefly discribed below.
Wh i t e 's m e d i u m : T h i s i s o n e o f t h e e a r l i e s t
plant tissue culture media developed for root
c u l t ur e .
MS medium : Murashige and Skoog (MS)
originally formulated a medium to induce
organogenesis, and regeneration of plants In
c u l t u r e d t i s s u e s .T h e s e d a y s , M S m e d i u m i s w i d e l y
used for many types of culture systems.
85 medium : Developed by Camborg, 85
m e d i u m w a s o r i g i n a l l y d e s i g n e df o r c e l l s u s p e n s i o n
and callus cultures. At present with certain
m o d i f i c a t i o n s ,t h i s m e d i u m i s u s e d f o r p r o t o p l a s t
c u l t ur e .
N6 medium : Chu formulated this medium ancr
it is used for cereal anther culture, besidesother
tissuecultures.
N i t s c h 's m e d i u m : T h i s m e d i u m w a s d e v e l o p e d
by Nitsch and Nitsch and frequently used for anther
cu ltures.
Among the media referredabove, MS medium is
most frequently used in plant tissue culture work
d u e t o i t s s u c c e s sw i t h s e v e r a l p l a n t s p e c i e s a n d
culture systems.
Synthetic and natural media : When a medium
is composed of chemically defined components,it
is referred to as a synthetic medium. On the other
hand, if a medium contains chemically undefined
compounds (e.9.,vegetableextract,fruit juice, plant
extract), it is regarded as a natural medium
517
518 B IOTECHNO LO CY

Components Amount (mg Fl )

Whitels Murashige and Skoog (MS) Gamborg (BS) Chu(N6) Nifsch's
Macronutrients
MgSO*.7HrO 750 370 250 185 185
KH2P04 170 400 68
NaHrPOo.HrO 19 150
KN03 60 't90c 2500 2830 950
NH4N03 1650 720
CaClr.2HrO 440 150 roo
(NH4)2.S04 134 463
Mi c ro n u tri e n ts
H3803 t.c 6.2 3 t.o
MnSOo.4HrO 5 22.3 4.4 25
MnSOo.Hr0 10 3.3
ZnSOo.THrO 3 8.0 2 t.3 10
NarMoOo.2HrO 0.25 0.25 0.25
CuSOo.5HrO 0 .01 0.025 0.025 0.025
CoClr.6HrO 0.025 0.025 0.025
KI 0.75 0.83 0.75 0.8
FeSOo.THrO 27.8 27.8 27.8
NarEDTA.2HrO 37.3 37.3 37.3
(g)
Sucrose 20 30 20 50 20
Organic supplements
Vitamins
Thlamine
HCI 0.01 0.5 10 1 0.5
(HCl)
Pyridoxine 001 0.5 1 0.5 0.5
Nicotinic
acid 0.05 0.5 1 0.5 4

Myoinositol 100 100 100

Others
Glycine 3 2 2
Foiicacid 0.5
Biotin 005
pH 5.8 58 5.5 5.8 5.8

Sirnthetic media have almost replaced the natural as mass values (mg/i or ppm or mg l-1;. However,
m c dia f or t is s ue c ult ur e. as per the recommendations of the International
Expression of concentrations in media : The Association of Plant Physiology,the concentrations
c onc ent r at ions of inor g a n i c a n d organrc of macronutrientsshould be expressedas mmol/l
c ons t it uent sin c ult ur e m edi a a r e u s u a l l v e x o r e s s e d and micronutrients as umol/l-
: r aDt er43 : P L A N TT IS SU E M ED IA
CULTURE 519

CONSTITUENT S O F M EDI A sources, sucrose is the most preferred. During the

course of sterilization (bv autoclavinq) of the
Ma ny ele ment s ar e needed f or plant nut r it i o n
m e d i r , r m ,s u c r o s e g e t s h y d r o l y s e d t o g l u c o s e a n c i
-:nd the ir ph vs iologic al f unc t ions . Thus , t he s e fructose The plant cells in culture first utilize
e leme ntsha ve to be s uoplied in t he c ult ur e m ediu m
glucose atrd therr fructose, In fact, glrrcose cr
to support adequate growth of cultures in vitro. A
frur:tosecan be directiv Lrspicl in ilre r:Lrlturernedia.
sele cte d list of t he elem ent s and t heir f unc t ions r n
It may be noted that irir energy supply, glr-rcoseis as
plants is given in Table 43.2.
efficient as sucrose r,r,hilefrr-rctoseis less effic!ent.
Th e cultL rrem edia us ual! y c ont ain t he f ollowi n g
constrtuents
.l
In org an ic nut r ient s
Tlsrs 43.2 A selected list of elemsnts and
2 Carbon and energy sources their functions in plants
3 Orga nic s uppiem ent s
Element Function(s)
4. Crowth regulators
5 Soliclifyingagents N i trogen Essential of pioteins,
component
nudei()
acidsandsome
6. p H o f medium
(Recuireii
coenzy!'nes in mcst
^L,.^l^^+
ouuirulill qudiluLy/
^,,^^+;1,,\
Inorganic nutYients

The in org anic nut r ient s c ons is t of

C al ci um of cellvrall,rnembrane
Synthesis
macranutrienfs (concentration >0.5 mmolil-) anci
{unction,
cellsignalling.
micronutrienfs (concentration <0.5 mmol/l-). A Maqnesium Cornponent
of chloroohyll.
cofactcr
ivide ra ng e of m iner al s alt s ( elem ent s )s upoly t h e forsomeenzymes
ma.ro - an d mic r onut r ient s The inor ganiq s ait s i n
\&/a ter u nd erg o dis s oc iat ion and ic niz at i o n .
Potassium Majorincrganic regulares
cation,
osmolicpotential
Consequently,one type of ion may be contributed
by more tha n o ne s alt For ins t anc e,in M S m ediu r n , PhosDhorus Component of nucleic
acidsand
K+ ion s a re co nt r ibut ed by KNO , and KHTP O , variousintermediates
in respiration
wh ile NO.,- io ns c om e f r om KNO , and NHoNOr . andphotosynthesis,
involvedin
Macronutrient elements : The six elements
enei'gy
transfer.
namefy nitrogen, phosphorus, potassium, calcium, S ul ur
f Componenl
ol certain
aminoacicis
magnesium and sulfur are the essential (methionine,
cysteine
andcvstine,
macron utrie nts f or t is s ue c ult ur e. The id e a l andsomecofactors).
concentration of nitrogen, and potassium is around
Manganese {orcertainenzymes
Cofactor
2 5 mmol l-1 wh ile f or c alc ium , phos phor us ,s u l f u r
.l
a nd mag ne siu m , it is in t he r ange of - 3 m m ol l - . l ron Component of cytochromes,
Fo r the sup oly o f nit r c lgenin t he m ediur n, nit r a t e s invclved
in electron
lransfer.
an d ammo niu m s alt s ar e t oget her us ed.
C hl ori ne in photosynthesis.
Participates
Micron utrie nt s : Alt hough t heir r equir em en t i s
C opper lnvolved
in electron
transfer
in min ute o ua niit ies , m ic r onut r ient s ar e es s en t i a l
reactions,
CoJactorforsome
for p lan t cells and t is s ues . Thes e inc lude ir o n ,
enzymes.
man ga ne se ,zinc , bor on, c of r per and m oly bdenu m
Among the microelements, iron requirement is C obal t Component
of vitamin
B,r.
very critical. Chelated forms of iron and copper are
Mol ybdenum Component of certain
enzymes
corn mon ly used in c ult ur e m edia.
(e.9.,nitrate
reductase),
cofactor
forsomeenzymes.
Carbon and energy sources
Zi nc forchlorophyll
Required
Plan t ce lls an d t is s uesin t he c ult ur e nr edium a r e
biosynthesis, for certain
cofactor
heterotrophic and therefore, are dependent on the
enzymes.
external carbon for energy. Among the energy
520 B IOTECHNO LO CY

lt is a common observationthat culfuresgrow yeast extract by L-asparagine;replacement of frurt

hetter on a medium with autoclavedsucrosethan extracts by L-glutamine.
o n a me d i u m w i th fi l te r-steri l i zed
sucrose Thi s
Activated charcoal : Supplementation of the
clearly indicatesthat the hydrolysedproductsof
m e d i u m w i t h a c t i v a t e d c h a r c o a l s t im u l a te s th e
sucrose(particularlyglucose)are efficientsources
growth and differentiation of certain plant cells
of energy.Direct use of fructosein the medium
(carrot, tomato, orchids). Some toxic/inhibitory
subjectedto autoclaving, is foundto be detrimental
compounds (e.g. phenols) produced by cultured
to th e g ro w tho f p l a n tc e l l s.
plants are removed (by adsorption) by activated
Be s i d e s s u c ro s e a n d gl ucose, other charcoal, and this facilitatis efficient cell growth rn
carbohydrates such as lactose,maltose,galactose, cu ltures.
raffinose,trehalose and cellobiose have been
Addition of activatedcharcoal to certain cultures
u s e d i n c u l tu re m e d i a b u t w i th a very l i mi ted
(tobacco, soybean) is found to be inhibitory,
success.
probably due to adsorption of growth stimuiants
such as ohvtohormones.
Organic supplements
Antibiotics : lt is sometimes necessaryto add
T h e o rg a n i c s u p p l e m e ntsi ncl ude vi tami ns,
antibioticsto the medium to prevent the growth of
a m i n o a c i d s , o rg a n i c a c ids, organi c extracts,
microorganisms. For this purpose, low
activatedcharcoaland antibiotics.
concentrations of streptomycin or kanamycin are
Vi ta m i n s: Pl a n tc e l l sa n d ti ssuesi n cul ture(l i ke used As far as possible, addition of antibiotics to
the natural plants) are capable of synthesizing t h e m e d i u m i s a v o i d e d a s t h e y h a v e a n i n h i b i to r y
vitaminsbut in suboptimalquantities, inadequate to i n f l u e n c e o n t h e c e l l g r o w t h .
supportgrowth.Thereforethe medium should be
supplemented with vitamins to achieve good Growth regulators
growth of cells.The vitaminsaddedto the media
Pl.ant hormones or phytohormones are a Broup
i n c l u d eth i a m i n eri, b o fl a v i nn,i aci n,pyri doxi ne,
fol i c
of natural organic compounds that promote growth,
a c i d ,p a n to th e n iacc i d ,b i o ti n,ascorbi caci d, myo-
development and differentiation of plants. Four
i n o s i to lp, a ra -a m i nboe n z o i caci d and vi tami nE .
broad classesof growth regulatorsor hormones are
Ami n o a c i d s: Al th o u g hthe cul turedpl antcel l s used for culture of plant cells-auxins, cytokinins,
can synthesizeamino acids to a certain extent, gibberellins (Fig. a3.1) and abscisic acid. fhey
media supplementedwilh amino acids stimulate promote growth, differentiation and organogenesis
cell growth and help in establishment of cells o f o l a n t t i s s u e si n c u l t u r e s .
lines. Further,organic nitrogen (in the form of
Auxins : Auxins induce cell division, cell
a m i n o a c i d ss u c h a s L -g l u tami ne,L-asparagi ne,L-
e l o n g a t i o n , a n d f o r m a t i o n o f c a l l u s i n cu l tu r e s.At
a rg i n i n eL, -c y s te i n ei s) mo re readi l ytakenup than
a low concentration,auxins promote root formation
i n o rg a n i cn i tro g e nb y th e p l ant cel l s.
w h i l e a t a h i g h c o n c e n t r a t i o n c a l l us fo r m a ti o n
O rg a n i c a c i d s : Ad d i ti on of K rebs cycl e o c c u r s . A s e l e c t e d l i s t o f a u x i n s u i ed i n ti ssu e
intermediates such as citrate,malate.succinateor cultures is given in Table 43.3.
fumarateallow the growthof plant cells. Pyruvate
Among the auxins, 2, 4-dichlorophenoxy acetic
s e g ro w tho f cul turedcel l s.
a l s oe n h a n c e th
acid is most effective and is widely used in culture
Organic extracts : lt has been a practiceto media.
supplementculture media with organic extracts
C y t o k i n i n s : C h e m i c a l l y , c y t o ki n i n s a r e
such as yeast, caseinhydrolysate,coconut milk,
d e r i v a t i v e s o f a p u r i n e n a m e l y a d e n i n e . Th e se
orangejuice, tomatojuice and potatoextract.
a d e n i n e d e r i v a t i v e s a r e i n v o l v e d i n c el l d i vi si o n ,
It is however,preferableto avoid the use of shoot differentiation and somatic embryo
naturalextractsdue to high variationsin the qualitv formation. Cytokinins promote RNA synthesisand
and quantityof growth promotingfactorsin them. t h u s s t i m u l a t e p r o t e i n a n d e n z y m e acti vi ti e s i n
In recentyears,naturalextractshavebeenreplaced t i s s u e s .T h e m o s t c o m m o n l v u s e d c vto ki n i n s a r e
b y s p e c i fi co rg a n i cc o mp o u nds
e.g.,repl acement
of eiven in Table 43.3.
Chaot er43 : PL A N TT IS SU E M ED IA
CULTURE 521

s p e c i e s .T h e y u s u a l l y i n h i b i t a d v e n t i t i o u sr o o t a n d
H N -C H "
shoot formation
Abscisic acid (ABA) : The callus growth of
cultures may be stimulated or inhibited by ABA.
This largely depends on the nature of the plant
species. Abscisic acid is an important growth
I regulationfor induction of embryogenesis.
H H
An auxin A cytokinin Solidifying agents
(lndole ^
(No-Methylaminopurine)
aceticacid)
F o r t h e p r e p a r a t i o no f s e m i s o l i d o r s o l i d t i s s u e
culture media, solidifying or gelling agent-s are
OH
r e q u i r e d . I n f a c t , s o l i d i f y i n g a g e n t se x t e n d s u p p o r t
t o t i s s u e sg r o w i n g i n t h e s t a t i c c o n d i t i o n s
Agar : Agar, a polysaccharide obtained frorn
seaweeds, is most commonly used as a gelling
agent for the follou'ing reasons(next page).

Tmrr 43.3 A selected list of plant growth

regulators used in culture media
Fig. 43.1 : Structures of selected plant growth
regulators. Crowth reg,ulator Chemical name
(abbreviation/name)

A uxi ns
Amon g th e c y t ok inins , k inet in and be n z y l - IAA Indole 3-aceticacid
a mino pu rine ar e f r equent ly us ed in c ult ur e m e d i a .
IBA Indole 3-butyric
acid
Ratio of auxins and cytokinins : The relative NAA 1-Naphthyl aceticacid
concentrationsof the growth factors namely auxtns 2, 4-D 2, 4-Dichlorophenoxyaceticacid
a nd cyto kin ins ar e c r uc ial f or t he m or phogen e s i so f 2, 4, 5-I 2, 4. S-Trichlorophenoxy
acetic
culture systems. When the ratio of auxins to acid
cytokinins is high, enibryogenesis, callus initiation 4--CPA 4-Chlorophenoxy aceticacid
and root initiation occur. On the other hand, for aceticacid
NOA 2-Naphthyloxy
axillary and shoot proliferation, the ratio of auxins
MCPA 2-Methyl4-chlorophenoxy acelic
to cvtokinins is low. For all practical purposes, it is
acid
considered that the formation and maintenance of
Dicamba 2-Methoxy 3, 6-dichlorobenzoic
ca llus cultu res r equir e bot h aux in and c y t o k i n i n ,
acr0
while au xin is neededf or r oot c ult ur e and c y t o k i n i n
Picloram 4-Amino 2. 5, 6-trichloropicolinic
for sh oo t cultur e.
acrd
The actual concentrations of the Srowth
Cytokinins
re gu lato rsin c ult ur e m edia ar e v ar iable depe n d i n g
BAP
on the typ e o f t is s ue ex plant and t he plant s p e c i e s .
aminopurine
6-Benzyl
BA Benzyl
adenine
Cib be rel!i ns : About 20 dif f er ent gibber e l l i n s 2 iP (rPA) adenine
N6-(2-isopentyl)
have been identified as growth regulators.Of these,
DPU Diphenyl
urea
gib be rellin43 ( CA3) is t he m os t c om m only us e d f o r
Kinetin 6-Furfuryl
aminopurine
tisue culture. CA, promotes growth of cultured
growth and induces dwarf
Zealin 4-Hydroxy
3-methyltrans
cells, enhances callus
plantlets to elongate. 2-butenyl
aminopurine
Thidiazuron 1-Phenyl yl)
3-(1,2, 3-thiadiazol-5
Cib be relli ns ar e c apable of pr om ot in g o r
urea
inh ibitin g tissue c ult ur es , depending on t he p l a n t
522
1. lt does not r eac t wit h m e d i a c o n s t i t u e n t s .
2 lt is not digested by plant enzymes and is
stable at culture temperature.
Agar at a c onc ent r at ion o f 0 . 5 t o 1 % i n t h e
m edium c an f or m a gel.
Celatin : lt is used at a high concentration(.10%)
wit h a lim it ed s uc c es s . This i s m a i n l y b e c a u s e
gelat in m elt s at low t em p e r a t u r e ( 2 5 'C ) , a n d
c ons equent lyt he gelling pr op e r t v i s l o s t .
O t her gelling agent s : Bio g e l ( p o l y a c r y l a m i d e
pellet s ) , phy t agel, gelr it e a n d p u r i f i e d a g a r o s e
ar e ot her s olidif y ing age n t s , a l t h o u g h l e s s
f r equent ly us ed. lt is in f ac t a d v a n t a g e o u st o u s e
s y nt het ic gelling c om pounds, s i n c e t h e y c a n f o r m
gels at a r elat iv ely low c onc e n t r a t i o n ( 1 . 0 t o 2 5
c l- 1)
pH of medium
The optimal pH for most tissue cultures is in the
range of 5.0-6.0. The pH generally falls by 0 3-0.5
unit s af t er aut oc lav ing.Bef or e s t e r i l i z a t i o n ,p H c a n
be adjus t ed t o t he r equir ed o p t i m a l l e v e l w h i l e
pr epar ingt he m edium . lt is us u a l l y n o t n e c e s s a r yt o
use buffers for the pH maintenance of culture
m edia.
At a pH higher t han 7. 0 an d l o w e r t h a n 4 5 , t h e
plant c ells s t op gr owing in c u l t u r e s . l f t h e p H
f alls dur ing t he plant t is s ue c u l t u r e , t h e n f r e s h
m edium s hould be pr epar ed.I n g e n e r a l , p H a b o v e
6. 0 giv es t he m edium har d a p p e a r a n c e , w h i l e
pH below 5. 0 does not a l l o w g e l l i n g o f t n e
m edium .
PREPARATI O N O F M EDIA
The gener al m et hodolo g y f o r a m e d i u m
pr epar at ion inv olv es pr epar at i o no f s t o c k s o l u t i o n s
.1
( in t he r ange of 0x t o 1 0 0 x c o n c e n t r a t i o n s )
us ing high pur it y c hem ic als a n d d e m i n e r a l i z e d
water. The stock solutions can be stored (in glassor
plas t ic c ont ainer s )f r oz en and u s e d a s a n d w h e n
required.
B IOTE CHNO LO CY
Most of the growth regulatorsare not soluble rn
water.They have to be dissolvedin NaOH or alcohol.
Dry powders in media preparation
The conventional procedure for media
p r e p a r a t i o ni s t e d i o u s a n d t i m e c o n s u mi n g N o w a
days, plant tissue culture media are commercially
prepared, and are available rn the market as dry
p o w d e r s .T h e r e q u i s i t em e d i u m c a n b e p r e p a r e db y
d i s s o l v i n g t h e p o w d e r i n a g l a s s d i sti l l e d o r
d e m i n e r a l i z e dw a t e r .S u g a r o
, r g a n i cs u p p l e m e n tsa n d
agar (melted) are added, pH adjusted and
t h e m e d i u r nd i l u t e d t o a f i n a l v o l u m e ( u s u a l l y1 l i tr e ) .
Sterilization of media
T h e c u l t u r e m e d i u m i s u s u a l l y s t e r i l i ze d i n a n
autoclave at 121"C and 15 psi for 2O minutes.
H o r m o n e s a n d o t h e r h e a t s e n s i t i ve o r g a n i c
c o m p o u n d s a r e f i l t e r - s t e r i l i z e d ,a n d a d d e d to th e
autoclaved medium.
SELECTION OF A SUITABLE M E D IU M
l n o r d e r t o s e l e c t a s u i t a b l e m e di u m fo r a
p a r t i c u l a r p l a n t c u l t u r e s y s t e m , i t i s c usto m a r y to
s t a r t w i t h a k n o w n m e d i u m ( e . g . M S me d i u m , 8 5
m e d i u m ) a n d t h e n d e v e l o p a n e w m e d i um w i th th e
desired characteristics.Among the constituentsof a
m e d i u m , g r o w t h r e g u l a t o r s( a u x i n s ,c y t o ki n i n s) a r e
h i g h l y v a r i a b l e d e p e n d i n g o n t h e c u l t u r e syste m .l n
practice, 3-5 different concentrations of growth
r e g u l a t o r s i n d i f f e r e n t c o m b i n a t i o n s a r e u se d a n d
the best among them are selected.
For the selection of appropriate concentrations
o f m i n e r a l s a n d o r g a n i c c o n s t i t u e nts i n th e
m e d i u m , s i m i l a r a p p r o a c h r e f e r r e d a b o ve , ca n b e
employed.
Medium.utmost important for culture
F o r t i s s u e c u l t u r e t e c h n i q u e s , i t i s a b so l u te l y
e s s e n t i a l t h a t t h e m e d i u m p r e p a ra ti o n a n d
c o n r p o s i t i o na r e c a r e f u l l y f o l l o w e d . A n y m i sta ke i n
t h e p r e p a r a t i o n o f t h e m e d i u m i s l i k el y to d o a
great harm to the culture system as a whole.
 f,) rotopl.rsts are naked plant cells without the cell viriety of studies Protoplastshave a wide range of
E wa ll, b trt they pos s es spr as m a m em or ane a n c l a p p l i c a t i o n s ,s o m e o f t h e m a r e l i s t e d b e l o w .
all oth er ce llula r c om ponent s . They r er pr es entth e .l
fu nction al p lan t c ells but f or ihe lac k of t he bar r i e 'r , The protoplast in culture can be regeneratecl
cell wall Protoplasts of different species can be into a whole plant.
fused to generate a hybrid and this process is 2 Hybrids can be developed from protoplast
referred to as somafic hybridization (or protoplast fusion
fr,rsion )Cyb ridizat ion is t he phenom enon of f usi o n
o f a rro rmal p rotoplas twit h an enuc leat ed( v r it h o u t 3 lt is easy to perform single cell cloning with
p roto plas tt hat : ' es r - r lt in
nu cle r,rs) s t he f or m at ion o f protoplasts.
a cybrid or cytoplast (cytoplasmic hybrids).
4 Cenetic transformations can be achieved
t h r o u g h g e n e t i c e n g i n e e r i n go f p r o t o p l a s t D N A .
F{istorical developments
.l 5. Protoplasts are excellent materials for
The term protoplast was introclucedin BB0 by
u ltrastrr:ctr-rra
I studies.
Han ste in. The f ir s t is olat ion of pr ot oplas t s v , , a s
ach ieved by Kler c l< er ( 1892) enr ploy ing a 5 l s o l a t i o no f c e l l o r g a n e l l e sa n d c h r o m o s o m e s
mecha nical me thod. A r eal beginning in pr ot opl a s t is easy from proloplasts.
resea rchwa s made in 1960 by Coc k ing who us e d
7 . P r o t o p l a s t sa r e u s e f u l f o r m e m b r a n e s t u d i e s
a n e nzyma tic m et hod f or t he r em ov al of c ell wa l l
(transportand uptake processes).
Rakab e a nd his as s oc iat es( 1971) wer e s uc c es s f u l
to a ch ieve th e re gener at ionof whole t obac c o pla n t B . l s o l a t i o n o f m u t a n t s f r o m p r o t o p l a s tc u l t u r e s
from protoplasts Rapid progress occurred after rs easy.
19 80 in p roto pl as tf us ion t o t m pr ov e plant gene t i c
a nd the dev elopm entof t r ans genicplan t s .
n.rate rial,

!]VIPORTANCE OF PROTOPLASTS ANP ISOLATIONOF PROTOPTASTS

THEIR CUL TU RES

The isola tion ,c ult ur e and f us ion of pr ot oplas t si s P r o t o p l a s t sa r e i s o l a t e d b y t w o t e c h n i q u e s

a fa scina ting field in plant r es ear c h. Pr ot op l a s t 1. M e c h a n i c a l m e t h o d
isola tion a nd their c ult ur es pr ov ide m illions o f
single cells (comparable to microbial cells) for a 2. Enzymaticmethod

523
524 B IOTECHNO LO CY

A plasmolysedcell Dissectionof a cell Protoolastreleased

Fig. 44.1 : A diagrammatic representation of mechanical method for the isolation of protoplasts.

M ECHANI CAL M ETHO D Enzymes for protoplast isolation

Pr ot oplas is olat ion by m e c h a n i c a l m e t h o d i s a
c r ude and t edious pr oc edu r e . T h i s r e s u l t s i n t h e
is olat ionof a v er y s m all num b e r o f p r o t o p l a s t s T
. he
technique involves the following slages(Fig. 44.1).
1. A s m all piec e of epid e r m i s f r o m a p l a n t i s
selected.
2. The c ells ar e s ubjec t e d t o p l a s m o l y s i s .T h i s
causes protoplasts to shrink away from the cell
walls .
3. The tissue is dissected to release the
protoplasts.
Mechanical method for protoplastisolation is no
m or e in us e bec aus e of t he f o l l o w i n g l i m i t a t i o n s
. Yield of pr ot oplas t sand t h e i r v i a b i l i t y i s l o w .
The enzymes that can digest the cell walls are
required for protoplast isolation. Chemically, the
p l a n t c e l l w a l l i s m a i n l y c o m p o s e d of ce l l u l o se ,
h e m i c e l l u l o s ea n d p e c t i n w h i c h c a n b e r e sp e cti ve l y
d e g r a d e d b y t h e e n z y m e s c e l l u l a s e , h e m i ce l l u l a se
and pectinase.The different enzymes for protoplast
isolation and the corresponding sources are given
in Table 44.1.
In fact, the various enzymes for protoplast
i s o l a t i o n a r e c o m m e r c i a l l y a v a i l a b l e .Th e e n zym e s
a r e u s u a l l y u s d d a t a p H 4 . 5 t o 6 . 0 , te m p e r a tu r e
2 5 - 3 0 'C w i t h a w i d e v a r i a t i o n i n i n c u b a ti o n o e r i o d
that may range from half an hour to 20 hours. The
enzymatic isolation of protoplasts can be carried
out by two approaches.

. lt is restrictedto certain tissueswith vacuolateo

c ells .
lns;'l- 44.1A selected llst of comnerclally
o The method is laborious and tedious.
avallable enrymes for protoplast isolation, and
However, some workers prefer mechanical thelr sources
methods if the cell wall degrading enzymes (of
enzymatic method) cause deleterious effects to Enzyme Source
protoplasts Cellulases
Cellulase R-10
onozuka Trichoderma viride
E N Z Y M AT IC M ET H O D
YC
Cellulase Trichoderma viride
Enzymatic method is a very widely used Cellulysin Trichoderma viride
technique for the isolation of protoplasts.The
Driselase lrpex lactus
advantages of enzymaticmethodincludegoodyield
o f v i a b l ec e l l s ,a n d mi n i malor no damageto the Hemocellulases
protoplasts. Hemicellulase Aspergillus
niger
Helicase Helixpomatia
Sources of protoplasts Rhozyme HP-150 Aspergillus
niger
Protoplasts can be isolatedfrom a wide variety Hemicellulase
H-2125 Rhizopus
sp
of tissuesand organsthat include leaves,roots,
s h o o t a p i c e s ,fru i ts , e m b r yos and mi crospores. Pectinases
A m o n g th e s e , th e m e s ophyl l ti ssue of ful l y Macerase Rhizopus
arrhizus
expandedleavesof youngplantsor new shootsare Pectolyase japonicus
Aspergillus
m o s t fre q u e n tl yu s e d . l n addi ti on, cal l us and Macerozyme R-10 Rhizopus
arrhizus
suspension culturesalso serveas good sourcesfor
Zymolyase luteus
Arlhrobacter
protoplastisolation
Chapt er44 : PR O T OP L ASCTU L T U R E
AN D S OMA TICH Y B R ID IZA TION 525

Leaf sterilization

Differentiation
of
callustissue
r Peeledleaf segment
\
@@
Plasmolysedcells
J

Cellsin
enzyme mixture
/
Ce ll wa ll
reseneration\a";5 (e @,
Cellwall digestion
^ 6 [-l and releaseof protoplasts

\=7*3p=^o--M
(,xo-{_
-.____'-_/
Platingof
*
Protoplasts
protoplasts lsolated Celldebris
protoplasts
Washingol
protoplasts

Fig. 44,2 : Major steps involved in protoplast isolation, culture and regeneration of plants.

1 . Two step or sequential method : The iissue lsolation of protopfasts from leaves
is first treated with pectinase (macerozyme) to
Leaves are most commonly used, for protoplast
sep ara tecell s by degr ading m iddle lam ella. T h e s e
i s o l a t i o n ,s i n c e i t i s p o s s i b l et o i s o l a t eu n i f o r m c e l l s
free cells are then exoosed to cellulase to release
in large numbers. The procedure broadly involves
protoplasts.Pectinasebreaks up the cell aggregat€s
the following steps (Fig. 44.2).
in to in dividu al c ells while c ellulas e r em ove s t h e
cell w,all proper. 1 . S t e r i l i z a t i o no f l e a v e s .

2. One step or simultaneous method : This is 2. Removal of epidermal cell layer.

the preferred method for protoplast isolation. lt
3. Treatmentwith enzymes.
involves the s im ult aneous us e of bot h t n e
enzymes- macerozyme and cellulase. 4. lsolation of protoplasts
526 B IOTECHNO LO CY

Bes ides leav es , c allus c u l t u r e s a n d c e l l suitable manipulations of nutritional and

s us oens ion c ult ur es c an als o b e u s e d f o r t h e p h y s i o l o g i c a lc o n d i t i o n s , t h e c e l l c o l o n i e s m a y b e
isolation of protoplasts. For this purpose, young g r o w n c o n t i n u o u s l y a s c u l t u r e s o r r e ge n e r a te dto
and ac t iv ely gr owing c ells ar e p r e f e r r e d . whole plants.

Purification of protoplasts P r o t o p l a s t sa r e c u l t u r e d e i t h e r i n s e mi so l i d a g a r
o r l i q u i d m e d i u m . S o m e t i m e s ,p r o t o p l astsa r e fi r st
The enz y m e diges t ed p l a n t c e l l s , b e s i d e s
allowed to develop cell wall in liquid medium, and
pr ot oplas t s c ont ain undige s t e d c e l l s , b r o k e n
then transferredto agar medium.
pi' ot oplas t sand undiges t edt is s u e s T h e c e l l c l u m p s
and undiges t edt is s uesc an be r e m o v e d b y f i l t r a t i o n .
Agar culture
This is f ollowed by c ent r if uga t i o na n d w a s h i n g s o f
the protoplasts.After centrifugation,the protoplasts Agarose is the mosf frequently used agar to
are recovered above Percoll. s o l i d i f y t h e c u l t u r e m e d i a . T h e c o n c e n t r a ti o no f th e
a g a r s h o u l d b e s u c h t h a t i t f o r m s a s o ft a g a r g e l
Viability of protoplasts w h e n m i x e d w i t h t h e p r o t o p l a s t s u s p e n si o n .Th e
p l a t i n g o f p r o t o p l a s t si s c a r r i e d o u t b y B e r g m a n n 's
I t is es s ent r al t o ens ur e t h a t t h e i s o l a t e d c e l l p l a t i n g t e c h n i q u e ( R e f e r4 2 . 8 ) l n a ga r cu l tu r e s,
protoplastsare healthy and viable so that they are t h e p r o t o p l a s t sr e m a i n i n a f i x e d p o s i ti o n , d i vi d e
c apable of under going s us t ai n e dc e l l d i v i s i o n s a n d a n d f o r m c e l l c l o n e s . T h e a d v a n t a g e w i th a g a r
regeneration.There are several methods to assess c u l t u r e i s t h a t c l u m p i n g o f p r o t o p l a s t sis a vo i d e d .
t he pr ot oplas tv iabilit y .
Liquid culture
I . Fluorescein diacetate (FDA) staining
m et hod- The dy e ac c um u l a t e s i n s i d e v i a b l e L i q u i d c u l t u r e i s t h e p r e f e r r e d me th o d fo r
protoplastswhich can be detected by Iluorescence o r o t o p l a s t c u l t i v a t i o n f o r t h e f o l l o w i n g r e a so n s.
mrcroscoov.
1 lt is easy to dilute and transfer.
2. Phenosafraninestain is selectively taken up
2 D e n s i t y o f t h e c e l l s c a n b e m a n i p u l a te d a s
by dead pr ot oplas t s( t ur n r ed) w h i l e t h e v i a b l e c e l l s
desired
r em ain uns t ained.
3 . F o r s o m e p l a n t s p e c i e s , t h e c e l l s ca n n o t
3. Ex c lus ion of Ev ans b l u e d v e b v intacr
d i v i d e i n a g a r m e d i u m , t h e r e f o r el i q u i d m e d i u m i s
meml]ranes.
the only choice.
4. Measurement of cell wall formation-
Calc of iuor whit e ( CFW ) s t ain b i n d s t o t h e n e w l y 4 . O s m o t i c p r e s s u r eo f l i q u i d m e d iu n .rca n b e
f or m ed c ell walls whic h em it f l u o r e s c e n c e . altered as desired.

5. Oxygen uptake by protoplasts can be MEDIA

CULTURE
measured by oxygen electrode.
T h e c u l t u r e m e d i a w i t h r e g a r d t o n u tr i ti o n a l
6. Photosynthetic activity of protoplasts.
components and osmoticum are briefly described.
7. The ability of protoplasts to undergo
c ont inuous m it ot ic div is ion s ( t h i s i s a d i r e c r Nutritional components
m eas ur e) .
I n g e n e r a l , t h e n u t r i t i o n a l r e q u i r e m e n ts o f
protoplasts are similar to those of cultured plant
c e l l s ( c a l l u s a n d s u s p e n s i o nc u l t u r e s ) .Mo stl y, M S
a n d B 5 m e d i a ( R e f e r C h a p t e r 4 3 ) w ith su i ta b l e
m o d i i i c a t i o n sa r e u s e d . S o m e o f t h e s p e ci a lfe a tu r e s
of protoplast culture media are listed below.
The very first step in protoplasiculture is the
developmentof a cell wall aroundthe membrane 1 T h e m e d i u m s h o u l d b e d e vo i d o f
o f th e p ro to p l a s t.T h i s i s fol l ow ed by the cel l a m m o n i u m , a n d t h e q u a n t i t i e s o f i r o n a n d zi n c
d i v i s i o n sth a t g i v e ri s e to a smal l col ony.W i th s h o u l d b e l e s s .
 44 : PRO TO PLASTCULTUREAND S O M A T I C H Y B R I D I Z A T I O N
Ch AOIET 527

2 . The con c ent r at ion of c alc ium s hould o e Feeder layer technique
2-4 -times h igh er t han us ed f or c ell c ult ur es .Th i s i s
For culture of protoplastsat low density feeder
needed for membrane stabiliiy.
l a y e r t e c h n i q u e i s p r e f e r r e d .T h i s m e t h o d i s a l s o
3 High a ux in/ k inet in r at io is s uit ablet o ind u c e i m p o r t a n t f o r s e l e c t i o no f s p e c i f i c m u t a n t o r h y b r i d
ce ll divisio ns while high k inet in/ aux in r at i o i s c e l l s o n p l a t e s .T h e t e c h n i q u e c o n s i s t so f e x p o s i n g
req uire d fo r re gener at ion. p r o t o p l a s tc e l l s u s p e n s i o n st o X - r a y s ( t o i n h i b i t c e l i
division with good nretabolic activity) and then
4 Clucose is the preferred carbon soulce by
plating them on agar plates.
pro top lasts al t hough a c om binat ion of s u g a r s
(glu co sean d suc r os e)c an be us ed.
Co-culture of protoplasts
-5 The vita m ins us ed f or pr ot opias tc ult ur e s a r e
th e same a s u s ed in s t andar dt is s ue c ult ur e m e d i a . Protoplasts of two different plant species (one
slow growing and another fast growing) can be co-
Osmoticum and osmotic pressure cultured. This type of culture is advantageoussince
the growing speciesprovide tlre growth factors and
Osmoticum broadly refers to Lhe reagents/ o t h e r c h e m i c a l s w h i c h h e l p s i n t h e g e n e r a t i o no f
chemicals that are added to increase the osmotic cell wall and cell division. The co-culture method
pressure of a liquid. i s g e n e r a l l y u s e d i f t h e t w o t y p e s o f p r o t o p l a s t sa r e
The isola tion ar r d c ult ur e of pr ot oplas t sr eq u i r e m o r p h o l o g i ra l l y d i s t i n c t
osrrroticp rote c t ion unt il t hey dev elop a s t r ong c e l l
wall. In fa ct, i f t he f r es hly is olat ed pr ot oplas t sa r e Microdrop culture
d irectly a dd ed t o t he nor m al c ult ur e m edium , t h e y
S p e c i a l l y d e s i g n e dd i s h e s n a m e l y c u p r a k d i s h e s
will bu rst Th us , addit ion of an os m ot ic um r s
with outer and inner chambers are used for
e ssen tialfor bot h is olat ion and c ult ur e m edi a o f
microdrop culture. The inner chamber carries
protoplast to prevent their rupture The osmotica
s e v e r a l w e l l s w h e r e i n t h e i n d i v i d u a l p r o t o p l a s t si n
a re o f two tvp es- non- ionic and ionic .
droolets of nutrient medium can be added. The
Non -ion ic os m ot ic a : The non- ionic s ubs t a n c e s o u t e r c h a m b e r i s f i l l e d w i t h w a t e r t o m a i n t a i n
mo st co mmon ly us ed ar e s oluble c ar bohy d r a t e s h u m i d i t y . T h i s m e t h o d a l l o w s t h e c u l t u r e o f f e w e r
such a s ma nnit ol, s or bit ol, gluc os e, f r uc t o s e , protoplastsfor droplet of the medium.
g ala cto se an d s uc r os e M annit ol, being
me tab olically iner t , is m os t f r equent ly us ed.

lon ic o smo t ic a : Pot as s ium c hlor ide, c alc i u m

chlo ride a nd m agnes ium phos phat e ar e t he i o n r c
sub sta ncesin us e t o m aint ain os m ot ic pr es s u r e .
When the protoplastsare transferredto a culture
med ium, the u s e of m et abolic ally ac t iv e os m o t r c
stab ilize rs (e.9. , gluc os e, s uc r os e) along w r t h
meta bo lica lly i ner t os m ot ic s t abiliz er s( m annito l ) i s
advantageous.As the growth of protopiasisand cell
wa ll reg en era t ionoc c ur s , t he m et abolic ally ac t i v e
comp ou nd s a r e ut iliz ed, and t his r es ult s in t h e
reduced osmotic pressureso that proper osmolarity
is main tain ed .
CULTURE M ETHO DS
The cu lture t ec hniquesof pr ot oplas t sar e al m o s t
the same th at a r e us ed f or c ell c ult ur e wit h s uit a b l e
mo dificatio ns Som e im por t ant as pec t s ar e br i e f l y
g rve n.
Protoplast regeneration which may also be
regarded as protoplast development occurs in two
srages.
.l
. Formationof cell wall
2. Development of callus/whole plant.
Formation of cell wall
The orocess of cell wall formation in cultureo
protopiastsstartswithin a few hours after isolation
thal mhy take two to several days under suitable
conditions. As the cell wall development occurs,
t h e p r o t o p l a s t s l o s e t h e i r c h a r a c t e r i s t i cs p h e r i c a l
shape. The newly developed cell wall by
p r o t o p l a s t sc a n b e i d e n t i f i e d b y u s i n g c a l c o f l u o r
w h i t e f l u o r e s c e n ts t a i n .
528 B IOTECHNO LO CY

The freshly formed cell wall is composed of

loos ely bound m ic r of ibr ils w h i c h g e t o r g a n i z e d t o T*sE 44.2 Selected examples of plant species
f or m a t y pic al c ell wall. Th i s p r o c e s s o f c e l l w a l l regeneYated from ProtoPlasts
development requires continuous supply of
Category Plant species
nutrients, particularly a readily metabolisedcarbon
source (e.g. sucrose). Cell wall development rs Cereals Oryzasativa
f ound t o be im pr oper in t h e p r e s e n c e o f i o n i c Zeanays
os m ot ic s t abiliz er sin t he m e d i u m . Hordeumvulgare
The protoplastswith proper cell wall development Vegetables Cucumissativus
under go nor m al c ell div is i o n . O n t h e o t h e r h a n d , Brassica
oleracea
protoplastswith ooorly regeneratedcell wall show Capsicumannuum
budding and f ail t o under g o n o r m a l m i t o s i s Woodytrees Larixeurolepsis
i^#^^
wvucd ^^^^^A^,^
.vat rvyuvr a
Development of callus/whole plant Prunus
aviun
As t he c ell wall f or m at i o n a r o u n d p r o t o p l a s t sl s Ornamentals Rosasp
c om plet e, t he c ells inc r ea s e i n s i z e , a n d t h e f i r s t Chrysanthemun
sp
div is ion gener ally oc c u r s w i t h i n 2 - 7 d a y s . Pelargonium
sp
Subs equentdiv is ions r es ul t i n s m a l l c o l o n i e s , a n d
by t he end of t hir d w e e k , v i s i b l e c o l o n i e s
Tubersand roots Betavulgaris
( m ac r os c opicc olonies ) ar e f o r m e d . T h e s e c o l o n i e s lponocabatatas
are then transferredto an osmotic-free(mannitol or Oil crops Helianthus
annrJces
sorbitol-free) medium for further development to Brassica
napus
f or m c allus . W it h induc t i o n a n d a p p r o p r i a t e Legumes Glycinemax
m anipulat ions ,t he c allus c a n u n d e r g o o r g a n o g e n i c
or embryogenic differentiation to finally form the
whole plant. A general view of the protoplast di sconti nuousgradi ents duri ng cent r if ugat ion.
isolation,culture and regenerationis representedin Exposureof protoplaststo cytochalasinB in
Fig. 44.2 (See p. 525). association is a betterapproach
with centrifugation
for fragmentationof protoplasts. There are three
Plant regeneration can be done from the callus
typesof sub-protoplasts(Fig.44.3).
obtained either from protoplasts or from the
culture of plant organs. There are however, certain 1. Mi ni protopl asts : Theseare a lso called as
differencesin these two calluses.The callus deriveo karyoplasts and contain the nucleus.
from plant organs carries preformed buds or Mi ni protopl astscan di vi de and are capable of
or ganiz ed s t r uc t ur es , wh i l e t h e c a l l u s f r o m regenerati on i nto pl ants.
protoplast culture does not have such structures 2. Cytoplasts : These are sub-protoplasts
The first successof regenerationof plants from contai ni ngthe ori gi nal cytopl asmicm at er ial( in
protoplast cultures of Nicotiana tabacum was part or ful l ) but l ack nucl eus.Thus,cyt oplastar
s e
ac hiev ed by Tak ebe et a/ ( i n 1 9 7 1 ) . S i n c e t h e n , nuclear-freesub-protoplastswhich cannot divide,
several speciesof plants have been regeneratedby but they can be used for cybridization
using protoplasls (Table 44.2) 3. Microprotoplasts; l-his term was suggested
that contain not all but a few
for sub-protoplasts
cnromosomes.

fhe fragments derived from protoplasfs that do

n o t c o n ta i n a l l th e c o n t entsof pl ant cel l s are
referredto as sub-protoplasts. lt is possibleto T h e c o n v e n t i o n a l m e t h o d t o i m p r o ve th e
experimentally inducefragmentation of protoplasts characteristicsof cultivated plants, for years, has
to form sub-protoplasts. This can be done by been sexual hybridization The major limitation of
applicationof differentcentrifugal forcescreatedby sexual hybridization is that it can be performed
 Chapt er44 : P RO T O P L ASCTU L T U R E
AN D SOMA TICH Y B R ID IZA TION
Fig. 44.3 : A diagrammatic representation of fragmentation ol protoplast to form sub-protoplasts
(cytoplasts and miniprotoplasts)
w ithin a pla nt spec ies or v er y c los ely r elat e d
species This restrictsthe improvementsthat can be
do ne in p lan ts.
The species barriers for plant improvenrent
en co un tere d in s ex ual hy br idiz at ion c an be
overcome by somatic cell fusion that can form a
viab le h yb rids. Som at ic hy br idiz at ion br oadl y
involves in vitro fusion of isolated protoplasts to
form a hybrid cell and its subsequent development
to form a hybrid plant. Plant protoplasts are of
immen se u tility in s om at ic plant c ell genet ic
ma nip ula tion s an d im por ov em ent of c r ops . Thus ,
protoplasts provide a novel opportunity to create
cells with n ew ge net ic c ons t it ut ion.And pr ot oplas t
fusion is a wonderful approach to overcome sexual
incompatibility between different speciesof plants
More de tails on t he applic at ions of s om at i c
hybridization are given later.
So matic h ub ridiz at ion inv olv es t he f ollowing
asDects:
'I
. Fusion of protoplasts
2. Sele ctio n o f hy br id c ells
3. lde ntificatio nof hv br id olant s .
F USION OF PR O TO PLASTS
As the isolated protoplasts are devoid of cell
walls, their in vitro fusion becomes relatively easy
T he re a re no b ar r ier s of inc om pat ibilit y ( at
Interspecific,intergenericor even at interkingdom
levels) for the protoplast fusion.
Pro top last fusion t hat inv olv es m ix ing of
protoplasts of two different genomes can be'
ac hie ve d by spo ntaneous ,m ec hanic al, or induc ed
fusion methods.
Biotechnology [34]
529
| (.])
\ \_-/ /
Cytoplast
t9
Miniprotoplas
Spontaneous fusaon
Cell fusion is a natural processas is observed in
case of egg fertilization. During the course of
enzymatic degradationof cell walls, some of the
adjoining protoplasts may fuse to form
homokaryocyfes thomokaryons).These fused cells
may sometimes contain high number of nuclei
( 2 - 4 0 ) . T h i s i s m a i n l y t r a c a u s eo f e x p a n s i o n a n c l
subsequent coalescence of plasmodermal
connections between cells. The freouencv of
homokaryon formation was found to be high in
p r o t o p l a s t si s o l a t e d f r o m d i v i d i n g c u l t u r e d c e l l s .
Spontaneously fused protoplasts, however,
cannot regenerate into whole plants, except
u n d e r g o i n ga f e w c e l l d i v i s i o n s .
Mechanical fusion
The protoplasts can be pushed together
mechanically to fuse. Protoplasts of Lilium and
Trillium in enzyme solutions can be fused by gentle
t r a p p i n g i n a d e p r e s s i o ns l i d e . M e c h a n i c a l f u s i o n
may damage protoplastsby causing injuries.
Induced fusion
Freshly isolated protoplasts can be fused by
induction. There are several fusion-inducing agents
which are collectively referred to as fusogense.g.
NaNOr, high pH/Ca2*, polyethylene glycol,
p o l y v i n y l a l c o h o l , l y s o z y m e ,c o n c a v a l i nA , d e x t r a n ,
dextran sulfate,fatty acids and esters,electrofusion.
Some of the fusogens and their use in induced
fusion are described. A diagrammatic
representationof protoplast fusion is depicted in
Fig. 44.4.
530 B IOTECHNO LO CY

T h e m e t h o d c o n s i s t so f i n c u b a t i n g p r oto p l a stsi n a
s o l u t i o n o f 0 . 4 M m a n n i t o l c o n t a i n i n g 0 .0 5 M
.l
CaCl, at pH 0 . 5 ( g l y c i n e - N a O H b u ffe r ) a n d
t e m D e r a t u r e 3 7 'C f o r 3 0 - 4 0 mi n u te s Th e
protoplasts form aggregates, and fusion usually
o c c u r s w i t h i n 1 0 m i n u t e s . B y t h i s m e th o d , 2 0 - 5 0 %
of the protoplastsare involved in fusion

Polyethylene glycol (PEG) treatment : This has

become the method of choice, due to its high
successrate,{or the fusion of protoplastsfrom many
Adhesion p l a n t s p e c i e s . T h e i s o l a t e d p r o t o p l asts i n cu l tu r e
m e d i u m ( . 1m l ) a r e m i x e d w i t h e q u a l vo l u m e ( .1m l )
o { 2 B - 5 6 'k P E C ( m o l . w t . 1 5 0 0 - 6 0 0 0 d a l to n s) i n a
t u b e P E C e n h a n c e sf u s i o n o f p r o t o p l a stsi n se ve r a l
s o e c i e s T h i s t u b e i s s h a k e n a n d t h en a l l o w e d to
settle The settled protoplasts are washed several
times with culture medium.
(O O)r"'on P E C t r e a t m e n t m e t h o d i s w i d e l y u se d fo r

Y-"J protoplast fusion as it has several advantages

o l t r e s u l t s i n a r e p r o d u c i b l e h i g h - fr e q u e n cy o f
heterokaryon formation.

e Low toxicity to cells.

{ a\ a-) I ruseoprotoptasr
V/ (heterokaryon)
\ V . Reduced formation of binucleate heterokaryons.
\_-./

I . P E C - i n d u c e df u s i o n i s n o n - s p e c i f i ca n d th e r e fo r e
c a n b e u s e d f o r a w i d e r a n g e o f p l a n ts

Hybrid
Fig. 44,4 : A diagrammatic representation of
protoplast fusion.
Treatment with sodium nitrate : The isolated
protoplasts are exposed to a mixture of 5.5%
NaNO , in 10% s uc r os e s o l u t i o n I n c u b a t i o n i s
c ar r ied out f or 5 nr inut es a t 3 5 'C , f o l l o w e d b y
centrifugation (200 xg for 5 min). The protoplast
pellet is kept in a water bath at 30'C for about 3C
m inut es , dur ing whic h pe r i o d p r o t o p l a s t f u s i o n
occurs. NaNO, treatment resultsin a low frequency
of heterokaryon formation,' particularly when
mesopfryll protoplastsare fused.
High pH and high Ca2* ion treatment : This
method was first used for the fusion of tobacco
protoplasts,and is now in use for other plants also.
E l e c t r o f u s i o n: I n t h i s m e t h o d , e l e ctr i ca l fi e l d i s
used for protoplastfusion. When the protoplastsare
o l a c e d i n a c u l t u r e v e s s e l f i t t e d w i th m i cr o -
electrodes and an electrical shock is applied,
p r o t o p l a s t s a r e i n d u c e d t o f u s e . El e ctr o fu si o n
t e c h n i q u e i s s i m p l e , q u i c k a n d e f f i c i e n t a n d h e n ce
preferred by many workers. Further, the cells
formed due to electrofusiondo not show cytotoxrc
responsesas is the case with the use of fusogens
(including PEC). The major limitation of this
method is the reguirement of specialized and
costly equipment.
Mechanism of fusion
T l r e f u s i o n o f p r o t o p l a s t si n v o l v e s th r e e p h a se s
a g g . l u t i n a t i o n , p l a s m a m e m b r a n e fu si o n a n d
formation of heterokaryons.
1 . Agglutination (adhesion) : When two
protoplasts are in close contact with each other,
a d h e s i o n o c c u r s . A g g l u t i n a t i o n c a n b e i n d u ce d b y
f u s o g e n se . g . P E C , h i g h p H a n d h i g h C a 2 +.
 A N D SOMA TICH Y B R ID IZA TION
Chapt er44 : P R OT OP L ASCTU L T U R E 531

2 Plasma membrane fusion : Protoplast

membranesget fused at localized sites at the points
o f ad he nsion . This leads t o t he f or m at ion o f
cytoplasmic bridges between protoplasts. The
plasma membrane fusion can be increased by high
pH and high Ca2+, high temperature and PEG, as
e xo lain ed b elo w.
(a) High pH and high Ca2+ ions neut r a l i s e
the surface charges on the protoplasts.
Th is allows c los er c ont ac t and nr em br a n e 2A 28
fusion between agglutinated protoplasts.
(b ) High t em per at ur e helps in the
II
inte rm ingling of lipid m olec ules o f
agglutinatedprotoplastmembranesso that
t
memb r ane f us ion oc c ur s .
/ /--\ /--\ \
(c) PEG c aus es r apid agglut inat ion a n d
formation of clumps of protoplasts.This
\UUl
re su lt sin t he f or m at ion of t ight adhes i o n s Homokaryon
( A +A )
of me m br anes and c ons equent ly t h e i r
fu sio n.
Fig. 44.5 : Fusion products of protoplasts.
3. Formation of heterokaryons : The fused
protoplastsget.rounded as a result of cytoplasmrc
b ridg es le ad ing t o t he f or m at ion of s pher i c a l on MS medium, but are sensitive to (inhibited
homokaryon or heterokaryon. by) actinomycin D. Petunia parodii protoplasts
( s p e c i e sB ) f o r m s m a l l c o l o n i e s , b u t a r e r e s i s t a n t o
SEL ECTION O F HYBRI D CELLS actinomycin D. When these two speciesare fuseo,
the fused protoplastsderive both the characters-
About 20-25'/o o! the protoplasts are actually
formation of macroscooic colonies and resistance
involved in the fusion. After the fusion process,the
to actinomycin D on MS medium. This helps in the
protoplast population consists of a heterogenous
selection of hybrids (Fig. 44.0. fhe parental
mixture of unfused chloroplasts,homokaryons and protoplasts
of both the species fail to grow.
heterokaryons (Fig. 44.5). lt is therefore necessary Protoplastsof P. parodii form very small colonres
to sele ct the hy br id c ells ( het er ok ar y ons ) .T h e while that of P. hybrida are inhibited by
commonly used methods employed for the a c t i n o m y c i n D .
selection of hybrid cells are biochemical, visual
Drug sensitivity technique was originally
and cytometric methods.
developed by Power et al (1976) lor the selection
of hybrids of Petuniasp (describedabove).A similar
Biochemical methods
orocedure is in use for the selection of other
The bio ch em ic al m et hodsf or s elec t ionof hy b r i d S o m a t i c h y b r i d s e . g , h y b r i d s b e t w e e n N i c o t i a n a
cells a re ba s ed on t he us e of bioc hem i c a t sylvestris and Nlcotian a knighti ana.
comp ou nd s in t he m edium ( s elec t ion m ediu m ) .
2. Auxotrophic mutants : Auxotrophs are
The se comp ou nds help t o s or t out t he hy br id a n d
m u t a n t st h a t c a n n o t g r o w o n a m i n i m a l m e d i u m a n d
parental cells based on their differences in the
thereforerequire specificcompoundsto be added to
expression of characters. Drug sensitivity and
the medium. Nitrate reductasedeficient mutants of
auxotroohic mutant selection methods are
tobacco (N. tabacum) are known. The parental
de scribe d be low
protoplastsof such speciescannot grow with nitrate
.l
. Drug sensitivity : This method is useful for a s t h e s o l e s o u r c eo f n i t r o g e nw h i l e t h e h y b r i d s c a n
th e sele ctio nhybr ids of t wo plant s pec ies ,if one o f grow. Two speciesof nitrate reductasedeficiency-
them is sensitive to a drug. Protoplastsof Petunia one due to lack of apoenzyme(nia-typemutant)and
hybride (speciesA) can form macroscopic callus the other due to lack of molvbdenum cofactor (cnx-
532 BIOTECHNOLOCY

SpeciesA SpeciesB There are two approaches in this direction-

(Petunia hybrida) (Petuniaparodii) g r o w t h o n s e l e c t i o n m e d i u m , a n d m e ch a n i ca l
I
I II isolation.

1. Visual selection coupled with differential

J J media growth: T h e r e e x i s t c e rta i n n a tu r a l
Protoplasts Protoplasts
differences in the sensitivity of protoplasts to the
n u t r i e n t so f a g i v e n m e d i u m . T h u s , s o me m e d i a ca n
selectively support the development of hybrids but
not the parental protoplasts.

A diagrammatic representation of visual

selection coupled with the growth of heterokaryons
on a selection medium is given in Fig. 44.8.

2 . M e c h a n i c a l i s o l a t i o n : T h e v i s u a l l y i d e n ti fi e d
heterokaryons under the microscope can be
No divisions
Smallcolonies Largecolonies and no growth i s o l a t e d b y m e c h a n i c a l m e a n s . T h i s i n vo l ve s th e
use of a special pipette namely Drummond pipette.
+
I The so isolated heterokaryons can be cloned to
Nofurther FurthergroMh finally produce somatic hybrid plants. The major
growth limitation of this method is that each type of hybrid

+
I
Callus

I
+
SpeciesA
(NR nia-)
SpeciesB
(NR cnx-)

Somatic
hybridplants
II II
Fig. 44.6 : A diagrammatic reprcsentation of drug
T J
Protoplasts Protoplasts
sensitivity method for the isolation of hybrid cells.

type mutant)are known. The parentalprotoplasts

c a n n o tg ro w o n n i tra temedi umw hi l e the hybri d
protoplastscan grow (Fig. 44.7).
The selectionof auxotrophicmutantsis possible Minimalculturemediumwith nitrate
o n l y i f th e h y b ri d c e l l s c an grow on a mi ni mal
m e d i u m.An o th e rl i m i ta ti o nof the techni quei s the
p a u c i tyo f h i g h e rp l a n ta u x otrophs. No divisions
No divisions Colonies and no growth
and no growth
Visual methods
Vi s u a sl e l e c ti o o
n f h y b ri dcel l s,al thoughtedi ous
Callus
is very efficient. In some of the somatic
h y b ri d i z a ti o ne x p e ri me n ts,chl oropl astdefi ci ent
(albinoor non-green) protoplasts of one parentare
fusedwith greenprotoplasts of anotherparent.This Somatic
facilitatesthe visualidentification of haterokaryons hybridplants
under light microscope. fhe heterokaryonsare
bigger and green in colour while the parental Fig. 44,7 : A diagrammatic representation of hybrid
protoplastsare either small or colourless.Further selection based on auxotrophic mutant (NR nia--
Nitrate reductase apoenzyme defieient; NR cnr-
identificationof these heterokaryonshas to be
Nitrate rcductase lacking molybdetum cofactor).
carriedout to developthe specifichybrid plant.
ChA O I CT A N D S OMA TICH Y B R ID IZA TION
44 : P R OT OP L ASCTU L T U R E 533

SpeciesA SpeciesB i d e n t i f i c a t i o no f h y b r i d p l a n t s a r e b r i e f l y d e s c r i b e d .
(Green) (Albinoor non-green)

+
I I
+ Morphology of hybrid plants
Protoplasts Protoplasts
Morphological features of hybrid plants which
usually are intermediate between two parents can
be identified. For this purpose, the vegetative and
floral charactersare considered.These include leaf
shape, leaf area, root morphology, flower shape, its
structure, size and colour, and seed capsule
Culturedon a selectionmedium morphology.

The somatic hybrids such as pomatoes and

topatoes which are the fused products of potato
and tomato show abnormal morphology, and thus
Smallgreencell Greencell Whitecell
colonies colonies colonies can be identified.

II II ,I A l t h o u g h t h e g e n e t i c b a s i so f t h e m o r p h o l o g i c a l
I
+ + + charactershas not been clearly known, intermediate
No further Greencallus Whitecallus morphological features suBgestthat the traits are
growth u n d e r t h e c o n t r o l o f m u l t i p l e g e n e s .l t i s p r e f e r r a b l e
II
t o s u p p o r t h y b r i d m o r p h o l o g i c a l c h a r a c t e r sw i t h
+ evidence of genetic data.
So m a tic
h yb r id p la n ts
lsoenzyme analysis of hybrid plants
Fig. 44.8 : A diagrammatic representation of visual
selection of hybrids coupled with growth on T h e m u l t i p l e f o r m s o f a n e n z y m e c a t a l y s i n gt h e
selection medium. same reaction are referred to as isoenzymes.
Electrophoreticpatterns of isoenzymes have been
widely used to verify hybridity. Somatic hybrids
c ell r equir esa s p e c i a l c u l tu re me d i u m fo r i ts posses specific isoenzymes (of certain enzymes) of
growth. This can be overcome by employing one or the other parent or both the parents
m ic r odr opc ultu reo f s i n g l ec e l l su s i n gfe e d e rl a yers s i m ul t a n e o u s l y .
( Ref erp. 506) .
T h e r e a r e m a n y e n z y m e s p o s s e s s i n gu n i q u e
Gytometric methods isoenzymes that can be used for the identificationof
somatic hybrids e g amylase, esterase,aspartate
Some workers use flow cvtometrv and aminotransferase, phosphodiesterase, isoperoxidase,
fluorescent-activated cell sortingtechniquesfor the and hydrogenases(of alcohol, lactate, malate). lf
analy s isof pla n tp ro to p l a s ts
w h i l e th e i rv i a b i l i tyrs t h e e n z y m e i s d i m e r i c ( h a v i n g t w o s u b u n i t s ) ,
m aint ained.Th e s a me te c h n i q u e sc a n a l s o be s o m a t i c h y b r i d s u s u a l l y c o n t a i n a n i s o e n z y m ew i t h
appliedfor sortingand selectionof heterokaryons.a n i n t e r m e d i a t em o b i l i t y p r o p e r t i e s .
T he hy br idc el l sd e ri v e dfro m s u c hs e l e c ti o nhave
s
proved useful for the developmentof certain The isoenzymes are often variable within the
s om at ichy br idp l a n ts . same plant. Therefore, it is necessaryto use the
same enzyme from each plant (parentsand somatic
|DE NT |F T CAT | ON OF H YB R T D (C E L L SI hybrids), from a specific tissue with the same age.
P LA NT S
Chromosomal constitution
The development of hybridcellsfollowedby the
generationof hybridplantsrequiresa clearproofof The number of chromosomes present in the
genetic countribution from both the parental hybrid cells can be directly counted. This provides
protoplasts.
The hybriditymustbe established only information on the ploidy state of the cells. The
from euploidand not fromaneuploidhybrids.Some somatic hybrids are expected to possess
of the commonly used approaches for the chromosomesthat are equal to the total number of
534 B IOTECHNO LO CY

ltetE 44.3 A selected list of interspecific hybrlds produced through protoplast fusion
along with the chromosomenumbers in the hybrids

Plant species with their chromosome number Chromosome nuntber(s) in the hybrid(s)

Petuniaparodii(2n= 48) + P. hybrida(2n= 1a) 44-48

Daturainnoxia(2n = 2\ + D. stramoniun (2n= 24) 46,48,72
Nicotiana
tabacum (2n= 48)
(2n= a8) + N. nesophila 96
tabacun(2n= 48)+ l,l. glutinosa
Nicotiana (2n= 24]l 50-58
Lycopersicon
esculentun (2n= 24)
(2n-- zal + L. peruvianum 72
Solanum (2n= 24,48)+ S chacoense
tuberosum (2n= M) 60
Brassica
oleracea (2n= 18)
(2n= 18)+ B. campestrrs Highly
variable
napus(2n= 38) + 8. juncea(2n= 36)
Brassica Highly
variable

c hr om c s om es or iginally pr e s e n t i n t h e p a r e n t a l s p e c i e so f p l a n t s r e s u l t i n f o r m a t i o n o f a sym m e tr i c
protoplasts.Sometimes, the hybrids are found to
contain more chromosonresthan the total of both the
par ent s .The pr es enc eof c h r o m o s o m a i m a r k e r s i s r e p l i c a t i o n o f D N A i n t h e f u s i n g p r o t o p l a sts.
gr eat lyus ef ulf or t he genet ica n a l y s i so f h y b r i d c e l l s
M olec ular t ec hniques
M any r ec ent dev elopm en t si n n r o l e c r - r l abri o l o g y
hav e im pr ov ed t he unde r s t a n d i n g o f g e n e t i c
c ons t it ut ionof s om at ic plant h y b r i d s .S o m e o f t h e m
are listed below.
l. Dit Ter enc es in t he r e s t r i c t i o n p a t t e r n s o f
c hlor oplas t and m it oc hondr i a l D N A s .
2. M olec ular m ar k er s s u c h a s R F L R A F L P ,
RAPD and m ic r os at ellit es .
3. PCR t ec hnology .
CHRO M O SO M E NUM BER IN
SO M ATI C HYRBI DS
The c hr om os om e num ber i n t h e s o m a t i c h y b r i d s
is generally more than the total number of both of
the parental protoplasts. However, wide variations
ar e r epor t ed whic h m ay be d u e t o t h e f o l l o w i n g
reasons
1 . Fusion gf more than two protoplasts.
2. lr r egular it iesin m it ot i c c e l l d i v i s i o n s .
3. I n f us ogen or elec t r o- i n d u c e df u s i o n s , a b o u t
one third of the fusions occur between more than
two protoplasts.
4. Differences in the status of protoplasts
( ac t iv ely div iding or quie s c e n t ) f r o m t h e t w o
hybrids
5 A s y m m e t r i c h y b r i d s m a y b e d u e to u n e q u a l
t i . P r o t o p l a s t i s o l a t i o n a n d c u l t u re m a y a l so
l e a d t o s o m a c l o n a l v a r i a t i o n s , a n d t h u s va r i a ti o n s
in chromosome number
A selected list of interspecifichybrids produced
t h r o u g h p r o t o p l a s tf u s i o n a l o n g w i t h t he n u m b e r o f
c h r o m o s o m e si n t h e h y b r i d s i s g i v e n i n Ta b l e 4 4 .3 .
Symmetric and asymmetric hybrids
lf Ihe chromosome number in the hybrid is the
sum of the chromosomes of the two parental
protoplasts, the hybrid is said ro be symmetric.
S y m m e t r i c h y b r i d s b e t w e e n i n c o m p a ti b l e sp e ci e s
a r e u s u a l l y 's t e r i l e .T h i s m a y b e d u e t o p r o d u cti o n
o f 3 n h y b r i d s b y f u s i n g 2 n o f o n e s p e ci e sw i th n o f
another species.
Asymmetric hybrids have abnormal or wide
variations in the chromosome number than the
exact total of trvo species.These hybrids are usually
formated with full somatic complement of one
p a r e n t a l s p e c i e s w h i l e a l l o r n e a r l y a l l o f th e
chromosomes of other parental species are lost
d u r i n g m i t o t i c d i v i s i o n s . A s y m m e t r i c h yb r i d s m a y
be regarded as cybrids but for the introgressed
8enes.
As given in Table 44.3, protoplast fusion
between N. tabacum (2n = 48) and N. nesophila
( 2 n = 2 4 ) r e s u l t s i n a s y m m e t r i c h yb r i d s, w h i l e
asymmetric hybrids are formed when B. napus and
B. iunea are fused.
 44 : P.R O T OP L AS
ChA P I CT CU A N D S O MA TICH Y B R ID IZA TION
T LTURE 535

fhe cytoplasmic hybrids where the nucleus is

derivecl from only ane parent and the cytoplasm is
derived from both the parents are referred to as
cybrid s.The p he nom enonof f or m at ion of c y br id s i s
regarded as cybridization. Normally, cybrids are
prduced when protoplastsfrom two phytogenetically
distinct species are fused Cenetically, cybrids are
hybrids only for cytoplasmic traits.

Hybrid s an d s om at ic inc om pat ibilit y

Man y a times , pr oduc t ion of f ull- pledgedhy b r i d s Nuclear

through fusion of protoplasts of distantly related fusion
higher plant species is rather Cifficult clue to
Y
instab ility o f the t wo dis s im ilar genom es in a ,=n\
/ | tv - \
commo n cytop las m . This phenom enon is r ef e r r e C
to as somatic incompatibility. HybriCs formed lu_o
Vn!,/
o
d espite so matic inc om pat ibilit y m ay ex h i b i t \\;_-/
stru ctu ralan d d ev elopm ent alabnor m alit ies .Sev e r a r Hybrid C;rtopiasr',ic'iybrids
(Cybrids)
g en era tion s ma y be r equir ed t o elim inat e t h e
un de sira blege nes Due t o t his lint it at ion in s om a t i c Fig. 44.9 : A diagrammatic representation of
hybrid iza tion , c y br idiz at ion inv olv ing pr ot op l a s t hybridization and cybridization (Note : The plastids
fu sio n for p ar t ial genom e t r ans f er is gain i n g
are shown as ] and Ql
imoortance in recent vears

Methodology of cybridization
X-rays irradiated protoplasts for more efficient
A diagrammatic representaiionof the formation formation of cybrids.
of hybrids and cybrids is given in Fig. 44.9.
4 . l t i s p o s s i b l et o s u p p r e s sn u c l e a r d i v i s i o n r n
As the formation of heterokaryonoccurs during some protoplasts and fuse them with normal
hybrid iza tion , the nuc lei c an be s t im ulat ed t o protoplasts.
segregateso that one protoplast contributes to tfre
cvto ola sm whil e t he ot her c ont r ibut es nuc le u s
Genetic recombination in
a lon e (o r bo th nuc leus and c y t oplas m ) .I n t his w a y
asexual or sterile plants
cybrid iza tion c an be ac hiev ed. Som e of th e
approachesof cybridization are given hereunder. There are many plants that cannot reproduce
s e x u a l l y .S o m a t i c h y b r i d i z a t i o n i s a n o v e l a p p r o a c h
1 . The protoplastsof cytoolasm donor species
through which two parental genomesof a sexual or
are irradiated with X-rays or Trays. This treatment
sterile plants can be brought together. Thus, by
re nd ers the pro t oplas t sinac t iv e and non- div idi n g ,
fusing parental protoplasts, fertiie dipioids and
b ut th ey are ef f ic ient donor s of c y t oplas m r c
polyploids can be produced.
constituentswhen fused with recipient protoplasts.

2. Normal protoplasts can be directly fused Overcoming barraers of sexual

with enucleated protoplasts Enucleated protoolasts
can be isolated by high-speed centrifugation.
3 Protoplasts are inactivated by metabolic
inhibitors such as iodoacetate. In practice,
iodoacetate treated protoplasts are fused with
incompatibility
Sexual crossing between two different specres
(interspecific) and two different genus (intergeneric)
is impossible by conventional breeding methods.
Somatic hybridization overcomes the sexual
536 B IOTECHNO LO CY

inc om pat ibilit y bar r ier s . Tw o e x a m p l e s a r e g i v e n

hereunder. Tlo'.r 44.4 Selected examples of sonatic hybrids
developed by interspecific protoplast fuslon
1 . Fusion between protoplasts of potato
(Solanum tuberosum) and tomato (Lycopersicon Common name Common nante
esculentum) has created pomato (Solanopersicon, a (botanical name) (botanical nante)
new genus ) .
Tomato Potato
2. Interspecificfusion of four different species (Lycopersicon
esculentun) (Solanum
tuberosuml
of rice (Oryza brachyantha, O. eichngeri, O.
Petunia Petunia
officinalis and O. perrieri) could be done to
parodil
(Petunia (Petuniaparvillora)
im pr ov e t he c r op.
Thorn
apple Deadlynightshade
A list of selected examples of somatic hybrids
(Datura
innoxia) (Atropabelladonna)
developed by interspecificprotoplastfusion is given
in Table 44.4. Turnip Thalecress
(Brassica
campestis) (Arabidopsis
thalianal
A novel approach for gene transfer
Som at ic hy br idiz at ion ha s m a d e i t p o s s i b l e t o
disease) transferred
could be successfully from one
transfer several desirable genetic charactersamong
speciesto another.For example, resistancehas
the plants (Table 44.5).
been introducedin tomatoagainstdiseasessuchas
TMV spottedwilt virus and insectpests.
Applic at ions of c y br ids
2. Environmental tolerance : The genes
Cy br idiz at ion is a wonde r f u l t e c h n i q u e w h e r e i n
responsible for the toleranceof cold, frostand salt
the desired cytoplasm can be transferred in a
coul d be successful li yntroducedthro ughsom at ic
single step. Cybrids are important for the transfer of
of cold t oler ance
e.g., i ntroducti on
hybri di zati on.
c y t oplas m ic m ale s t er ilit y ( C M S ) , a n t i b i o t i c a n d
gene i n tomato.
her bic ide r es is t anc ein agr ic u l t u r a l l y u s e f u l p l a n t s .
3. Quality characters: Somatichybridsfor the
Some of the genetic triats in certain plants are
of hi ghni coti necontent,and low er ucic
producti on
c y t oplas m ic ally c ont r olled. T h i s i n c l u d e s s o m e
acid have been developed(Tahle44.5).
types of male sterility, resistance to certain
antibiotics and herbicides. A selected list of 4. Cytoplasmicmale sterility : A modification
agronomic characterstransferredthrough cybrids rs of hybri di zati oni n the form of cybridizat ionhas
given in Tahle 44,5 (along with somatic hybrids). made i t possi bl eto transfercytop lasm icm ale
steri l i ty.D etai l sgi ven i n the preceed ing
sect ion.
Cybridization has been successfully used to
transfer CMS in rice. Cybrids oI Brassicaraphanus Other application of
that contain nucleus of B. napus, chloroplasts of somatic hybridization
atrazinc resistant B. campestris and male sterility
1. Somatichybridizationhas helped to study
from Raphanus sativas have been developed.
the cytoplasmicgenesand their functions.In fact,
the i nformati oni s successful l yused in plant
breedi ngprogrammes.
2 P rotopl ast fusi on w i l l help in t he
combi nati onof mi tochondri aand chlor oplasttso
Sonr at ic hy br idiz at ion h a s o p e n e d n e w resul t i n a uni que nucl ear-cytoplasmgenet
ic ic
possibilites for the in vitro genetic manipulation of combi nati on.
plants to improve the crops. Some of the practical 3. S omati chybri di zati on can be do ne in plant s
applic at ion ar e br ief ly giv en that are sti l l i n j uveni l ephase.
.l
. Diseaseresistance: Severalintersoecificand 4. Protoplasttransformation(with traits like
intergeneric hybrids with disease resistance have nitrogenfixationby incorporating exogenousDNA)
been created. Many disease resistancegenes (e.g., fol l ow ed by somati c hybri di zati o nwill yield
tobacco mosaic virus, potato virus X, club rot i nnovati vepl ants.
 AN D S OMA TICH Y B R ID IZA TION
Chaot er44 : P R OT OP L ASCTU L T U R E 537

Tloru 44.5 A selected llst of genctlc tralts transferred through protoplast

fuslon In crop plant species

Somatic hybrids Genetic trait in new plant/

resistance(or tolerance) trait
Nicotiana + N. nesophila
tobaccum Tobacco mosaic virus
Tobacco hornworm
Solanuntuberosum + S. chacoense PotatovirusX
Solanumtuberosum + S. comnersounii Frosttolerance
Solanum tuberosum + S. brevidens Potatoleafrollvirus
Solanunochranthum + Lycopersiconesculentum Coldtolerance
Brassica oleracea+ B. napus Blackrot
lanalus+ Cucumis
Citrullus melo Clubrot
Raphanus sativus+ Brassicanapus Beetcystnematode
Hordeum vulgare+ Daucuscarota Frostandsalttolerance

Somatic hvbrids Quality character

Nicotiana + N. rustica
tabacum Highnicotine
content
Brassicanapus+ Erucasativa Lowerucicacid

Cytoplasmic hybrids (cybrids) Agronomic character

Nicotiana
tabacum + N. sylvestris Streptomycin
resistance
Nicotiananigrum+ Solanumtuberosum Triazine
resistance
Brassicanigra+ B. napus Hygromycinresistance
Solanum nigrum+ S. tuberosum Triazine
resistance
Nicotianatabacun+ N. sylvestris Cytoplasmic
malesterility
Brassicanapus+ B. tournefortii Cytoplasmic
malesterility
Brassicacanpestris+ B. napus Cytoplasmic
malesterility
Lycopersiconesculentun + Solanunacaule Cytoplasmic
malesterility
Brasicanapus + B. canpestris+ Raphanussativa Cytoplasmic
maiesterilityandlriazineresistance

LIMITATIONS OF 4. Protoplast fusion between different species/

SOMATIC HYBRI DI ZATI O N g e n u s i s e a s y ,b u t t h e p r o d u c t i o n o f v i a b l e s o m a t i c
hvbrids is not oossible in all instances.
Alth ou gh so m at ic hy br idiz at ion is a no v e l
approach in plant biotechnology, there are several 5. Some of the somatic hybrids, particularly
oro ble ms a nd lim it at ions . The s uc c es s of t h e when produced by the fusion of taxonomically
te ch niq ue larg ely depends on ov er c om ing t h e s e different partners, are unbalanced and not viable.
limita tion s, so m e of whic h ar e lis t ed below. 6 . T h e r e a r e l i m i t a t i o n si n t h e s e l e c t i o nm e t h o d s
.l
. Somatic. hybridization does not always of hybrids, as many of them are not efficient.
produce plants that give fertile and visible seeds. 7 There is no certainity as regards the
2. Regenerated plants obtained from somatic expression of any specific character in somatic
hybridization are often variable due to somaclonal h y b r i di z a t i o n .
variations, chromosomal elimination, organelle B. Somatic hybridizationbetween two diploids
segregatronetc. r e s u l t si n t h e f o r m a t i o n o f a n a m p h i d i p l o i d w h i c h i s
3. Protoplast culture is frbquently associated not favourable. For this reason, haploid protoplasts
with g en etic inst abilit y are recommendedin somatic hybridization.
 aploid plants are characterizedby possessing
l__l
| | only a single set of chromosomes(gametophytic
num ber of c hr om os om esie. n ) i n t h e s p o r o p h y t e .
This is in c ont r as tt o diploids w h i c h c o n t a i n t w o s e r s
(2n) of chromosomes.Haploid plants are of great
significancefor the production of homozygous lines
(homozygous plants) and for the improvement of
plant s ir r plant br eedingpr ogr a m m e s .
Brief history
The ex is t enc e of haploid s w a s d i s c o v e r e d ( a s
szply as 1921) by Bergner in Datura stramonium.
Plant breeders have been conducting extensrve
r es ear c ht o dev elop haploids . T h e I n d i a n s c i e n t i s t s
Guha and Maheswari (1964) reported the direct
development of haploid embryos and plantlets
from microspores of Datura innoxia by the cultures
of ex c is ed ant her s . Subs eq u e n t l y , B o u r g i n a n d
Hit s c h ( . 1967) obt ained t he f ir s t f u l l - p l e d g e dh a p l o i d
plants from Nicotiana tabacum. Thereafter,much
progress has been made in the anther cultures of
wheat , r ic e, m aiz e, pepper a n d a w i d e r a n g e o f
ec onom ic ally im por t ant s pec i e s .
G r ouping of haploids
Haploids m ay be div id e d i n t o t w o b r o a d
cateSofles.
.l
. Monoploids (monohaploids) : These are the
haploids t hat pos s es s ha l f t h e n u m b e r o f
c hr om os om es f r om a diploid s p e c i e s e . g . m a i z e ,
bar ley
538
2 P o l y h a p l o i d s : T h e h a p l o i d s p o sse ssi n gh a l f
t h e n u m b e r o f c h r o m o s o m e s f r o m a p o l yp l o i d
s p e c i e s a r e r e g a r d e d a s p o l y h a p l o i d s e .g . w h e a t,
Dotato.
It may be noted that when the term haploid is
g e n e r a l l y u s e d i t a p p l i e s t o a n y p l a n t o r i g i n a ti n g
f r o m a s p o r o p h y t e ( 2 n ) a n d c o n t a i n i ng h a l f th e
number (n) of chromosomes
IN VTVA AND VITRO APPROACHES
'fV
T h e i m p o r t a n c e o f h a p l o i d s i n t h e f i el d o f p l a n t
b r e e d i n g a n d g e n e t i c sw a s r e a l i s e dl o n g a g o Th e i r
practical application, however, has been restricted
d u e t o v e r y a l o w f r e q u e n c y ( <0 . 0 0 1 % ) o f th e i r
f o r m a t i o n i n n a t u r e . T h e p r o c e s s o f ap o m i xi s o r
parthenogenesis (development of embryo from an
unfertilized egg) is responsible for the spontaneous
natural production of haploids. Many attempts
were made, both by in vivo and in vitro methods to
d e v e l o p h a p l o i d s .T h e s u c c e s sw a s m u ch h i g h e r b y
in vitro techniques.
ln vivo techniques for haploid
production
There are several methods to induce haploid
production in vivo. Some of them are listed below.
.l
. Androgenesis : Development of an egg cell
containing male nucleus to a haploid is referredto
as androgenesis. For a successful in vivo
a n d r o g e n e s i st,h e e g g n u c l e u s h a s t o b e i n a cti va te d
or eliminated before fertilization.
 ChA P t Cr
45 : P R O D U C T IO N
O F H AP L O IDPL A N TS
2. Gynogenesis : An unfertilized egg can lre
m an ipu late d (by delay ed pollinat ion) t o dev el o p
in to a h ap loid p lant .
3. Distant hybridization : Hybrids can be
prod uced b y e lim inat ion of one of t he par en t a l
ge no mes a s a r es ult of dis t ant ( int er s pec if ic o r
inte rge ne riccro s s es )hy br idiz at ion.
4lrra dia tion ef f ec t s : Ult r a v iolet r ay s o r
X-ravs mav be us ed t o induc e c hr om os om a l
brea ka ge a nd t heir s ubs equent elim inat ion t o
prod uce h ap loid s .
5 Ch emical t r eat m ent : Cer t ain c hem ic a l s
( e g ., ch lora mph enic ol, c olc hic ine, nit r ous ox id e ,
m ale ic hydra z ide) c an induc e c hr om os om a l
elimin atio n in s om at ic c ells whic h m ay r es ult r n
hao loid s.
ln vitro techniques for haploid
production
In th e p lan t b iot ec hnologypr ogr am m es ,haplo i d
production is achieved by two methods
1 . And rog en es is : Haploid pr oduc t ion oc c u r s
thro ug h an the r or pollen c ult ur e, and t hey a r e
referred to as androgenic haploids.
2. Gynogenesis : Ovary or ovuie culture that
re su lts in th e p r oduc t ion of haploids , k nown a s
gynogenic haploids.
In a nd rog en es is , t he m ale gam et ophyt e
(m icrospo reor im m at ur e pollen) pr oduc es haplo i d
pla nt Th e ba s ic pr inc iple is t o s t op t h e
development of pollen cell into a gamete (sex cell)
and force it to develop into a haploid plant.
There are two approaches in androgenesis-
a nth er cu lture a nd pollen ( m ic r os por e) c ult ur e .
You ng pla nts, gro wn under opt im al c ondit ions o f
ligh t, temp era tur e and hum idit y , ar e s uit able f o r
androg en esi s.
ANTHER CULT URE
The selected flower buds of young plants are
surface-sterilizedand anthers removed along with
the ir filame nts. The ant her s ar e ex c is ed unde r
aseoticconditions,and crushed in 17oacetocarmine
to test the stage of pollen development. lf they are
539
at the correct stage,each anther is gently separated
(from the filament) and the intact anthers are
inoculated on a nutrient medium. Injured anthers
should not be used in cultures as they result in
callusingof anther wall tissue.
T h e a n t h e r c u l t u r e sa r e m a i n t a i n e d i n a l t e r n a t i n g
p e r i o d s o f l i g h t ( 1 2 - 1 8 h r ) a n d d a r l <n e s s( 6 - 1 2 n r s t
at 28"C. As the anthers proliferate, they produce
callus which later forms an embryo and then a
haploid plant (Fig. 45.1).
POLLEN (MTCROSPORE) CULTURE
Haploid plantscan be produced from immature
pollen or microspores(male gametophytic cells).
The pollen can be extracted by pressing and
s q u e e z i n gt h e a n t h e r s w i t h a g l a s s r o d a g a i n s t t h e
s i d e s o f a b e a k e r .T h e p o l l e n s u s p e n s i o ni s f i l t e r e d
to remove anther tissue debris. Viable and large
pollen (smaller pollen do not regenerate) are
concentrated by filtration, washed and collected.
These pollen are cultured on a solid or liquid
medium The callus/embryoformed is transferredto
a suitable medium to finally produce a haploid
plant (Fig. 45.1), and then a diploid plant (on
colchicine treatment).
Gomparison between anther
and pollen cultures
Anther culture is easy, quick and practicable.
Anther walls act as conditioning factors and
promote culture growth. Thus, anther cultures are
reasonably efficient for haploid production. The
malor limitation is that the plants not only originate
from pollen but also from other parts of anther.This
r e s u l t si n t h e p o p u l a t i o n o f p l a n t s a t d i f f e r e n tp l o i d y
levels (diploids, aneuploids). The disadvantages
associatedwith anther culture can be overcome by
pollen culture.
Many workers prefer pollen culture, even
though the degree of successis low, as it offers the
following advantages
. Undesirable effectsof anther wall and associateo
t i s s u e sc a n b e a v o i d e o .
. A n d r o g e n e s i s ,s t a r t i n gf r o m a s i n g l e c e l l , c a n b e
better regulated.
. l s o l a t e dm i c r o s p o r e s( p o l l e n ) a r e i d e a l f o r v a r i o u s
genetic manipulations (transformation,
mutaSenesrs)
. The yield oi haploid plants is relatively higher.
540 B IOTE CHNO LO CY

D E V E LOP ME N T OF A N D R OGE N IC
H A P LOID S
The processof in vitro androgenesis for the
o{ hapl oi dpl antsi s d epict edin
ul ti mateproducti on
-----__}
J-- Fig. 45.2.
n
ww
n
Anthers
ss
Anthers
The cul turedmi crosporesmai nl y f ollow f our
distinctpathwaysduringhe initial stagesof in vitro
androgenesi s.
J J Pathway | : The uninucleate microspore
undergoesequal division to form two daughter
cellso( equal size e.g. Daturainnoxia.
Platingof Pathwayll : In certainplants,the microspore
anthers Extractionof pollen
di vi desunequal l yto gi vebi ggervegeta t ive cell and
J J a smallergenerativecell. lt is the vegelativecell
,d_\ that undergoesfurtherdivisionsIo form callusor
r^€&\
qry embryo.The generati ve cel l , on the o t her hand,
degeneratesafter one or two divisions-e.g.,
Proliferating Nicotianatabacum,Capsicumannuum.
anthers
Pollencollected Pathway lll : In this case, the microspore
J (afterwashing)
undergoesunequal division. rhe embryos are
_1')-a
q ', <
(ry
I formed from the generative cell while the
vegetativecell does not divide at all or undergoes
Callus \i limitednumberof divisionse.g. Hyoscyamus niger.
P athw ayl V : The mi crospore di vi desunequally
I Pollenculture
(solid/liquid
medium) as i n pathw aysl and l l . H ow ever,i n thi scase,bot h
t\

l'w I vegetativeand generative cells can further divide

u
1 ,.1l

Embryo \i
and contribute to the development of haploid
plant e.g. Datura metel, Atropa belladonna.
development \__-/ At the initial stages,the microsporemay follow
Pollen
embryo
any one of the four pathwaysdescribedabove.As
the cel l s di vi de, the pol l en grai n becom es
and burstopen.Thi smul ti celluar
mul ti cel l ul ar m ass
may form a calluswhich laterdifferentiates into a
Haploid plantlet plant (through callus phase). Alternately,the
massmay producethe pl antt hr ough
mul ti cel l ul ar
I cot.hi"in"
direct ernbryogenesis(Fig. 45.1).
J treatment
FA C TOB S A FFE C TIN G A N D R OGENESI S
A good knowledgeof the variousfactorsthat
i nfl uenceandrogenesiwsi l l hel p to i mpr ovet he
productionof androgenichaploids.Someof these
factorsare brieflydescribed.

Genotype of donar plants

The successof antheror pol l en cul t ur elar gely
diploidplant
Homozygous
dependson the genotypeof the donor plant. lt is
thereforeimportantto selecfonly highly responsive
Fig, 45.1 : Diagrammatic reptesentation of anthel
genotypes. Some workers choose a breeding
and pollen cultures for the production of haploid
and diploid plants.
approachfor improvement of genotypebeforethey
are usedi n androeenesi s.
Chapt er45 : P R O D U C T IO N
O F H AP L O IDPLA N TS 541

(A)

FI RST MITOSIS

(B)

e n r \ r , i ^ h \ / i; /h a r r l n i ,l \

Fig. 45.2 : Diagrammaticrepresentationof miuoscope divrsionsleading to the formationol a multtcelluar

pollen grain (A), followedby the formationof haploid sporophyte(B) (Note : l, ll, lll and lV indicaterespective
pathways).
542 B IOTECHNO LO G Y

Stage of microspore or pollen Effect of light

The selection of anthers at an ideal stage of ln general, the production of haploids is better
microspore development is very critical for haploid in light. There are however, certain plants which
production. In general, microspores ranging from can grow well in both light and dark.
tetrad to binucleate stages are more responsive. lsolated pollen (not the anther) appears to be
Anthers at a very young stage (with microspore s e n s i t i v e t o l i g h t . T h u s , l o w i n t e n si ty o f l i g h t
mother cells or tetrads) and late stage (with promotes development of embryos in pollen
binuc leat e m ic r os por es )ar e u s u a l l y n o t s u i t a b l ef o r cultures e.g. tobacco.
androgenesis.However, for maximum productron
of andr ogenic haploids , t h e s u i t a b l e s t a g e o f Effect of culture medium
m ic r os por e dev elopm ent is de p e n d e n t o n t h e p l a n t
T h e s u c c e s so f a n t h e r c u l t u r e a n d a n d r o g e n e si s
species, and has to be carefully selected
i s a l s o d e p e n d e n t o n t h e c o m p o s i ti o n o f th e
medium. There is, however, no single medium
Phy s iologic al s t at us of a d o n a r p i a n t
s u i t a b l ef o r a n t h e r c u l t u r e so f a l l p l a n t sp e ci e s Th e
The plant s gr own u n d e r b e s t n a t u r a l c o m m o n l y u s e d m e d i a f o r a n t h e r c u l t ur e s a r e M 5 ,
env ir onm ent al c ondit ions ( l i g h t , t e m p e r a t u r e , Wh i t e 's , N i t s c h a n d N i t s c h , N 6 a n d 8 5 . Th e se
nut r it ion, CO , et c . ) wit h goo d a n t h e r sa n d h e a l t h y m e d i a i n f a c t a r e t h e s a m e a s u s e d i n p l a n t ce l l a n d
m ic r os por es ar e m os t s uit a b l e a s d o n o r p l a n t s . tissue cultures. ln recent years, some workers have
Flowers obtained from young plants, at the d e v e l o p e d s p e c i a l l y d e s i g n e d m e d i a fo r a n th e r
beginning of the flowering season are highly cultures of cereals.
responsive.The use of pesticidesshould be avoided S u c r o s e , n i t r a t e , a m m o n i u m s a l t s , a m i n o a ci d s
at leas t 3- 4 week s pr ec eedin g s a m p l i n g . a n d m i n e r a l s a r e e s s e n t i a l f o r a n d r o g e n e si s. In
some species, growth regulators- auxin and/or
Pretreatment of anthers cytokinin are required for optimal growth.
The bas ic pr inc iple of nat i v e a n d r o g e n c s i si s t o l n c e r t a i n p l a n t s p e c i e s ,a d d i t i o n o f g l u ta th i o n e
stop the conversion of pollen cell into a gamete, and ascorbic acid promotes androgenesis.When
and f or c e it s dev elopm ent in t o a p l a n t . T h i s i s r n t h e a n t h e r c u l t u r e m e d i u m i s s u p p l e m e n te d w i th
f ac t an abnor m al pat hway i n d u c e d t o a c h i e v e a c t i v a t e d c h a r c o a l , e n h a n c e d a n d r o g e n e si s i s
in vitro androgenesis. Appropriate treatment of observed. lt is believed that the activated charcoal
ant her s is r equir ed f or good s u c c e s s o f h a p l o i d removes the inhibitors from the medium and
production. Treatment methods are variable and f a c i l i t a t e sh a o l o i d f o r m a t i o n .
lar gely depend on t he donor p l a n t s p e c i e s .
.l
Chemical treatment : Certain chemicars
ar e k nown t o induc e par t hen o g e n e s ies . g . 2 - c h l o r o -
et hy lphos phonic ac id ( et hr e l ) . Wh e n p l a n t s a r e
Haploid plants can be developed from ovary or
t r eat ed wit h et hr eal, m ult in u c l e a t e d p o l l e n s a r e
ovule cultures. lt is possible to trigger female
pr oduc ed. Thes e pollens whe n c u l t u r e d m a y f o r m
gametophytes (megaspores) of angiosperms to
embryos.
develop into a sporophyte.The plants so produced
2. Tem per at ur e inf luenc e : I n g e n e r a l , w h e n are r€ferred ro as gynogenic haploids.
the buds are treated with cold temperatures(3-6"C) Gynogenic haploids were first developed by San
f or about 3 day s , induc t ion o c c u r s t o y i e l d p o l l e n Noem (1976) from the ovary cultures of Hordeum
embryos in some plants e g. Datura, Nicotiana. v u l g a r e .T h i s t e c h n i q u e w a s l a t e r a p p l i e d fo r r a i si n g
Further, induction of androgenesis is better if haploid plants of rice, wheat, maize, sunflower,
anthers are stored at low temperature, prior to sugar beet and tobacco.
c ult ur e e. g. m aiz e, r y e.
In vitro culture of unpollinatedovaries(or ovules)
There are also reports that pretreatment of i s u s u a l l y e m p l o y e d w h e n t h e a n t h e r cu l tu r e s g i ve
ant her s .of c er t ain plant s at h i g h e r t e m p e r a t u r e s unsatisfactoryresultsfor the production of haploid
( 35' C) s t im ulat esandr ogenesi se . g . s o m e s p e c i e so f plants. The procedure for gynogenic hdploid
Brassica and Capsicum. oroduction is brieflv described.
 O F H AP L O IDPLA N TS
Chaot er45 : P R OD U C T IO N 543

The flower buds are excised 24-48 hr prior to

anthesisfrom unpollinated ovaries.After removal of
calyx, corolla and stamens, the ovaries (see
Fig. a53) are subjectedto surfacesterilizatibn.The
ovary, with a cut end at the distal part of pedicel,
is inserte d in the s olid c ult ur e m edium . W hen e v e r Style
a liq uid med ium is us ed, t he ov ar ier ar e plac e d o n
Anther
a filter p ap er o r allowed t o f loat ov er t he m ed i u m
with pedicel inserted through filter paper. The
commo nly used m edia ar e M S, W hit e' s , N6 a n d
Nitsch, supplemented growth factors.
Production of gynogenic haploids is particularly Calyx
useful in plants with male sterile genotype. For
su ch pla nt sp ec ies , t his t ec hnique is s uper io r t o
an the r cu lture t ec hnioue.

Limitations of gynogenesis
In practice,productionof haploid plantsby ovary/
ovule cultures is not used as frequently as anther/ Fig. 45.3 : A diagrammatic representation of
po llen cultu res in c r op im pr ov em ent pr ogr am m e s . important pafts in a flower.
Th e majo r limit at ionsof gy nogenes isar e lis t ed .
1 . The diss ec t ion of unf er t iliz ed ov ar ies a n d
ovule s is ra the r dif f ic ult . The above markers have been used for the
2 . The p resenc eof only one ov ar y per f low e r i s development of haploids of maize.
another disadvantage.In contrast, there are a large It may be noted that for the detection of
n umb er o f mic r os por esin one ant her . androgenic haploids, the dominant gene marker
However, the future of gynogenesismay be more should be presentin the female plant.
p romising with im pr ov ed and r ef ined m et hods .

Two approaches based on morphology and
genetics are commonly used to detect or identify
ha plo ids.
MORPHOLOGICAL APPROACH
The vegetativeand floral parts,and the cell sizes
o f h ap loid p lant s ar e r elat iv ely r educ ed w h e n
comp are d to diploid plant s . By t his way hap l o i d s
can be de tec t ed in a populat ion of diplo i d s .
Morphological approach, however, is not as
effective as genetic approach.
GENETIC APPROACH
Cenetic markersare widely used for the specific
identificationof haoloids.Severalmarkersare in use'
. 'a.,' ma rke r f or br own c olour ed aleur one.
. 'A' marker for purple colour.
. 'Lg' marker for ligulelesscharacter.
As described in the preceeding pages, haploid
plants are obtained either by androgenesis or
gynogenesis.These plants may grow up to a flowering
stage, but viable gametes cannot be formed due to
lack of one set of homologous chromosomes.
Consequently,there is no seed formation.
Haploids can be diploidized (by duplication of
chromosomes)to produce homozygousplants.There
are mainly two approaches for diploidization-
c o l c h i c i n e t r e a t m e n ta n d e n d o m i t o s i s .
COLGHICINE TREATMENT
Colchicine is very widely usedfor diploidization
of homologous chromosomes.lt acts as an inhibitor
of spindle formation during mitosis and induces
chromosome duplication. There are many ways of
colchicine treatment to achieve diploidization for
production of homozygous plants.
544 BIOTECHNOLOCY

1. Whenthe plantsare mature,colchicinein the l n d u c t i o n o f m u t a t i o n s

formof a pasteis appliedto the axilsof leaves.NoW
In general, majority of induced mutations are
the main axis is decapitated. This stimulatesthe
recessiveand thereforeare not expressedin diploid
axillarybudsto growintodiploidandfertilebranches.
c e l l s ( d u e t o t h e p r e s e n c e o f d o m i na n t a l l e l e ) .
2. The young plantletsare directlytreatedwithHaploids provide a convenient system for the
c o l c h i c i n e s o l u ti o n , w a s h ed thoroughl y and
induction of mutationsand selectionof mutantswith
replanted.This resultsin homozygousplants. desiredtraits.In fact, the haploid cells can be cultured
3. The axillarybuds can be repeatedlytreated a n d h a n d l e d i n a f a s h i o n s i m i l a r t o m i c ro o r g a n i sm s.
with colchicinecotton wool for about 2-3 weeks. Mutants from several plant species that are
resistant to antibiotics, toxins, herbicrdes etc. have
EN D OMIT OS IS been developed.When the haploid cells of tobacco
Endomitosis is the phenomenonof doublingthe plant (Nicotiana tabacum) were exposed to
number of chromosomes without division of tne m e t h i o n i n e s u l f o x i m i n e( a m u t a g e n ) ,m uta n tsw h i ch
, unstabl e showed lower level of infection to Pseudomonas
n u c l e u sT. h e h a p l o i dc e l l s ,i n generalare
in c u l tu rew i th a te n d e n c y
to undergoendomi tosi s. tabaci were produced.
This property of haploid cells is exploited for
pl ants.
n p ro d u c eh o mozygous
d i p l o i d i z a ti o to Production of disease
resistance plants
T h ep ro c e d u re i n v o l v e sg r ow i nga smal lsegment
o f h a p l o i d p l a n t s te m i n a sui tabl e medi um Disease resistance genes can be introduced
supplementedwith growth regulators (auxin and w h i l e p r o d u c i n g h a p l o i d s . T h e s o d e ve l o p e d
c y to k i n i n ).T h i s i n d u c e sc a l l usformati onfol l ow ed haploids are screened for the desired resistance,
b y d i ffe re n ti a ti o nD. u ri n g t he grow th of cal l us, a n d t h e n d i p l o i d i z e d . S o m e e x a m p l e s o f d i se a se
c h ro m o s o madlo u b l i n go c c u rsby endomi tosi Thi s. s resistanceplants are listed.
re s u l tsi n th e p ro d u c ti o no f di pl oi d homozygous .l
. Hwansambye, a rice variety resistantto leaf
c e l l sa n d u l ti m a te l yp l a n ts .
b l a s t ,b a c t e r i a ll e a f b l i g h t a n d r i c e s t r i p ete n u i vi r u s.

2. Barley accession Q21681 resistant to stem

rust, leaf rust and powdery mildew.

More examples of disease resistancecrops are

!n vitro production of haploids is of great given in Table 45.1.
significancein plant breeding programmes. Some
of them are listed. Production of insect resistance plants

Some varieties of rice resistantto insects have

Development of homozygous lines
been developed e.g. Hwacheongbyeo resistantto
lt is now possibleto develophomozygouslines brown plant hopper. Other varietiesof rice that are
within a span of few months or a year by resistantto pests have also been produced.
e mp l o y i n ga n th e r/p o l l ecnu l t ure Thi s i s i n contrast
to the conventionalplant breedingprogrammethat Production of salt tolerance plants
might take severalyears (6-10 yrs). ln this way,
The plant specieswith salt tolerance are needed
productionof haploidsis highlyusefulfor research
f o r t h e i r c u l t i v a t i o n i n s o m e a r e a s .A n th e r cu l tu r e s
relatedto plant geneticsand breeding.
have resulted in some varieties of rice and wheat
with good salt tolerance e.g. wheat Hua Bain
Generation of exclusive male plants
124-4.
By the processof androgenesisto produce
haploids,followed by chromosomedoubling,it is Gytogenetic research
p o s s i b l eto d e v e l o p e x c l u si vemal e pl ants.The
Haoloids are usefuI in severaI areas of
m a l e p l a n ts a re p a rti c u l arl yuseful w hen thei r
cytogenetic research.These include
p ro d u c ti v i ty are much morethan
a n d a p p l i c a ti o ns
fe m a l ep l a n ts . . Productionof aneuploids
 O F H A PL OIDPLA N TS
Chaot er45 : P R OD U C T IO N 545

TABrr45.1 A selected llst of inproved varietles of crops developed by using anther culture

Crop Varieties Improvements made

Wheal(Triticum
aestivum) L u n g h u1a, Z i n gH ual ,Zi ngH ua2 Highyield,rustresistance,
cold
Huapei 1, Florin, Ambitus, Jingdan2288 resistance,
largespikes,moretlllers.
Rice(Oryzasativa\ Tangfong
1, XinXiu,ZhogHua8 Highyield,goodquality, resistance
disease
ZhongHua9, Huayu 1, HuaYu2,
HuapeiShanyou63,Zhekeng66,
TaBe 78,Nonhua 5, Hirohikari,
Hirohonami.
Tobacco(Nicotiana 2, Tanyu
tabacum) Tanyu1,Tanyu 3, F 211 Mildsmoking,
disease
resislance
HaiHua19,HaiHua30
Rraccieq nantrc Jaikisan Lowerucicacid

o Dete rmina tion of t he nat ur e of ploidy

o Determination of basic chromosome number
Evalu atio nof or igin of c hr om os om es .
' It has been possibleto raise severalhaploid
pl antsthroughanthercul tures.H ow ever,hapl oi d
In du ctio n o f genet ic v ar iabilit y
breeding programmes have not yielded the
Beside sth e d ev elopm ent of haploid m ut ant s, i t expected and desired results, despite heavy
is also p ossib le t o pr oduc e plant s wit h v ar io u s investments madeduringthe past3 decades.There
p loid y levels th r ough andr ogenes is . are many probl ems encounteredw i th hapl oi d
production.Someof them are listedbelow.
Doubled haploids in genome mapping
1. The frequency of haploid production is very
Genome mapping, a recent development in low, hence selectionis often difficult.
r role cu lar bio logy , c an be m or e c onv enien t l y 2. The operati onof ti ssuecul tureto devel op
a ch ieved by u si ng doubled haploid plant s pec ie s . haploids requires high level expertise and
manaS ement
Evolutionary studies
3. Besideshaploids,different ploidy levels are
A co mpa riso n of dihaploids ( doubled haploid s ) also produced.
,\ ith dip loid wild plant s pec ies will be us ef u l
4. Haploids with deleterioustraits frequently
io trace the evolutionary origin of various
developin cultures.
olants.The close evolutionarv relationshipbetween
:omato and potato has been evaluated by this 5. The cal l us deri vedfrom anther cul ture i s
eo Droa cn .
usual to hapl oi dproducti on.
l ydetri mental
6. l sol ati onof hapl oi dsfrom cul turesi s often
CROPS DEVELO PED THBO UG H difficult since polyploids outgrow haploids.
ANTHER CULT URES 7. fhe embryosderivedfrom haploidsoftenget
aborted.
An the r cultu re has m ade it pos s iblet o dev elo p
many new varietiesof plants, a selectedlist is given B . H apl oi dproducti oni s associ atedw i th hi ' h
in Table 45.1. i nci denceof al bi no (col ourl ess)
pl ants.

ln g en era l, the new c r ops ar e high- y ieldin g 9 The doubling of haploids (diploidization)
varietieswith diseaseresistance.Some of them are may not always lead to the formation of
resistance to cold, salt etc. Many plant breeders homozygousplant.
t he se d ays u se ant her c ult ur es in t heir r egul a r 10 ln vitro haploid production is not
bree din g p rog ram m es . economi cal lvi
y abl edue to very l ow success
rate.

Biotechnology [35]
-f- he genetic variations found in the in vitro
I cultured cells are collectivelyreferredto as
. he pl ants deri ved from
s o ma c l o n a vl a ri a ti o n sT
such cells are referredto somaclones.
Some authors use the Ierms calliclones and
protoclones to represent cultures obtained from
c allus and pr ot oplas t sr es p e c t i v e l y .
The growth of plant cells in vitro is an asexual
pr oc es s inv olv ing only m i t o t i c d i v i s i o n o f c e l l s .
Thus , c ult ur ing of c ells is t h e m e t h o d t o c l o n e a
particular genotype lt is therefore expected that
plant s ar is ing f r om a giv en t i s s u ec u l t u r e s h o u l d b e
t he ex ac t c opies of t he p a r e n t a l p l a n t . T h e
oc c ur r enc e of phenot y pi c v a r i a n t s a m o n g t h e
r egener at edplant s ( f r om t i s s u e c u l t u r e s ) h a s b e e n
known for several years. These variations were
ear lier dis m is s edas t is s ue c u l t u r e a r t e f a c t s .
The t er m s om ac lonalv a r i a t i o n sw a s f i r s t u s e d b y
Lar k in and Sc owc r af t ( 198 1 ) f o r v a r i a t i o n s a r i s i n g
due to culture of cells. i.e., variability generated by
a tissue culture. This term is now universallv
accepted.
As describedelsewhere(Chapter 42\, the explant
us ed in t is s ue c ult ur e m ay c o m e f r o m a n y p a r t o f
t he plant or gans or c ells T h e s e i n c l u d e l e a v e s ,
r oot s , pr ot oplas t s , m ic r o s p o r e s a n d e m b r y o s .
Som ac lonal v ar iat ions ar e r e p o r t e d i n a l l t y p e s o f
plant t is s ue c ult ur es .
ln recent years/ the term gametoclonal
variations is used for the variations observed in the
546
regeneratedplants from gametic cells (e.g., anther
c u i t u r e s ) . F o r t h e p l a n t s o b t a i n e d f r o m p r o to p l a st
cultures, protoclonal variations is used.
BASIS OF SOMACLONAL V A RIATION S
S o m a c l o n a l v a r i a t i o n s o c c u r a s a r e su l t o f
genetic heterogeneity (change in chromosome
n u m b e r a n d / o r s t r u c t u r e ) i n p l a n t t i ssu e cu l tu r e s.
This may be due to
. E x p r e s s i o no f c h r o m o s o m a l m o s a i c i smo r g e n e ti c
d isorders
. S p o n t a n e o u sm u t a t i o n sd u e t o c u l t u r e co n d i ti o n s.
T h e g e n e t i c c h a n g e sa s s o c i a t e dw i th so m a cl o n a l
variations include polyploidy, a n e u p l o i d y,
c h r o m o s o m a lb r e a k a g e ,d e l e t i o n ,t r a n sl o ca ti o n a , nd
g e n e a m p l i f i c a t i o n s , b e s i d e s s e v e r a l m u ta ti o n s In
fact, the oresence of several chromosomai
a b e r r a t i o n s - r e c i p r o c a l t r a n s l o c a t i o n , d e l e ti o n s,
i n v e r s i o n s , c h r o m o s o m a l b r e a k a g e, m u l ti ce n tr r c,
acentric fragments have been found among the
s o m a c l o n e so f b a r l e y , g a r l i c a n d o a t.
T h e o c c u r r e n c e o f m u t a t i o n s i n cu l tu r e s ts
r e l a t i v e l y l o w . M u t a t i o n s m a y b e d u e va r i e d
n u t r i e n t s ,c u l t u r e c o n d i t i o n s a n d m u ta g e n i c e ffe cts
o f m e t a b o l i c p r o d u c t s t h a t a c c u m u l a te i n th e
medium.
S o m a c l o n a l v a r i a t i o n s d u e t o tr a n sp o sa b l e
e l e m e n t s ,m i t o t i c c r o s s i n go v e r a n d ch a n g e s i n th e
cytoplasmic genome have also been reported.
Chapter46 : SOMACLONALVARIATIONS 547

Nome nclatu re of s om ac lones

The somaclonesthat are regeneratedfrom tissue

cultu res d irectly a r e r egar ded as Ro or R plant s .
Explant(e.9.,leaf)
The self-fertilized progeny of Ro plants represent
R t pla nts. R2, R3, R4 et c . plant s ar e t he
subsequent generations. Some workers use other
nomen cla ture- so m ac lones( SC, = Ro) , SC2, SC3,
SC, etc for subsequentgeneratrons.

Callus

There are two procedures commonly used for

ob ta inin g th e cr op plant s wit h s om ac lona l
varia tion s
.l
Without in vitro selection
Shoot regeneration
2. With in vitro selection.

+
I
WITHOUT IN V'TRC SELECTION

An explant (leaf, stem, root etc.) is cultured on

a su itab le me diu m , s upplem ent ed wit h gr owt h
regula tors.Th e un or ganiz edc allus and c ells do not
con tain a ny se lec t iv e agent ( t ox ic or inhibit or y
sub sta nce) The s e c ult ur es ar e nor m ally
sub cu lture d, a nd t r ans f er r ed t o s hoot induc t ion
medium for regenerationof plants.The so produced
plants are grown in pots, transferredto field, and
analyzed for somaclonal variants (Fig. a6J). Plant
Somaclonal Variantsof several crops have been +
I
successfu lly ob tai ned by t his appr oac h e. 9. , Transfer
to field
sugarcane,potato, tomato, cereals etc. A selected I
\P
list of disease resistant crop plants obtained from
Screenfor desiredtraits
somacfonal variations without in vitro selection
alo ng with th e pa t hogenic or ganis m s is giv en in +
I
Table 46.1. Selectionof
somaclonalvariants
Limitations of without in vitro +
I
selection ap pro ac h Agronomrc
trials
There is no directed and specific approach for Fig. 46.1 : A diagrammatic representation of
the isolation of somaclones witl.rout in vitro isolation of somaclones without in vilro selection.
. electio n. Con se quent ly , t he appear anc e of a
:jesire d tra it is pu r ely by c hanc e. Fur t her , t his
proced ure is tim e c ons um ing and r equir es c u l t u r e s( p r o t o p l a s t c, a l l u s ) l i k e m i c r o o r g a n i s m sa n d
scr e en ingof man y plant s . s e l e c t i o no f b i o c h e r n i c a lm u t a n t s .T h e c e l l l i n e s a r e
screened from plant cultures for their ability to
WITH VITBO SELECTION survive in the presence of a toxic/inhibitory
'TV
lsolation of somaciones with in yitro selection s u b s t a n c ei n t h e m e d i u m o r u n d e r c o n d i t i o n s o f
metho d ba sically in v olv es handling of plant c ells in environmentalstress.
548 B IO TECHNO LO CY

Environmental stress tolerance

Trru 46.1 A selected list of disease reslstant
crop plants obtained from somaclonal H i g h s a l t c o n c e n t r a t i o n i n t h e s o i l i s th e m a j o r
variations without ln vitro selectlon, c o n s t r a i n tl i m i t i n g t h e c r o p d e v e l o pm e n ta n d yi e l d .
along with the pathogenicorganisnrs Many plants with salt tolerance (salinity)have been
developed e.g. rice, tobacco.
Crop Pathogeni c o rgan i sm (s)
Several attempts are being made by plant
Rice Helninthosporiumoryzae biotechnologists to develop cold tolerant crops,
Maize Helninthosporiunnaydis although the successrate has been very limiteo.
Barley Rhynchosporiumsecalis
Sugarcane Puccinianelanocephala, Plant Pathogenicorganism
Sclerospora
saccharii II
Helminthosporium Fijivirus
saccharii, + +
Culture
Potato Streptomycesscabie,Alternaria Explant
solani,Phytophthorainfestans, II J
Purifyculturefiltrate
PotatovirusX andY +
Tomato Pseudomonas solanacearum, Callus J
Fusarium oxysporum I lsolationof toxin

Tobacco Phytophthoraparasitica
Proliferation
+
and
J
Toxicitydetermination
Apple Phytophthoracactorun maintenanceof callus for lethalconcentration
Banana Fusariunoxysporum
Lettuce Lettuce
mosaicvirus
Alfalfa Fusariumsolani
+I
A diagrammatic representation of in vitro f Selection
protocol for the isolation of disease resistance + cycles
plants with in vitro selection approach is given in
J
lsolationof tolerantcalli
Fig. 46.2.
J
Regeneration
The differentiated callus, obtained from an
ex plant is ex pos ed in t he m e d i u m t o i n h i b i t o r s l i k e
t ox ins , ant ibiot ic s , am ino a c i d a n a l o g s . S e l e c t i o n
J
Plantlet
cycles are carried out to isolate the tolerant callus
cultures and these calli are regeneratedinto plants.
J
vlvoscreeningagainst
/n
The plants so obtained are in vitro screenedagainst toxin/pathogen(R6 or SC1 plants)
t he t ox in ( or pat hogen or a n y o t h e r i n h i b i t o r ) . T h e I
plants resistantto the toxin are selectedand grown Self I Vegetative
pollinationI propagation
further by vegetativepropagationor self pollination. +
The subsequent generations are analysed for Progenyclonesfrom each plant
disease resistant plants against the specific +
pathogenic organism. Testfor diseaseresistance
A selected list of disease resistant crop plants +
I
obtained from somaclonal variations by in vitro Generationof disease
resistantplants(R1 or SC2 plants)
s elec t ion along wit h pa t h o g e n i c o r g a n i s m s a n d
selection agents is given in Table 46.2. +
I
Besides the disease resistant olants (described Agronomictrials
above), plants with herbicide resistance and
Fig. 46.2 : A diagrammatic representation of isolation
antibiotic resistancehave also been developed with
of disease resistant,plants with in vitro selection.
in vitro selection approach.
 Chapter46 : SOMACLONALVARIATIONS 549

o f r a c h i s a n d p e t i o l e a r e m u c h h i g h e r ( - 5 0 %)
Tewe 46.2 A selccted list of disease resistant compared to those regenerated from callus of
crop plants obtained from somaclonalvariations l e a v e s ( - 1 2 %) .
with in yifro selection along with pathogenic
organismsand selectionagents
Duration of eell culture
Crop Pathogenic Selection I n g e n e r a l ,f o r m a n y p l a n t c u l t u r e s ,s o m a c l o n a l
organism(s) agent variations are higher with increased duration of
Hice Xanthomonas oryzae Bacterial
cells cultures. For example, it was reported that
Wheat Helninthosporium genetic variability increased in tobacco protoplasts
sativum Crudetoxin
from 1.5 to 6"/o by doubling the duration of
Pseudononas syringae Syringomycin
cu ltures.
Barley Fusarium sp Fusaricacid
Heln intho
sporiun sativun Crudetoxin Growth hormone effects
Maize Helminthosporium naydis HmT toxin
T h e p l a n t g r o w t h r e g u l a t o r si n t h e m e d i u m w i l l
SugarcaneHelminthosporium sacchari Toxin influence the karyotypic alterations in qultured
Potato Fusariunoxyspotum Culturefiltrate cells, and therefore development of somaclones.
Ercoiniacarotovora Pathogen Crowth hormones such as 2, 4-dichlorophenoxy
Tomato Pseudononas solanacearumCrudefiltrate acetic acid (2, 4-D) and naphthalene acetic acid
(NAA) are frequently used to achieve chromosomal
Tobacco mosaic virus Virus
variability.
Alfalfa Foxysporum sp Crudeliltrate
Tobacco Tobacco mosaic virus Virus Besidesthe factors discussedabove, selection of
Pseudononas somaclones with in yifro selection are dependent
syringae Methionine
on the following parameters.
sulfoximine
a S e l e c t i o n p r o p a g u l e ( c e l l s , p r o t o p l a s t s ,c a l l i ) .
AdvantaEes of with in vitra selection a Selection agent (toxin, herbicide, amino acio
a p p r aoc h analogue).

The major advantageof with in vitro selection
methodis the specificselectionof the desiredtrait
ratherthan a generalvariationfound at the plant
l e v el.T hispr oc edu rei s l e s sti m e c o n s u mi n g
when
comparedto without in vitro selectionapproach.
a Technique used for selection.
a Stability of resistantsubstance.
o ln vivo testing procedure.
a Ability for regenerationof plants.

FA CT O RS A F F E C T IN G PR O D U C T IO N O F
SO M A CLO NA L V AR IAN T S
Some of the importantfactorsthat influence
Jevelopment of somaclonalvariantsby without ln
: itro selectionand with in vitroselectionare briefly
:escribed. Somaclonal variations (and also gametoclonal
variations, described later) are highly useful in
Ge n ot y pe and e x p l a n t s o u rc e plant breeding programmes.The genetic variations
with desirable (or improved characters),besidesthe
The natureof genotypeof the plants influences
existing favourable characters can be introduced
the frequency of regeneration and frequency of
into the plants In general, Ihe methodology
production of somaclones.
adopted for induction of somaclonal variations are
Erplantscan be takenfrom any part of plant- simpler and easier compared to recombinant DNA
..,es, roots,internodes,ovariesetc. The sourceof technology. Hence, they are preferred by some
n 'r ant is v er y c r it i c a lfo r s o ma c l o n avl a ri a ti o n s . workers. The important applications of somaclonal
:,- instance,potatoplantsregenerated from callus variations are briefly described.
550 B IO TECHNO LO CY

Resistance to diseases
Tlslr 46.3 A selected list of somaclonal variants
Somaclonal variations have largely contributed
obtained from different cop species with their
morphological characters towards the development of disease resistancein
many crops e.g. rice, wheat, maize, sugarcane/
Crop Character(s) tobacco, apple, tomato.

Bice period,
Flowering panicle
size;tillernumber; Selected crops somaclonal variants, with
plantheight; shapeandcolour; increaseddisease resistancedeveloped, without ln
leaflength,
fl:gy:l_.t
{ lg.tiLg
tgg9: TYl?lll
:lglil't)/ vitro selection and with in vitro selection are
respectivelygiven in Tables 46.1 and 46.2.
Wheat graincolour;
tillernumber;
Plantheight;
seedstorageprotein.
Resistance to abiotic s t r e sse s
Maize Reduced pollenfertility
andmalesterility;
I t h a s b e e n p o s s i b l e t o d e v el o p b i o ch e m i ca t
twinsfalksfroma singlenooe.
mutants with abiotic stressresistance.
Sugarcane Highsugaryield;increased
stalklength,
. F r e e z i n gt o l e r a n c e e . g . w h e a t .
diameter,
weightanddensity.
Barley Increased heading . S a l t t o l e r a n c e e . 9 . , r i c e , m a i z e , to b a cco .
grainyield;leafshape;
date;ashcontent. o A l u m i n i u m t o l e r a n c e e . 9 . , c a r r o t, so r g h u m ,
Oats Plantheight; headingdate;morphology
and tomato.
fertility.
Resistance to herbicides
Soybean Vaiiable
height; seedprotein
maturity; and
oilconteni. C e r t a i n s o m a c l o n a l v a r i a n t s w i th h e r b i ci o e
resistancehave been developed. Selectedexamples
Potato yield;growth
Higher habit,maturity
and
are Srven
m0rpn0r0gy.
. Tokraccoresistantto glyphosate,sulfonylureaand
Tomato Dwar{habit;earlyflowering;
orangefrLiil
c0t0ur. oicloram.

Carrot Higher
carotene
content. . Carrot resistantto glyphosate.

Pineapple Foliage widthandspine o Lotus resistant to 2, 4-dichlorophenoxy acetic

lealcolour,
density;
fcrmation. acid (2, 2-D).

Brassica Multiple
branching
stem;alteredleaf;slow quality
lmproved seed
growth;
failure
to floweror delayin
largepollengrains.
flowering; A new variety oI Lathyrussativa seeds (Lathyrus
Bio L 2.12) with a low content of neurotoxin has
Tobacco yield;plantheight;
Increased leafnumber,
been developed through somaclonal variations
widthandyield;typeof inflorescence
shape,
( N o t e : L a t h y r i s m i s a c r i p p l i n g d i se a seca u se d b y
andvield.
c o n s u m p t i o n o f L a t h y r u s s e e d s i .e . ke sa r i d a l , i n
many parts of central India).

Pr oduc t ion of agr on o m i c a l l y

LIMITATIONS O F S O M A C L ON AL
us ef ul plant s
VARIATIONS
As a r es ult of s om a c l o n a l v a r i a t i o n s , s e v e r a l
Despite several applications of so m a cl o n a l
novel variants of existing crops have been
variations, there are certain Ii m i ta ti o n s/
dev eloped. e. g. , pur e t h o r n l e s sb l a c k b e r r i e s .
d i s a d v a n t a g e sa l s o .
In Table 46.3, somaclonal variations in a
. Most of the somaclonal variations may not be
selected list of crops with trseful and improved
useful.
morphological characters are given. The crops
include rice, wheat, maize, sugarcane, potato, o T h e v a r i a t i o n s o c c u r i n a n u n p r e d i cta b l e a n d
carrot etc. uncontrolled manner.
ChAPtCr46 : SOMACLONAL VARIATIONS 551

. Many a times the genetic traits obtained by

somaclonal variations are not stahle and Ttst'. 46.4 A selected list of plants regencrated
heritable. from gametoclonal variations

. S o maclon al va riat ions ar e c ult iv ar - depenc ien t Crop (ha. racteristics

wh ich is freq uent ly a t im e c ons um ing
process Oryzasativa Plantheight;
tirneol flowering;
seed
sizeandproteincontent; levelof
. S o maclon es ca n be pr oduc ed in only tillering;
waxymutant; chloroplast
those species which regenerate to complete c0ntent.
p lan ts.
Nicotianatabacun Plantsize;leafshape;number of
. M a ny ce ll lin es ( c alli) m ay not ex hibit leaves; conlent;
alkaloid virus
reg en era tionp ote nt ial. resisrance:
timeof ilowerino
napus
Brassica Leafshapeandcolour;timeto
flower; glucinolate
typeof flower;
podsizeandshape.
conlent;
Hordeumvulgare Plantheight; grain
daysto maturity;
yield;fertility
fhe variations observed while culturing the
gametic cells are regarded as gametoclonal
varia tion s. Th is is in c ont r as t t o t he s onr ac lonar F r o d u *t i o n of garnetoclcnes
varia tion s d ete cte d in t he c ult ur es of s om at ic
t rssue s. Cametoclones can be developed by culturing
male or female gametic ceils. The cultures of
The term gametoclones(in place of somaclones) a n t h e r s o r i s o l a t e d m i c r o s p o r e s
are widely used.
is used for the products of gametoclonal (For details, Refer
Chapter 43).
varia tion s.
A selected list of plants regenerated from
As th e so matic c ells div ide by m it os is , t he gametoclonal variations is given in Tahle 45.4.
gene tic ma teria l i s equally dis t r ibut ed io t he lmprovements have been made in several plant
daughter cells In contrast, the gametes, being the speciesthrough development of gametoclonese.g
,
products of meiosis, possessonly half of the parent rice, v;heat, tobacco.
cell g en etic mate rial.
Ssurce of gametoclonal variations
The gametoclonal variations d iffer from
somaclonal variations by three distinct features. There are three sources of eenetic variations in
the gametoclones.
1. Mu tan ts o bt ained f r om gam et oc lonal
varia tion sgive rise t o haploid plant s s inc e a s ingle 1. Cell culture technique may induce genetic
set of chromosomes are oresent. variations.

2 Meiotic crossing over is the recombination 2. Variations may be induced while doubling
process observed in gametoclonal variations. the haploid chromosomes.

3. The ga meto c lones c an be s t abiliz ed by 3. Cenetic variations may ocCur due to

doublin g the chro m os om e num ber . h e t e r o z y g o s i t yo f t h e d i p l o i d s .
 - ?lant s c an be pr opa g a t e d b y s e x u a l ( t h r o u g h . C e n e b a n k s c a n b e m o r e e a s il y e sta b l i sh e db r
i: generation of seeds) or asexual (through c l o n a l l y p r o p a g a t e dp l a n t s .
m ult iplic at ion of v eget a t i v e p a r t s ) m e a n s . C l o n a l
propagation refers to the process of asexual ite v-j:fc: *!G:':.:ri SirtpaE,*tiGi"r
reproduction by multiplication of genetically The in vivo clonal propagation of plants is
identical copies of indlvidual plants. The term tedious, expensive and frequently unsuccessful ln
clone is used to represent a plant population vitro clonal propagation through tissue culture is
der iv ed f r om a s ing l e i n d i v i d u a l b y a s e x u a l referred to as micropropagation. Use of tissue
r eor oduc t ion c u l t u r e t e c h n i q u e f o r m i c r o p r o pa g a ti o n w a s fi r si
Asexual reproduction through multiplication of started by Morel (1960) for propagation of orchids
vegetative parts'is the only method for the in vivo and is now applied to se ve r a l p l a n ts
propagation of certain plants, as they do M i c r o p r o p a g a t i o n i s a h a n d y t e bh n i q u e fo r r a p i c
not pr oduc e v iable s e e d s e g . b a n a n a , g r a p e , multiplication of plants
f ig, c hr y s ant hem um . C l o n a l p r o p a g a t i o n h a s
been successfully applied for the propagation of 9 Aq;[r EVf *ft{J'H riF Ei gSfr *$lFt$F -tr,,,sjd'j {.'e
apple, potato, tuberous and several ornamental Micropropagation is a complicated process anc
Dlant s . m a i n l y i n v o l v e s 3 s t a g e s ( 1 , l l a n d l l l ) . So m e
authors add two more stages (stage 0 and lV) tc'
i !.L1'; . ! :,c r=! r.? i'*;geiat|lne pr$fiF g-!titli j
-:i more comprehensive representation of micro-
propagation. All these stages are represented r:-
Asexual (vegetative)propagation of plants has
Fig. 47.1, and briefly described hereunder.
certain advantagesover sexual propagation.
o Fas t er m ult iplic at ion- l a r g e n u m b e r o f p l a n t s S t a g e 0 : T h i s i s t h e i n i t i a l ste p i n m i cr c-
propagation,and involves the selection and grorri-
c an be pr oduc ed f r o m a s i n g l e i n d i v i d u a l i n a
short period o f s t o c k o l a n t s f o r a b o u t 3 m o n t h s u n d e r co n tr o l l e :
c o n di t i o n s .
. Pos s iblet o pr oduc e g e n e t i c a l l y i d e n t i c a l p l a n t s .
S t a g e I : l n t h i s s t a g e , t h e i n i ti a ti o n a - .
. Sex ually- der iv ed sterile hybrids can be
e s t a b l i s h m e n to f c u l t u r e i n a s u i ta b l e m e d i u m
propagated.
achieved. Selection of appropriate explants :
. Seed- r ais edplant s p a s st h r o u g h a n u n d e s i r a b l e i m p o r t a n t . T h e m o s t c o m m o n l y use d e xp l a n ts a r -
juv enile phas e whic h i s a v o i d e d i n a s e x u a l o r g a n s , s h o o t t i p s a n d a x i l l a r y b ud s. Th e ch o se -
propagailon. explant is surfacesterilizedand washed before us-
 (MICROPROPAGATION)
Chapter47 : CLONALPROPACATION 553

Stage Methodology involved 2. Mul ti pl i cati on

by adventi ti ous
shoots.

Stage0 Selectionof motherplant Besidesthe above two approaches,the planf

and its maintenance regeneration processesnamely organogenesisand
I
.k
somatic embryogenesismay also be treated as
Q t eno I lnitiationand establishment micropropagation,hencethey are alsodescribedin
of culture this chapter.
+
I 3. Organogenesi: sTheformati onof i ndi vi dual
Stage ll Multiplication
of shootsor organs such as shoots, roots, directly from an
rapidsomaticembryo
formation explant(lackingpreformedmeristem)or from the
I cal l usand cel l cul turei nducedfrom the expl ant.
+
S t a g e l ll /n vlfro germinationof 4. Somaticembryogenesis : The regeneration
of
somaticembryosand/or embryosfrom somaticcells,tissuesor organs.
rootingof shoots

+
I MU LTIP LIC A TION B Y A X ILLA R Y B U D S
StagelV Transferof plantletsto A N D A P IC A L S H OOTS
sterilizedsoil for hardening
undergreenhouse
environmenl Quiescent or actively dividing meristems are
presentat the axillaryand apicalshoots(shoottips).
The axillarybuds locatedin the axilsof leavesare
Fig. 47.1 : Major stages involved in micropropagation.
capable of developing into shoots. ln the in vivo
state,how everonl y a l i mi ted numberof axi l l ary
meristems can form shoots. By meansof induced
Stage ll :lt is in this stage,the major activity of
in vitro multiplicationin micropropagation, it is
micropropagation occurs in a defined culture
possibleto developplantsfrom meristemand shoot
me diu m. Sta gell m ainly inv olv es m ult iplic ati o n o f
tip culturesand from bud culture!s.
shoots or rapid embryo formation from the explant.

Stage lll : This stage involves the transfer of Meri stem and shoot ti p cul tures
shoots to a medium for rapid development into
Apical meristemis a dome of tissuelocatedat
shoots. Sometimes,the shoots are directly planted
the extremetip of a shoot. The apical meristem
in soil to develop roots. ln vitro rooting of shoots
alongwith the young leaf primordiaconstitutes
the
is p refe rredw hile s im ult aneous lyhandling a l a r g e
shoot apex. For the developmentof disease-free
n umb er of spec ies .
plants,meristemtips shouldbe cultured.
Stage lV : This stage involves the establishment
Meristemor shoottip is isolatedfrom a stemby
o f p lan tletsin s oil. This is done by t r ans f er r i n gt h e
a V-shapedcut. The size (frequently0 2 to 0.5
plantlets of stage lll from the laboratory to the
mm) of the ti p i s cri ti calfor cul ture.In generalthe
,
environment of greenhouse. For some plant
larger the explant (shoottip), the better are the
species, stage lll is skipped, and unrooted stage ll
chancesfor culture survival. For good resultsof
shoots are planted in pots or in suitable compost
micropropagation, explantsshould be taken from
mixture .
the acti vel ygrow i ngshootti ps,andthe i dealti mi ng
The different stages described above for is at the end of the plantsdormancyperiod.
, micropropagation are particularly useful for
The mosi widely used media for meristem
comparison between two or more plant systems,
cul tureare MS medi umand W hi te' smedi um. A
besides better understanding. lt may however, be
diagrammatic representationof shoot tip (or
noted that not all plant species need to be
meri stem) i s gi ven i n
cul turei n mi cropropagati on
propagated in vitro through all the five stages
Fig 47.2, and brieflydescribedhereunder.
referred above.
In stagel, the cultureof meristemis established.
Micropropagation mostly involves in vitro clonal
Addition of growth regulatorsnamely cytokinins
propagation by two approaches.
(ki neti n,B A )and auxi ns(N A A or IB A )w i l l support
1 . Multiplication by axillary buds/apicalshoots. the growth and development.
 554 B IOTECHNO LO G Y

Pottedplantlet Plant
/
I
/

Rootedplantlet Primaryculture

Rootingof Shootdevelopment
shoots (withaxillaryshoot
proliferation)

Fig. 47.2 : A diagrammatic representation of shoot tip (or meristem) culture in micropropagation
(Note : l, ll and lll represent stages in micropropagation)

In stage Il, shoot development along with Bud cultures

axillary shoot proliferation occurs. High levels of
cytokinins are required for this purpose.
Stage lll is associated with rooting of shoots and
further growth of plantlet. The root formation is
f ac ilit at ed by low c y t ok i n i n a n d h i g h a u x r n
concentration. This is opposite to shoot formation
I
s inc e high lev el of c y t ok ini n s i s r e q u i r e d ( i n s t a g e
ffi
riil
4i
ll). Consequently, stage II medium and stage lll
fl medium shauld be different in composition.
i The optimal temperature for culture is in the
range of 2O-28oC (for majority 24-26oC). Lower
light intensity is more appropriate tor good
m ic r opr opagat ion.
The plant buds possessquiescentor active
meristems dependingon the physiologicalstateof
the plant. Two types of bud culturesare used-
si ngl enode cul tureand axi l l arybud cult ur e.
Singlenode culture : This is a naturalmethod
propagationof plantsboth in in vivo
for vegetative
and in vitro conditions.The bud found in the axil
of leaf is comparableto the stemtip, for its ability
i n mi cropropagati on.A bud al ongw i t h a pieceof
stem is isolatedand cultured to develop into a
pl antl et. C l osed buds are used to r educe t he
chancesof infections.
 (MIC R OP R OP A C A TION )
C hapt er47 : CLO N ALPR O P AC AT ION 555

Fig. 47.3 : A diagrammatic representation of micropropagation by single node technique.

A dia gra mmat ic r epr es ent at ionof s ingle nod e a r e s u l to f t h i s , a p i c a l d o m i n a n c e s t o p s a n d a x i l l a r y

culture is d ep icted in Fig 47. 3. I n s ingie nod e
culture , no cvtokinin is added.
Axillary bud culture : In this method, a shoot tip
alo ng with a xilla r y bud is is olat ed. The c ult ur esar e
carried ou t with high c y t ok inin c onc ent r at ion. A s
buds develop. A schematic representation of
axillary bud culture for a rosette plant and an
elongate plant is given in Fig 47.4.
For a good axillary bud culture, the cytokinin/
auxin ratio is around 10 :1. fhis is however,

(A)

Meristem Shoot Axillary

(orshoottip)isolated development branching
Rosetteplant andcultured

(B)

Shoot Axillary Rootingof

development branching axillaryshoot
Elongateplanl

Node isolated
and cultured

Fig, 47.4 : A diagrammatic representation of micropropagation of plants by axillary bud (or shoot tip)
method (A) Rosette plant (B) Elongate plant.
 556 B IOTECHNO LO CY

v ar iable and depends on t h e n a t u r e o f t h e p l a n t 4. Culture environment

speciesand the developmental stage of the explant
us ed. I n gener al, juv enile e x p l a n t s r e q u i r e l e s s l-ight : Photosynthetic pigment in cultured
t i s s u e sd o a b s o r b l i g h t a n d t h u s i n f l u e n ce m i cr o -
cytokinin compared to adult explants. Sometimes,
propagation. The quality of light is also known to
the presenceof apical meristem may interferewith
influence in vitro growth of shoots. e.g blue light
ax illar y s hoot dev elopm ent I n s u c h a c a s e , i t h a s
induced bud formation in tobacco shoots
to be removed
Variationsin diurnal illuminationalso influence
M ULTI PLI CATI O N AY micropropagation. In general,an illumination of 16
ADVENTI TI O US SI { O O TS hours day and B hours night is satisfactoryfor shoot
proliferation.
The stem and leaf structuresthat are naturally
formed on plant tissues located in sites other than
Temperature:Majority o f t h e cu l tu r e fo r
t he nor m al leaf ax il r egi o n s a r e r e g a r d e d a s
micropropagation requires an optimal temperature
adventitious shoots. There are many adventitious
around 25oC. There are however, some exceptions
s hoot s whic h inc lude s t em s , b u l b s , t u b e r s a n d
e.g Begonia X Cheimantha hybrid tissue grow at a
rhizomes. The adventitiousshoots are useful for rn .l
low temperature (around BoC).
vivo and in vitro clonal propagation.
Composition of gas phase : The constitution
The meristematic regions of adventitious shoots
o f t h e g a s p h a s e i n t h e c u l t u r e v e sse l s a l so
c an be induc ed in a s uit able m e d i u m t o r e e e n e r a t e
i n f l u e n c e sm i c r o p r o p a g a t i o n . U n o r g a n i ze dg r o w th
to plants.
of cells is generally promoted by ethylene, O2, COz
ethanol and acetaldehyde.
FACTO RS AFFECTI NG
M I CRO PNO PAG ATI O N
Factors affecting in vitro rooting
For a successful in vitro clonal propagation
(micropropagation),optimization of several factors A general description of the factors affecting
m i c r o p r o p a g a t i o n ,p a r t i c u l a r l y i n r e l a t i o n to sh o o t
is needed. Some of these factors are briefly
multiplication is given above.
described.

1 . Genotype of the plant : Selectionof the right
genotype of the plant species (by screening) is
necessary for improved micropropagation In
general, plants with vigorous germination and
branching capacity are more suitahle for micro-
propagation.
2. Physiologicalstatus of the explants : Explants
(plant materials) from more recently produced parts
of plants are more effective than those from older
r egions . Cood k nowledge of d o n o r p l a n t s ' n a t u r a l
propagation process with special reference to
gr owt h s t age and s eas onalin f l u e n c e w i l l b e u s e f u l
in s elec t ingex plant s .
3. Cult ur e m edia : The s t a n d a r d o l a n t t i s s u e
c ult ur e m edia ar e s uit able f o r m i c r o p r o p a g a t i o n
during stage I and stage il. However, for stage lll,
c er t ain m odif ic at ions ar e r e q u i r e d . A d d i t i o n o f
growth regulators (auxins and cytokinins) and
alt er at ions in m iner al c om p o s i t i o n a r e r e q u i r e d .
This is lar gely dependent on t h e t y p e o f c u l t u r e
(meristem, bud etc).
For efficient in vitro rooting during micro-
propagation, low concentration of salfs (reduction
to half to one quarter from the original) is
advantageous lnduction of roots is also promoted
by the presence of suitable auxin (NAA or IBA).
ORGANOGENESIS
Organogenesisis the process of morphogenesis
involving the formation of plant organs i.e. shoots,
roots, flowers, buds from explant or cultured plant
tissues. lt is of two types- direct organogenesis
and indirect organogenesis.
Direct organogenesis
Tissues from leaves, stems, roots and
inflorescences can be directly cultured to produce
plant organs. In direct organogenesis,the tissue
undergoesmorphogenesiswithout going through a
c a l l u s o r s u s p e n s i o nc e l l c u l t u r e s t a g e. Th e te r m
direct adventitious organ formation is also used for
direct organogenesis.
 Chapter47 : CLONALPROPACATION
(MICROPROPACATTON) 557

Ind uction of adv ent it ious s hoot f or m a t i o n

directly on roots, leavesand various other organs of
intact plants is a widely used method for plant
propagation.This approach is particularly useful for
herbaceousspecies.

For appropriateorganogenesisin culture sysrem,

exogenous addition of growth regulators-auxin
an d cytokini n is r equir ed. The c onc ent r at iono f t h e
plant
growth promoting substance depends on the age
and nature of the explant, besides the growth
+
I
co nd ition s.
/'7>\
!n dire ct or ganogenes is
lsLeaf
When the organogenesisoccurs through callus II
or suspension cell culture formation, it is regarded +
as indirect organogenesis(Fig 7.5 B and Q. Callus
growth can be established from many explants
(leaves.roots, cotyledons, stems,flower petals etc.)
for subsequentorganogenesis

The explants for good organogenesisshould be

mitotically act iv e im m at ur e t is s ues . I n gener a l ,t h e
bigger the explant the better the chances for
o bta inin g via ble c allus / bells us pens ionc ult ur e s l t
is advantageousto select meristematictissues(shoot
tip, leaf, petiole)for efficient indirect organogenesis.
This is because their growth rate and survival rate
are much better.

For indirect organogenesis,the cultures may be

grown in liquid medium or solid medium. Many
culture media (MS, 85 White's etc.) can be used in
'of
organogenesis. The ' concentration growth
regulators in the medium is critical for
organogenesis. By varying the concentrations of
auxins and cytokinins, in vitro organogenesiscan be
ma nio ula ted .

. Low auxin and low cytokinin concentration will

in du ce callu s f or m at ion.

. L ow au xin and high c y t ok inin c onc ent r at ionw i l l

promote shoot organogenesisfrom callus

. Hi8h a uxin a nd low c y t ok inin c onc ent r at ionw i l l

ind uce roo t f or m at ion.

SOMATIG E M BRYO G ENESI S Plant

The process oI regeneration of embryos from
somatic cells, tissues or organs is regarded as Fig. 47.5 : Micropropagation of plants by
organogenesis (A) Direct organogenesis (B) lndirect
somatic (or asexual) embryogenesis. Somatic
organogenesis through callus (C) lndirect
embryogenesismay result in non-zygotic embryos
organogenesis through suspension cell culture.
or somatic embryos (directly formed from somatic
558 B IOTE CHNO LO CY

organs), parthogenetic embryos (formed from

r-rnfertilizedegg) and androgenic embryos (formed
from male gametophyte) In a general usage,when
t he t er m s om at ic em br y o is us e d i t i m p l i e s t h a t i t i s
formed from somatic tissues u nder in vitro
conditions. Somatic embryos are structurally
similar to z.ygotie (sexually formed) embryos, and
t hey c an be ex c is ed f r om t h e p a r e n t t i s s u e s a n d
induc ed t o ger m inat e in t is s u e c u l t u r e m e d i a
Development of somatic embryos can be done
in plant cultures using somatic cells, particularly
epider m is , par enc hy m at ous c e l l s o f p e t i o l e s o r
s ec ondar y r oot phloem . So m a t i c e m b r y o s a r i s e
f r onr s ingle c ells loc at ed wi t h i n t h e c l u s t e r s o f
m er is t em at icc ells in t he c allu s o r c e l / s u s p e r r s i o n .
Fir s t a pr oem br y o is f or m ed w h i c h t h e n d e v e l o p s
int o an em br y o, and f inally a p l a n t .

Two routes of somatic embryogenesis are

known - direct and indirect (Fig a7.6).

Direct somatic ennbryogenesis

When the somatic embryos develop directly on
the excised plant (explant) without undergoing
callus formation, it is referred to as direct somatic
embryoglenesis(Fig 47.64). This is possible due to
the presence of pre-embryonic determined cells
(PEDQ found i,n certain tissues of plants. The
characteristic features of direct somatic
em br y ogenes is is av oiding t h e p o s s i b i l i t y o f
int r oduc ing s om ac lonal v a r i a t i o n s i n the
propagated plants.

Indirect somatic embryogenesis

I n indir ec t em br y ogenes is,th e c e l l s f r o m e x p l a n t
(excised plant tissues)are made to proliferate and
form callus, from which cell suspension cultures
Plantletwith Plantletwith
can be raised. Certain cells referredto as induced shootsand roots shootsand roots
embryogenic determined cells (IEDO from the
\,,-
c ell s us pens ion c an f or m s o m a t i c e m b r y o s . \//
\
Em br y ogenes isis m ade pos s ib l eb y t h e p r e s e n c eo f
growth regulators (in appropriate concentration)
and under s uit able env ir onm e n t a l c o n d i t i o n s .

Som at ic em br y ogenes is( di r e c t o r i n d i r e c t ) c a n

be c ar r ied on a wide r ange o f m e d i a ( e . g . M S ,
W hit e' s ) . The addit ion o f t h e a m i n o a c i d
L-glutamine promotes embryogenesis The presence Plant
of aux in s uc h as 2, 4- dic hlor o - p h e n o x ya c e t i c a c i d
of plantsby
Fig. 47.6 : Micropropagation
is es s ent ialf or em br y o init iat io n .O n a l o w a u x i n o r
embryogenesis(A) Direct embryogenesis(B) lndirect
no aux in m edium , t he em br y o g e n i cc l u m p s d e v e l o p
emDryogenes6
into mature embryos.
 (MIC R OP R OP A C A TION )
47 : C L O N ALPR O P AC AT ION
ChA P I CT
Ind irect som at ic em br y ogenes isis c om m er c i a l l y Production
rery attractive since a large number of embryos
ca n b e ge ne r at ed in a s m all v olum e of c u l t u r e
nredium. The somatic embryos so formed
are synchronous and with good regeneratron
cao ab ilitv.
Artificial seeds from somatic embryos
Artificial seeds can be made by encapsulation of
somatic embryos. The embryos, coated with
sod ium a lgin at e and nut r ient s olut ion, ar e dip p e d
in calcium chior ide s olut ion. The c alc ium i o n s
ind uce ra pid c r os s - link ing of s odium alginat e t o
p rod uce sma ll gel beads , eac h c ont aining a n
e ncap su late d em br y o. Thes e anif ic ial se e d s
(e ncap su late dem br y os ) c an be m aint ained i n a
viab le sta te till t hey ar e plant ed.
APPL ICATION S OF
MICROPROPA G ATI O N
Micro pro pa gat ion has bec om e a s uita b l e
alternative to conventional methods of vegetative
propagation of plants. There are several advantages
of micropropagation
High rate of plant propagation
Through micropropagation, a large number of
plants can be grown from a piece of plant tissue
within a short period. Another advantage is that
micropropagation can be carried out throughout
the year, irrespective of the seasonal variations.
Further,for many plants that are highly resistantto
conventional propagation, micropropagation is the
su itab le a ltern at iv e.
Th e small s iz ed pr opagules obt ained i n
micropropagation can be easily stored for many
years (germplasm storage),and transported across
international boundaries.
Production of disease-free plants
It is possible to produce disease-free plants
through micropropagation. Meristem tip cultures
are generally employed to develop pathogen-free
plants (details given later in this chapter). ln fact,
micropropagation is successfully used for the
production of virus-free plants of sweet potato
(lpomea batatus), cassava \Manihot esculenta) and
vam (Discorea rotundata).
s59
of seeds in some crops
Micropropagation, through axillary bud
proliferationmethod, is suitable for seed production
in some plants This is required in certain plants
where the limitation for seed production is high
degree of genetic conservation e.g. cauliflower,
on ton.
Gost-effective process
Micropropagation requires minimum growing
space. Thus, millions of plant species can be
maintained inside culture vials in a small room In
a nursery.
T h e p r o d u c t i o n c o s t i s r e l a t i v e l yl o w p a r t i c u l a r i y
in developing countries (like India) where the
manpower and labour charges are low
Automated micropropagation
It has now become oossible to automate
microprcpagation at various stages. In fact, bio-
reactors have been set up for large scale multi-
plication of shoots and bulbs. Some workers
e m p l o y r o b o t s ( i n p l a c e o f l a b o u r e r s )f o r m i c r o -
propagation, and this further reduces production
cost of plants.
DISADVANTAGES OF
MICROPROPAGATION
Gontamination of cultures
During the course of micropropagation,several
slow-growing microorganisms (e.9. Eswinia sp,
Bacillus sp) contaminate and grow in cultures. The
microbial infection, can be controlled by addition
of antibiotics or fungicides. However, this will
adversely influence propagation of plants.
Brewing of medium
Micropropagation of certain plants (e.g. woody
oerennials) is often associatedwith accumulation
o f g r o w t h i n h i b i t o r y s u b s t a n c e si n t h e m e d i u m .
Chemically, these substances are phenolic
c o m p o u n d s ,w h i c h c a n t u r n t h e m e d i u m i n t o d a r k
colour. Phenolic compounds are toxic and can
inhibit the growth of fissues.
Brewing of the medium can be preventedby the
addition of ascorbicacid or citric acid or polyvinyl
pyrrolidone to the medrum.
 560
Genetic variability
When micropropagation is carried out through
s hoot t ip c ult ur es , genet ic v a r i a b i l i t y i s v e r y l o w .
However, use of adventitious shoots is often
associatedwith pronounced genetic variability.
Vit r if ic at ion
During the course of repeated in vitro shooL
m ult iplic at ion, t he c ult ur esexh i b i t w a t e r s o a k e d o r
almost translucentleaves.Such shoofs cannot grow
and even may die. This phenomenon is referredto
as vitrification. Vitrification may be prevented
by increasing the agar concentration (from 0.6
t o1% ) in t he m edium . How e v e r , i n c r e a s e d a g a r
concentration reduces the erowth rate of tissues.
Gost factor
For s om e m ic r opr opa g a t i o n t e c h n i q u e s ,
ex pens iv e equipm ent , s ophis t i c a t e df a c i l i t i e s a n d
t r ained m anoower ar e needed . T h i s l i m i t s i t s u s e .
PRO DUCTI O N O F
DI SEASE. FREE PLANTS
Many plant species are infected with
pat hogens - v ir us es , bac t er ia , f u n g i , m y c o p l a s m a
and nem at odes t hat c aus e s y s t e m i c d i s e a s e s .
Although these diseasesdo not always result in the
death of plants,they reduce the quality and yield of
plants. The plants infected with bacteria and fungi
frequently respond to chemical treatment by
bactericides and fungicides. However, it is very
difficult to cure the virus-infected plants. Further,
v ir al dis eas e ar e eas ily t r a n s f e r r e d i n s e e d -
propagatedas well as vegetativelypropagatedplant
species. Plant breeders are always interested to
dev elop dis eas e- f r ee plant s , p a r t i c u l a r l y v i r a l
disease-freeplants. This have become a reality
through tissue cultures.
Apic al m er is t em s wit h l o w
c onc ent r at ion of v ir us es
In general,the apical meristemsof the pathogen
inf ec t ed and dis eas e har bou r i n g p l a n t s a r e e i t h e r
free or carry a low concentration of viruses,for the
f ollowing r eas ons .
. Abs enc e of v as c ular t is s u e i n t h e m e r i s t e m s
t hr ough whic h v ir us es r ead i l y m o v e i n t h e p l a n t Meristem-tip culture
body.
. Rapidly div iding m er is t em a t i c c e l l s w i t h h i g h adopted for meristem and shoot tip cultures has
metabolic activity do not allow virusesto multiply. been described (see Fig 47.2).
B IOTECHNO LO CY
. Virus replication is inhibited by a high
concentration of endogenous auxin in shoot
a p i c e s . T i s s u e c u l t u r e t e c h n i q u e s e m p l o yi n g
meristem-tips are successfully used for the
production of disease-free plants, caused by
several pathogens- viruses, bacteria, fungi,
mvcoptasmas.
METHODS TO ELIMINATE
VIRUSES IN PLANTS
In general,plants are infectedwith many viruses;
the nature of some of them may be unknown. The
u s a g ev i r u s - f r e ep l a n t i m p l i e s t h a t t h e g i ve n p l a n t i s
f r e e f r o m a l l t h e v i r u s e s ,a l t h o u g h t h i s m a y n o t b e
a l w a y s t r u e . T h e c o m m o n l y u s e d m e t h od sfo r vi r u s
elimination in plants are listed below, and briefly
described next.
r Heat treatment of plant
. Meristemtio culture
. C h e m i c a l t r e a t m e n to f m e d i a
o Other in vitro methoos
l.leat treatment (therrnotherapyf
of plants
In the early days, before the advent of meristem
cultures, in vivo eradication of viruses from plants
was achieved by heat treatment of whole plants.
T h e u n d e r l y i n g p r i n c i p l e i s t h a t m a n y vi r u se s tn
p l a n t t i s s u e s a r e e i t h e r p a r t i a l l y o r co m p l e te l y
i n a c t i v a t e d a t h i g h e r t e m p e r a t u r e sw i t h m i n i m a l
injury to the host plant. Thermotherapy (at
temperatures 35-40oC) was carried out by using
hot water or hot air for elimination viruses from
growing shoots and buds.
There are wvo limitations of viral elimination by
heat treatment
1 . M o s t o f t h e v i r u s e s a r e n o t s e n s iti veto h e a t
treatment.
2. Many plant species do not survive after
thermotherapy.
With the above disadvantages,heat treatment
h a s n o t b e c o m e p o p u l a r f o r v i r u s e l i m i n a ti o n .
A general description of the methodology
 (MICROPROPACATION)
Chapter47 : CLONALPROPACATTON 561

Terla 47.1 A selected list of the plants wlth vlrus elimitation by neristem cultures

Plant species Virus eliminated

Solanum tuberosum (potato) Leafroll,potatoviruses- A, X, Y, S
Nicotiana tabacum (tobacco) Tobacco mosaic virus
Saccharun (sugar
officinarum cane) - Mosaic virus
Allium sativum (garlic) Mosaic virus
Anenas sativus (pineapple) Mosaic virus
Brassica oleracea (caulillower) Cauliflower/mosaicvirusturnipmosaicvirus
lponoea batata (sweetpotato) Fealtherymottlevirus
Ribesgrassularia Veinbanding virus
Humulus lupulus Hoplatentvirus
Armoracia rusticena Turnipmosaic virus
Musa sp(Banana) Cucumber mosaic virus
Hycinthus sp Hycinthmosaic virus
Dahlia sp Dahliamosaic virus
Chrysanthenun sp VirusB
Petunia sp Tobacco mosaic virus
lrissp lrismosaicvirus
Cymbidium sp Cymbidium mosaicvirus
Fragaria sp virus,yellow
Pallidosis viruscomplex
Freesia sp Freesiamosaic virus

Fo r vira l e lim inat ion, t he s iz e of t he m e r i s t e m Ghemical treatment of media

u se d in cu ltur es is v er y c r it ic al. This is due to t h e
fact that most of the viruses exist by establishinga
gradient in plant tissues. In general, the
regeneration of virus-free plants through cultures is
inversely proportional to the size of the meristem
used . The m er is t em - t ip ex plant us ed f or v i r a l
e limina tion cult ur es ar e t oo s m all. A s t er eo s c o p i c
microsco pe i s us ually em ploy ed f or t his pur p o s e .
Me riste m - t ip c ult ur es ar e inf luenc ed b y t h e
following factors.
. Physicilog ic alc ondit ion of t he ex plant - ac t i v e l y
growing buds are more effective.
o Thermotherapy prior to meristem-tip culture-
fo r certa in plant s ( pos s es s ingv ir us es i n t h e
meristematicregions),heat treatment is first given
and then the meristem-tios are isolated and
cu ltured.
. Cu lture m edium - M S m edium wit h l o w
con ce ntra t ionsof aux ins and c y t ok inins is i d e a l .
A selected list of the plants from which viruses
h ave b ee n el im inat ed by m er is t emc ult ur es is g i v e n
in Table 47.1 .
Some workers have attempted to eradicate
virusesfrom infectedplantsby chemicaltreatment
of the ti ssuecul turemedi a. The commonl yused
(e.g.cytokinins)
chemicalsare growth substances
(e.gthi ouraci lacetyl
and anti metabol i tes , sal i cyl i c
acid). There are however,conflictingreportson
the el i mi nati onvi rusesby chemi caltreatmentof
the medi a. For i nstance,addi ti on of cytoki ni n
suppressed the mul ti pl i cati onof certai n vi ruses
w hi l e for someothervi ruses,i t actual l ysti mul ated.
Other in vitro methods
Besidesmeristem-tipculture, other in vitro
methodsare also usedfor raisingvirus-freeplants.
ln this regardcallus cultures have been successful
to someextent.The callusderivedfrom the infected
tissuedoesnot carrythe pathogens throughoutthe
cel l s. In fact, the unevendi stri buti onof tobacco
mosaicvirus in tobacco leaveswas exploitedto
developvirus-freeplantsof tobacco.
Somatic cell hybridization, gene transformation
and somaclonal variations also useful to raise
disease-free
olants.

Biotechnology [36]
562 B IOTECHNO LO CY

ELI M I NATI O N O F PATHOG E N S . Wh e n t h e e m b r y o s r e m a i n d o r m a n t fo r l o n g

O THER THAN VI RUSES r:eriods.
. Low survival of embryos in vivo.
Bes idest he elim inat ion of v i r u s e s , m e r i s t e m - t i p
. T o a v o i d i n h i b i t i o n i n t h e s e e d f o r g e r m i n a ti o n .
c ult ur es and c allus c ult ur es a r e a l s o u s e f u l f o r
. F o r c o n v e r t i n g s t e r i l e s e e d st o v i a b l e se e d l i n g s.
er adic at ion bac t er ia, f ungi a n d m y c o p l a s m a s .
Som e ex am ples ar e giv en S e e d d o r m a n c y i n p l a n t s p e c i e s i s a co m m o n
. The fungus Fusarium roseum has been o c c u r r e n c e .T h i s m a y t r e c j u et o c h e m i ca l i n h i b i to r s
s uc c es s f ullyelim inat edt hr o u g h m e r i s t e mc u l t u r e s or mechanical resistanceexerted by the structures
f r om c ar nat ion plant s . c o v e r i n g t h e e m b r y o S e e d d o r r n a ncy ca n b e
s u c c e s s f u l l yb y p a s s e db y c u l t u r i n g t h e e m b r yo s r n
o Certain bacteria (Pseudomonas carophylli,
vitrc
Pectobacterium parthenii) are eradicated from
c ar nat ion plant s by us ing m e r i s t e m c u l t u r e s . E m b r y o c u l t u r e i s r e l a t i v e l ye a s y a s th e y ca n b e
B r o w n o n a s i n r p l e i n o r g a n i c m e d i u m su p p l e -
M ERI TS AND DEM ERI TS O F m e n t e d w i t h e n e r g y s o u r c e ( u s u a l l y s ucr o se ) .Th i s
DI SEASE, FREE PLANT P R O D U C T I O N is possible since the mature embr'.vosexcised from
t h e d e v e l o p i n g s e e d s a r e a u t o t r o p h i c i n n a tu r e ,
Anrong the culture techrriques, meristem-tip
culture is the most reliable method for virus and
EMBRYO RESCUE
ot her pat hogenelim inat ion T h i s , h o w e v e r , r e q u i r e s
good knowledge of plant pathology and tissr,re Embryo rescue involves the crrlfure of immature
c u lt ur e. embryos to rescue them from unripe or hybrid
seeds which fail to germinafe. This approach rs
Vir us - f r ee plant s ex hibit i n c r e a s e d g r o w t h a n d very useful to avold embryo abortion arrd produce
v igour of plant s ,higher y ield ( e . 9 .p o t a t o ) ,i n c r e a s e d a v i a b l e p l a n t .
flower size (e.9. Chrysanthemum),improved rooting
of siem cuttings (e.g. Pelarg,onium) Wi l d h y b r i d i z a t i o n i n v o l v i n g c r o s si n g o f tw o
different species of plants from the same genus or
Virus-free plants are more susceptible to the d i f f e r e n t g e n e r a o f t e n r e s u l t s i n f a i l u r e . Th i s i s
same virus when exposed again. This is the major mainly becausethe normal development of zygote
limitation. Re,infectionof disease-freeplants can a n d s e e d i s h i n d e r e d d u e t o g e n e ti c b a r r i e r s
be m inim iz ed wit h good k no w l e d g e o f g r e e n h o u s e C o n s e q u e n t l y ,h y b r i d e n d o s p e r m f a i l s to d e ve l o p
m aint enanc e. l e a d i n g t h e a b o r t i o n o f h y b r i d e m b r yo . Th e
e n d o s p e i m m a y a l s o p r o d u c e t o x i n s t h at u l ti m a te r y
kill the embryo.
I n t h e n o r m a l c i r c u m s t a n c e s ,e n d osp e r m ftr st
develops and supports embryo development
Embryo culture deals with the sterile isolation nutritionally. Thus, majority of embryo abortions
and in vitro growth of a mature or an immature a r e d u e t o f a i l u r e i n e n d o s p e r m d eve l o p m e n t.
embryo with an ultimate objective of obtaining a Embryo abortion can be avoided by isolating and
v iable plant . Conv ent ional l y , t h e t e r m e m b r y o c u l t u r i n g t h e h y b r i d e m b r y o s p r i o r t o a b o r ti o r r .
culture refers to the sexually produced zygolic
embryo culture. This is different from the somatrc The most important application of embryo
em br y ogenes is alr
, eady des c r i b e d i n t h i s c h a p t e r rescue is the production of interspecific and
intergeneric hybrids from wild plant species
There are two types of embryo culture - mature
em br y o c ult ur e and im m a t u r e e m b r y o c u l t u i " e Gulture technique for embryo rescue
(embryo rescue).
The isolation of immature embryos often poses
some difficulty. The aseptically isolated embryos
M ATURE EM BRYO CULT U R E
c a n b e g r o w n i n a s u i t a b l e m e d i u m u n d e r o p ti m a l
Mature embryos are isolatedfrom ripe seedsand c o n d i t i o n s . I n g e n e r a l , a c o m p l e x n u t r i en t m e d i u m
cultured in vitro. Mature embryo cultures are i s r e q u i r e d f o r c u l t u r e m e i h o d s i n v o l vi n g e m b r yo
c ar r ied out in t he f ollowine c o n d i t i o n s . rescue.
 (MICROPROPAGATION)
Chapter47 : CLONALPROPACATION 563

Endosperm
with failed Normal
development enoosperm
'Hybrid Normal
embryo emoryo
Ovulewithhybridembryo Ovule with normalembrvo
j

I
J
t
Hybridembryo

Normalendosoerm

Fig. 47.7 : Embryo-endosperm transplant technique used in embryo rescue (or immature embryo culture).

F or ade q u a ten u tri ti o n a sl u p p o rto f i m mature 1 . Heterotrophic phase : This is an early phase
em br y osemb
, ry o -e n d o s p e trarm n s p l a nits used. and the embryo is mostly dependent on the
endosperm and maternal tissuesfor nutrient supply.
Embryo-endosperm transplant: The endosperm
t r ans plantt e c h n i q u eu s e d fo r c u l tu ri n gi mmature 2. Autotrophic phase : This phase is
embryosis givenin Fig.47.7,and brieflydescribed characterized by the metabolic capability of the
below. embryo to synthesize substances required for its
growth which slowly makes it independent.
T he hy b ri d e mb r:y ofro m th e o v u l e i n w hi ch
endospermdevelopmenthasfailed is takenout by The critical stage is the intervening phase
excision.Another normallvdevelopedovule with between the heterotrophic and autotrophic phases.
endos per me n c l o s i n ga n e m b ry o i s th o s e n. Thi s The nutrient supply is highly variable at this phase
ovule is dissected and the normalembryois pressed which mostly depends on the plant species.
w i th a n exi t
out . T his le a v e sa n o rm a le n d o s p e rm
hole. Now, the hybridembryocan be insertedinto In general,the composition of the medium for
t he nor m a l e n d o s p e rmth ro u g h e x i t h o l e . Thi s culturing immature embryos is more complex than
rm n s p l a nwt h ich can
r es ult sin em b ry o -e n d o s p e tra that required by mature embryos which can grow
be c ult ur edi n a s u i ta b l em e d i u m. o n a s i m p l e i n o r g a n i c m e d i u m F u r t h e r ,t h e t r a n s f e r
of embryos from one medium to another is
By using embryo-endosperm transplant,many frequently needed in order to achieve full
interspecificand intergeneric plants have been develooment of embrvos.
r ais ede. g. ,h y b ri dp l a n tso f l e g u m e s .
Composition of the medium : Some salient
NUT RI T I O N A L R EOU IR E M EN T S OF features of medium and culture conditions are
E M B RY O C U L T U R E S listed below

Therearetwo phasesin the embryodevelopment . Inorganic constituents of MS, B5 or White's

l q u i re me nvta ri e sa c c ordi ngl y
and t he nut ri ti o n are media are adequate.
56,4 BIOTECHNOLOCY

Tlow 4:7.2A selectedlist of dlstant plant speciessossed and the reslstancctralts developed
throughembryorescuetechnique
Distant plant species crossed Resistancetrait(s)
Oryza sativax O.minuta Bacterial andblast
blight
Solanum tuberosum x S. etuberosum leafrollvirus
Potato
Solanun melanogena x S. khasianum Brinjal
shoot andlruitborer
Brassicanapus x B. oleracea vduudvs
^^hL^^^ ^^hiillu
oPl

Lycopersiconesculentumx L. peruvianum fungiandnematodes

Virus,
Hordeun sativum x H. vulgare Powderymildewandspot blotch
Triticum
aestivum x Thinopyrunscipeum Salttolerance

. Sucrose is most commohly used energy source 2. Overcomingseeddormancy: Seeddormancy

. Ammonium nitrate is the preferred source of
n itrogen.
. Cas ein hy dr oly s at e,r ic h in v a r i o u s a m i n o a c i d s i s
frequerrtlyused.
o Certain natural plant extracts with embryo factor
pr onlot e em br y o c ult ur es e . g I i q u i d e n d o s p e r m
of coconut milk. The embryo factor is believed to
s upply c er t ain am ino ac i d s , s u g a r s , g r o w t h
regulatorsetc.
. ln general, growth regulatorsare not required, as
t hey induc e c allus f or m at io n .
. Embryos grow
well in the pH range of
5- 7 . 5.
o An incubation temoerature of 24-26"C is idear.
. Bettergrowth of embryos is observed in darkness
which are then transferred to lieht for
ger m inat ion.
During the culture conditions, the embryos are
grown into plantlets,and then transferredto sterile
soil for full-pledged growth to maturity.
1 Seedsterilityis mostlyassociated
APPLI CATI O NS O F EM BR Y O C U L T U R E
.l
. Prevention of embryo abortion : Incompati-
bilit y bar r ier s in int er s pec i f i c a n d i n t e r g e n e r i c
hy br idiz at ion pr ogr am m es l e a d i n g t o e m b r y o
abortion can be successfullyovercome by embryo
rescue. In fact, many distant hybrids have been
obtained through embryo rescue techniques. A
selected list of distant plant speciescrossedand the
resistancetraits developed by employing embryo
rescue technique is given in Table 47.2.
is causedby severalfactors-endogenous inhibitors,
embryo immaturity,specificlight and temperature
etc. Further,
requirements, dry storagerequirements
in someplantsthe naturalperiodof seeddormancy
itself is too long. Embryoculture is successfully
appliedto overcomeseeddormancy, andto produce
i n thesepl antspeci es.
vi abl eseedl i ngs
3. Shorteningof breedingcycle : Sonreof the
pl ants i n thei r naturalstate have l on g br eeding
cycl es. Thi s i s mostl y due to seed dor m ancy
attributedto seed coat and/or endosperm.The
enrbryoscan be excisedand cultured in vitro ro
devel op i nto pl ants w i thi n a short p er iod. For
i nstance, H ol l i es,a C hri stmas plantcan
decorati on
l>e grown in 2-3 weeks time through embryo
cultures in contrast to 3 years period required
throughseedgermination.
4. Productionof haploids: Embryoculturehas
been successfullyused for the production of
haploid(or monoploid)plantse.g. barley.
5. Overcoming seed sterility : Certain plant
speciesproducesterileseedsthat do not Serminate
e.g.earlyripeningvarietiesof cherry,apricot,plum'
with incomplete
embryo development which leads to the death of
the germinating embryo. Using embryo cultures,it
is possibleto raiseseedlingsfrom sterileseedsof
earl y ri peni ngfrui tse.g. apri cot,pl um.
6. Clonal propagation: Embryosare ideally
suitedfor in vitroclonalpropagation. This is due to
the fact that embryosare juvenile in naturewith
high regenerative potential.Further,it is possibleto
induce organogenesis and somaticembryogenesis
from embryonictissues.
 ermplasnr broadly refers to the hereditary
I material (total content ot genes)transmittecltr-r
the offspr ing t hr ough ger m c ells Ce r m p l . r s n r
pro vid esth e r aw m at er ialf or t he br eec lert o d e v e l o p
va riou s cr ops . 1- hus , c ons er v at ion of ge r m p l a s r l r
assu mess ignif ic anc ein all br eeding pr ogr a m m e s .
As th e pr im it iv e m an lear nt about t he u t i l i t y o f
pla nts for f ood and s helt er ,he c ult iv at ed t h e h a b i t
of saving selected seeds or vegetative propagules
fro m on e seas ont o t he nex t one. I n ot he r w o r d s ,
this may be regardedas primitivebut conventional
germplasm preservation and management, which
is hig hly v aluable in br eedr ng pr ogr am m e s
The very objective of germplasm conservation
(or storage) is to preserve the genetic diversity of a
particular plant or genetic stock for its use at any
time in future In recent years, many new plant
sp ecies wi t l. r des r r ed and im pr ov ed c har a c t e r i s t r c s
h ave star t ed r eplac ing t he pr im it ive a n d
co nven tionally us ed agr ic ult ur al plant s . l t r s
imp orta nt t o c ons er v e t he endanger ed p l a n t s o r
else so me of t he v aluable genet ic t r ait s pr e s e n t I n
the prtmttive plants may be lost.
A global body namely lnternational Board of
Plant Genetic Resources (IBPGR) has been
e sta blishe df or ger m plas m c ons er v at ion. It s n r a i n
o bie ctive is t o pr ov ide nec es s ar y s upp o r t f o r
colle ctio n, c ons er v at ion and ut iliz at ion o f p l a n t
genetic resourcesthroughout the world
There are two approaches f or germpl.rsm
co nservati onof plant genet ic m at er iais
I /n-situ conservation
'2 Fx silrr conservation
The conservation oi germplasm in their natural
environment by establishingbiosphere reserves(or
national parks/gene sanctuaries) is regarded as
irr-.slfuconservation This approach is particularly
u s e f L rflo r p r e s e r v a t i o n
o f l a n d p l a n t si n a n e a r n a t u r a l
h a b i t a ta l o n g w i t h s e v e r a lw i l d r e l a t i v e sw i t h g e n e t i c
diversity fhe in-situ conservationis consideredas a
h i g h p r i o r i t y g e r m p l a s mp r e s e r v a t i o np r o g r a m m e
The major limitations of in-situ conservationare
listed below
. The risk of losing germplasrr due to
e n v i r o n m e n t a ]h a z a r d s
. T h e c o s t o f m a r n t e n a n c eo f i r l a r g e n u m b e r o f
B e n o t y p e si s v e r y h i g h .
r:i jl ; ij :ri-..r ili :+Pi i il fl *l.f.I! Tilr, I I
Ex-situconservation is the chief method for the
preservationof germplasntobtainecJfrom r:ultivated
and wild plant materials.The genetic materials in
the form of seeds or from in vitro cultures (plant
cells, tissuesor organs) can be preserved as gene
banks for long term storage under suitable
c o n d i t i o n s . F o r s u c c e s s f u l e s t a b l i s h m e n to f g e n e
b a n k s , a d e q u a t e k n o w l e d g e o f g e n e t i c s t r u c t u r eo f
plant populations,and the techniques involved tn
sampling,regenerationm , a i n t e n a n c eo f g e n e p o o l s
etc .lre essentral.
565
t
I

566 B IO TECHNO LO CY

Germplasm conservation There are mainly three approaches for the ln

in the form of seeds vitro conservation of germPlasm
1. Cryopreservation(freeze-preservation)
Usualfy, seeds are the most common and
convenient materials to conserve plant germplasm. 2. Cold storage
This is becausemany plants are propagatedthrough 3. Low-pressureand low-oxygen storage
seeds, and seeds occupy relatively small space'
Further, seeds can be easily transported to various
places.

There are however, certain limitations in the

Cryopreservation (Creek, krayos-frost) literally
conserVationof seeds
means preservation in the frozen state. The
. Viability of seeds is reduced or lost with passage principle involved in cryopreservation is to bring
of time. the plant cell and tissue cultures to a zero
metabolism or non-dividing state by reducing the
. Seeds are susceptible to insect or pathogen
temperature in the presence of cryoprotectants.
attack, often leading to their destruction.
Cryopreservationbroadly means the storage of
o This approach is exclusively confined to seed germplasm at veri low temperatures.
propagating plants, and therefore it is of no use
o Over solid carbon dioxide (at -79"C)
for vegetatively propagated plants e.g. potato,
Ipomoea, Dioscorea. . Low temperature deep freezers (at -80"C)
. In vapour phase nitrogen (at -1 50'C)
. lt is dif f ic ult t o m aint ai n c l o n e s t h r o u g h s e e d
conservation. i I n l i q u i d n i t r o g e n ( a t - 1 9 6 'C )

Certain seeds are heterogeneousand therefore, Among these, the most commonlY used
are not suitable for true genotype maintenance. cryopreservation is by employing liquid nitrogen.
At the temperatureof liquid nitrogen (-1 96'C), the
c e l l s s t a y i n a c o m p l e t e l y i n a c t i v e s ta te a n d th u s
In vitro methods for germplasm
can be conserved for long periods. In fact,
conservation
cryopreservationhas been successfullyapplied for
In vitro methods employing shoots, meristems germplasm conservation of a wide range of plant
and embryos are ideally suited for the conservation s p e c i e s e . g . r i c e , w h e a t , p e a n u t, ca ssa va ,
of germplasm of vegetatively propagated plants. sugarcane,straberry,coconut. Severalplants can be
The plants with recalcitrant seeds and genetically regenerated from cells, meristems and embryos
engineered materialscan also be preservedby this stored in cryoPreservation
in vitro approach.
Mechanism of cryoPreservation
There are several advantagesassociatedwith in
The technique of freeze preservationis based on
v itro germplasm conservation
the transfer of water present in the cells from a
. Large quantities of materialscan be preservedin liquid to a solid state. Due to the presenceof salts
s m all s pac e a n d o r g a n i c m o l e c u l e s i n t h e c e l l s , th e ce l l w a te r
requires much more lower temperature to freeze
. The ger m plas m pr es er v e dc a n b e m a i n t a i n e d t n
(even up to -68'C) compared to the freezing point
an environment, free from pathogens
of pure water (around 0"C). When stored at low
r lt can be protected against the nature's hazards temperature, the metabolic processes and
b i o l o g i c a l d e t e r i o r a t i o n si n t h e c e l l s/ti ssu e sa l m o st
r From the germplasm stock, large number of
come to a standstill.
plants can be obtained whenever needed.
Precautions/limitations for successful
. Obstaclesfor their transportthrough national and
cryopleservation
int er nat ional bor der s a r e m i n i m a l ( s i n c e t h e
ger m plas m is m aint a i n e d u n d e r a s p e c t i c Cood technical and theoretical knowledge of
c ondit ions ) . I i v i n g p l a n t c e l l s a n d a s w e l l a s c r yo p r e se r va ti o n
 AND CRYOPRESERVATION
CONSERVATION
Chaoter48 : CERMPLASM 567

t2 ------l
tr
Shoottips

Pregrownon medium
with DMSO

in culturemedium

Ampoulethawing

Ampoulestored
in liquidnitrogen
Shoottips in ampoule
frozenin liquidnitrogen

Fig. 48.1 : An outline of the protocol for cryopreservation of shoot tip (DMSO-Dimethyl sulfoxide).

technique are essential. Other precautions (the cryopreservationof plant cell culture followed by
limita tion stha t s hould be ov er c om e)f or s uc c es s f u l the regeneration of plants broadly involves the
cryopreservationare listed below following stages
.l
. Forma tion ice c r y s t als ins ide t he c ells s hould b e . Development of sterile tissue cultures
prevented as they cause injury to the organelles 2. Addition of cryoprotectantsand pretreatment
a nd th e cell.
3. Freezing
. Hig h in tracel lular c onc ent r at ion of s olut es m a y
4. Storage
a lso da mag e c ells .
5. Thawing
o Sometimes,certain solutesfrom the cell may leak
o ut d urin g fre ez ing- 6. Reculture

. Cryoprotectants also affect the viability of 7 . M e a s u r e m e n to f s u r v i v a l / v i a b i l i t y

cells. B. Plant regeneration.
. The physiological status of the plant material is The salient features of the above stages are
also imoortant. briefly described.

TECHNIOUE O F CRYO PRESERVATI O N Development of sterile tissue culture

An outline of the protocol for cryopreservation The selection of plant species and the tissues
of shoot tip is depicted in Fig. 48.1 . The with particular referenceto the morphological and
568 B IOTECHNO LO CY

phy s iologic alc har ac t er sla r g e l y i n f l u e n c et h e a b i l i t y freezing technique is used for the cryopreservation
of the explant to survive in cryopreservalion.Any of shooi tips and somatic embrYos.
tissue from a plant can be used for cryopreservation 3. Stepwise freezing method I This is a
, br y os ,en d o s p e r n t so, v u l e s ,s e e d s , combination of slow and rapid freezing procedures
e. g. m er is t em sem
c ult ur ed plant c ells , pr oto p l a s t s ,c a l l u s e s . A m o n g (rvith the advantagesof both), and is carried out tn
these, meristematic cells and suspension cell a s t e p w i s em a n n e r .T h e p l a n t m a t e r i al i s fi r st co o l e d
cultures,in the late lag phaseor log phaseare most t o a n i n t e r m e d i a t e t e m p e r a t u r e a nd m a i n ta i n e d
suitable. t h e r e f o r a b o u t 3 0 m i n u t e s a n d t h e n r a p i d l y co o l e d
b y p l u n g i n g i t i n t o l i q u i d n i t r og e n . Ste p w tse
; \ odlt ion of c r y opr ot ect a n t s and freezing rnethod has been successfully used for
pretrelatment
c r y o p r e s e r v a t i o n o f s u s p e n s i o n c u l tu r e s, sh o o t
Crvoprotectants are the compounds that can aoices and buds
prevent the damage caused to cells by freezing or 4. Dry freezing method : Some workers have
thawing. The freezing point and supercooling point reported that the non-germinated dry seeds can
of water are reduced bv the presence ot survive freezing at very low temperature in contrast
cryoprotectantsAs a result,the ice crystalformation t o w a t e r - i m b i b i n g s e e d s w h i c h a r e su sce p ti b l eto
is retarded during the process of cryopreservation c r y o g e n i c i n j u r i e s . I n a s i m i l a r f a s h i o n , d e h yd r a te d
Ther e ar e s ev er al c r y opr o t e c t a n t sw h i c h i n c l u d e cells are found to have a better survival rate after
dim et hy l s ulf ox ide ( DM S O ) , g l y c e r o l . e t h y l e n e , cryopreservatton.
pr opy lene,s uc r os e,m ann o s e ,g l u c o s e , p r o l i n e a n d
acetanride. Among these, DMSO, sucrose and Storage
glycerol are most widely used. Cenerally, a mixture
M a i n t e n a n c eo f t h e f r o z e n c u l t u r e sa t th e sp e ci fi c
of cryoprotectantsinstead of a single one, is used
temperatureis as important as freezing. ln general,
for more effectivecryopreservationwithout damage
the frozen cells/tissues are kept for storage at
to cells/tissues.
temperatures in the range of -70 to -196'C.
However, with temperatures above -1 30'C, ice
Fl a+ ; illr g
c r y s t a l g r o w t h m a y o c c u r i n s i d e t he ce l l s w h i ch
The serrsitivityof the cells to low temperature is r e d u c e sv i a b i l i t y o f c e l l s S t o r a g ei s i d e a l l y d o n e i n
v ar iable and lar gely depe n d s o n t h e p l a n t s p e c i e s . l i q u i d n i t r o g e nr e f r i g e r a t o r - a t 1 5 0 'C i n th e va p o u r
Four different types of freezing methods are used. p h a s e ,o r a t - 1 9 6 'C i n t h e l i q u i d p ha se .

1 . Slow-freezing method : The tissue or the
r equis it e plant m at er ial is s l o w l y f r o z e n a t a s l o w
cooling rates of 0.5-5oC/min from 0'C to -100"C,
and t hen t r ans f er r ed t o l i q u i d n i t r o g e n . T h e
advantage of slow-freezing method is that some
amount of water flows from the cells to the outside.
This pr om ot es ex t r ac ellu l a r i c e f r o m a t i o n r a t h e r
t han int r ac ellularf r eez in g . A s a r e s u l t o f t h i s , t h e
plant c ells ar e par t ially d e h y d r a t e d a n d s u r v i v e
better. The slow-freezing procedure is successfully
used for the cryopreservation of suspension
cultures.
2. Rapid freezing method : This technique is
quit e s im ple and inv olv e s p l u n g i n g o f t h e v i a l
c ont aining plant m at er i a l i n t o l i q u i d n i t r o g e n .
During rapid freezing, a decrease in temperature
-300" to -1000"C/min occurs. The freezing process
is c ar r ied out s o quic k ly th a t s m a l l i c e c r y s t a l sa r e
f or m ed wit hin t he c ells . F u r t h e l t h e g r o w t h o f
int r ac ellular ic e c r y s t als i s a l s o m i n i m a l . R a p i d
The ultimate obiective of storage is to stop all
t h e c e l l u l a r m e t a b o l i c a c t i v i t i e sa n d m a i n ta i n th e i r
r,iability For long term storge, temperature at
-196"C in liquid nitrogen is ideal. A regular and
c o n s t a n t s u p p l y o f l i q u i d n i t r o g e n to th e l i q u i d
nitrogen refrigeratoris essential.lt is necessaryto
c h e c k t h e v i a b i l i t y o f t h e g e r m p l a s mp e r i o d i ca l l y i n
some samples.
Proper documentation of the germplasm storage
has to be done. The documented information must
b e c o m p r e h e n s i v ew i t h t h e f o l l o w i ng p a r ti cu l a r s.
. T a x o n o m i c c l a s s i f i c a t i o no { t h e ma te r i a l
o History of culture
. Morphogenic potential
o C e n e t i c m a n i p u l a t i o n sd o n e
. Somaclonalvariations
. Culture medium
o Growth kinetics
ChAPICT
48 : CERMPLASM
CONSERVATION
AND CRYOPRESERVATION 569

Thawing
Tlrrr 48.1 A selected list of plants in varlous
Thawingis usuallycarriedout by plungingthe
forms that are successfullycryopreserved
frozen samples in ampoules into a warm water
(temperature 37-45"C)bathwith vigorousswirling. PIant material Plant species
By this approach,rapidthawing(atthe rateof 500-
750" C m in- l) o c c u rs ,a n d th i s p ro te c tsth e c el l s Cellsuspensions 4^,-^
vr yzd ^^t:.,^
JdIIva

from the damagingeffectsice crystalformation. Glycinenax

As the thawingoccurs(icecompletelymelts)the Zeanays
ampoulesare quicklytransferred to a waterbath at Nicotiana tabacun
temperature2O-25"C. This transferis necessary
Capsicum annun
sincethe cellsget damagedif left for long in warm
(37-45'C\ water bath. Callus Oryzasativa
Capsicun annum
For the cryopreservedmaterial (cells/tissues)
where the water contenthas been redur-edto an Saccharum sp
optimal level before freezing, the process of Protoplast Zeamays
t howingbec om e sl e s sc ri ti c a l . Nicotiana tabacum
Meristems Solanun tuberasum
Beculture
Cicerarielinun
In general,thawedgermplasmis washedseveral
times to removecrvoorotectants. This materialis
Zygotic
embryos Zeamays
t hen r ec ult ure di n a fre s h me d i u m fo l l o w i ng Hordeum vulgare
standardorocedures. Manihot esculenta
Some workers prefer to directly culture the Somatic
embryos Citrussinensis
thawed materialwithout washing.This is because Daucus carota
certain vital substances,releasedfrom the cells
Coffeaarabica
during {reezing,are beiievedto promote in vitro
cultures. Pollen
embryos Nicotianatabacum
Citrussp
Measurement of survival/viability Atropabelladona
The viability/survival of the frozencells can be
measuredat any stageof cryopreservation or after
thawingor r ec u l tu re . appropriate plant growth and regeneration, the
cryopreserved cells/tissues have to be carefully
The techniquesemployedto determineviability
nursed, and grown. Addition of certain growth
of cryopreserved cellsarethe sameas usedfor cell promoting substances, besides maintenance of
cultures (Refer Chapter 42). Staining techniques
appropriate environmental conditions is often
us ingt r iphenylte tra z o l i u mc h l o ri d e(T T C ),E v an' s
necessaryfor successfulplant regeneration.
blue and fluorescein diacetate(FDA)are commonly
used. A selected list of plants (in various forms) that
have been successfullyused for cryopreservationis
T he bes t ind i c a to rto m e a s u reth e v i a b i l i tyof
siven in Table 48.1 .
c e l l s i s th e i r e n try i n to c e l l d i v i s i on
c r y opr es er v ed
and regrowthin culture.This can be evaluatedby
t he f ollowingex p re s s i o n ^
No. of c e l l s /o rg a nBsro w i n B
* ,O O
No. of cells/organs thawed
Cold storage basically involves germplasm
conservation at a low and non-freezing
Plant regeneration temperatures(l-9"C). The growth of the plant
The ultimate purpose of cryopreservation of materialis sloweddown in cold storagein contrast
the desiredplant. For to completestoppagein cryopreservation.
germplasmis to regenerate Hence,
t.-

570 B IOTECHNO LO CY

Other
gases (2%)

z
E
c)
o
6 seo
o
o-
0

Normalatmospheric LOW-pressure LOW-oxygen

storage slorage storage

Fig. 48.2 : A graphic representation of tissue culture storage under normal atmospheric pressure,
low-Pressure,and low-oxYgen'

cold storage is regarded as a slow growth c o n s e r v a t i o n . A g r a p h i c r e p r e s e n t a t i o no f ti ssu e

germplasm conservation method The major culture storageunder normal atmospheric pressure,
advantageof this approach is that the plant material Iow-pressure and low-oxygen is depicted in
is not subjected to cryogenic injuries.
(cells/tissues) Fig. 48.2.

Long-term cold storage is simple, cost-effective

and y ields ger m plas m wit h g o o d s u r v i v a l r a t e .
Many in vltro developed shoots/plantsof fruit tree
s pec ies haVe been s uc c es s f u l l y s t o r e d b y t h i s
approach e.g. grape plants, strawberryplants.Virus-
free strawberry plants could be preserved at 10'C
for about 6 years, with the addition of a few drops
of m edium per iodic ally ( o n c e i n 2 - 3 m o n t h s ) .
Several grape plants have been stored for over 1 5
years bv cold storage(at around 9'C) by transferring
t hem v ear lv t o a f r es h m edi u m .
Low.pressure storage (LPSI
storage,
In low-pressure pressure
theatmospheric
s u r r o u n d i n g t h e p l a n t m a t e r i a l i s r e d u ce d . Th i s
resultsin a partial decreaseof the pressureexerted
by the gases around the germplasm. fhe lowered
partial pressure reduces the in vitro growth of
plants (of organized or unorganizedtissues).
Low-pressure storage systems are useful for
short-termand longterm storageof plant materials.
T h e s h o r t - t e r m s t o t a g e i s p a r t i c u l a rl y u se fu l to
i n c r e a s et h e s h e i f l i f e o f m a n y p l a n t m a te r i a l se .g .
f r u i t s , v e g e t a b l e s ,c u t f l o w e r s , p l a n t c u tti n g s. Th e
g e r m p l a s mg r o w n i n c u l t u r e sc a n b e s t or e dfo r l o n g
term under low pressure.

As alternatives to cryopreservation and cold B e s i d e s g e r m p l a s m p r e s e r v a t i o n , LPS r e d u ce s

storage,Iow-pressurestorage(LPS)and low-oxygen t h e a c t i v i t y o f p a t h o g e n i c o r g a n i s m s a n d i n h i b i ts
storage(LOS) have been d'evelopedfor germplasm s p o r e g e r m i n a t i o n i n t h e p l a n t c u l t u r e syste m s.
 AND CRYOPRESERVATION
CONSERVATION
Chaoter48 : CERMPLASM
Low'oxygen storage (LOSI
In the Iow-oxygen storage, the oxygen
con ce ntra tion is r educ ed, but t he at m os phe r i c
pre ssure(2 50 mm Hg) is m aint ainedby t he addit i o n
o f in ert g ases(p ar t ic ular lynit r ogen)
The partial pressureof oxygen below 50 mm Hg
re du ce s p lan t t is s ue gr owt h ( or ganiz ed o r
u no rga nizedtiss ue) .This is due t o t he f ac t t hat wi t h
r e du ce d availa bilit y of O r , t he pr oduc t ion of CO,
is low. As a consequence, the photosynthetic
activity is reduced, thereby inhibiting the plant
tissue growth and dimension.
Limitations of LOS : The long-term conservation
of plant materialsby low-oxygen storageis likely to
in hib it the pla nt gr owt h af t er c er t ain dim ens ion s .
The germplasm storage has become a boon to
plant breeders and biotechnologists.Some of the
ap plicatio ns a re br ief ly des c r ibed.
1 . Maintenance of stock cultures : Plant
mate rials(ce ll/tis s uec ult ur es )of s ev er als pec iesc a n
be crvooreservedand maintained for severalyears,
an d used as a nd when needed. This is in c ont r a s t
to an in vitro cell line maintenancewhich has to be
subcultured and transferredperiodically to extend
via bility. Th us, ger m plas m s t or age is an id e a l
meth od to a vo id s ubc ult ur ing, and m aint ain c e l l s /
tissu esin a viab le s t at e f or m any y ear s .
571
2. Cryopreservationis an ideal method for long
term conservation of cell cultures which produce
secondary metabolites (e.9. medicines).
3 Disease (pathogen)-freeplant materials can
be frozen, and propagated whenever required.
4. Recalcitrant seeds can be maintained for
ron8.
5. Conservation of somaclonal and game-
toclonal variations in cultures.
6. Plant materials from endangered species can
be conserved.
7. Conservation of pollen for enhancing
longevity.
B Rare germplasmsdeveloped through somatic
h y b r i d i z a t i o n a n d o t h e r g e n e t i c m a n i p u l a t i o n sc a n
be stored
9. Cryopreservation is a good method for the
s e l e c t i o n o f c o l d r e s i s t a n tm u t a n t c e l l l i n e s w h i c h
could develop into frost resistantplants.
10, Establishment of germplasm banks for
exchange of information at the international level.
LIMITATIONS OF GERMPLASM
STORAGE
The maior limitations of germplasm storage are
the expensive equipment and the trained
personnel. lt may, however, be possible in the near
future to develop low cost technology for
c r y o p r e s e r v a t i o no f p l a n t m a t e r i a l s .
 , y the conventional plant breeding techniques, '
-c, ." . 6..' ." '
s ignif ic ant ac hiev em ent s h a v e b e e n m a d e i n
I t i s o n l y t h r o u g h t h e r e c o m b i n a n t a p p r o a ch
the improvement of several t,ood crops. These age-
( g e n e t i c e n g i n e e r i n g )o f b i o t e c h n o l o g y,n e w g e n e s
old c las s ic al m et hods , inv o l v i n g g e n e t r a n s f e r
with desired characters (that may or may not be
through sexual and vegetative propagation, fake
p r e s e n t i n o t l . r e rp l a n t s )c a n b e i n t r o d u ce d i n to th e
very long time. For instance, about 6-8 years may
p l a n t s F u r t h e r , i t i s p o s s i b l e t o m a n i p u l a te th e
be required to develop a new rice or a wheat by
e x i s t i n g g e n e s t o m a k e t h e p r o t e i n s w i th su i ta b l e
s ex ual pr opagat ion. Rapid a d v a n c e s i n g e n e
a l t e r a t i o n s e . g . i n c r e a s e i n t h e c o n te n t o f a n
s t r uc t ur e and f unc t ion, c oup l e d w i t h t h e r e c e n t
e s s e n t i a la m i n o a c i d . T h e n r o s t i m p o r ta n t r e a so n s
dc v elopm ent s m ade in lhe g e n e t i c e n g i n e e r i n g
for developing transgenic planfs are listed
t ec hniques hav e dr am at ic all y i m p r o v e d t h e p l a n t
br eeding m et hods t o y ield t h e d e s i r e d r e s u l t s i n a o T o i m p r o v e a g r i c u l t u r a l , h o r t i cu l tu r a l o r
s hor t oer iod. ornamentalvalue of plants.
Plant genet ic t r ans f o r m a t i o n t e c h n o l o g v . To develop plant bioreactors for inexpensive
basically deals with the transfer of desirable m a n u f a c t u r eo f c o m m e r c i a l l y i m p o r t an t p r o d u cts
gene(s) from one plant species to another (or e.g. p r o t e i n s , m e d i c i n e s , p h a r m a ce u ti ca l
insertion of totally new genes) with subsequent compounos.
int egr at ionand ex pr es s ionof t h e f o r e i g n g e n e ( s )i n
o T o s t u d y t h e a c t i o n o f g e n e s i n p l a n ts d u r i n g
the host Benome. The term transgene is useci to
d e v e l o p m e n t a n d v a r i o u s b i o l o g i c a l p r o ce sse s.
represent the transferued gene, and the genetic
transformation in plants is broadly referred to as
Genetic traits ir;trorlueed intei
plant transgenesis fhe genetically transformed
transgemi* n*aile'.;
new plants are regarded as transgenic plants.
S i n c e p l a n t c e l l s a r e t o t i p o t e n t ( i .e . a si n g l e
The dev elopm ent of t r an s g e n i c p l a n t s i s t h e
p l a n t c e l l c a n r e g e n e r a t ei n t o a w h o l e p l a n t) , th e
out c om e of an int egr a t e d a p p l i c a t i o n o t
g e n e t i c a l l y e n g i n e e r e d c e l l s w i t h n e w g e n e ( s)ca n
r ec om binant DNA ( r DNA ) t e c h n o l o g y , g e n e
p r o d u c e a t r a n s g e n i cp l a n t T h i s p l a n t ca r r yi n g th e
t r ans f er - m et hods and t is s ue c u l t u r e t e c h n i q u e s .
d e s i r e d t r a i t w i l l g i v e r ai s e t o su cce ssi ve
(Note : The reader must refer Chapters 6 and 7 for
generations. Many genetic traits have been
bas ic inf or m at ion on genet ic e n g i n e e r i n g ,a n d f o r
introduced into plants through genetic engineering
c om m on t ec hniques em p l o y e d in rDNA
t ec hnology ) . R e s i s t a n c et o h e r b i c i d e s

572
Chapt er49 : C EN ET ICEN C IN EE R INO
CF PLA N TS -ME TH OD OLOC Y 573

Tmu 49.1 Gene transfer {DIiA delivery) nethods ln plants

h4ethod Salienl features

l. Vector-mediatedgene transfer
(Tiplasmid)-mediated
Agrobacterium genetransfer Veryefficient, groupof plants
butlimitedto a selected
Plantviralvectors Ine{fective
method,
hencenotwidelyused
ll. Direct or vectorlessDNA transfer
(A) Physical methods
Electroporation Mostlyconfinedto protoplasts
thatcanbe regenerated to
viableplants.Manycerealcropsdeveloped.
(particle
Microprojectile bombardment) Verysuccessful method usedfor a widerangeof plantsr
Riskof genereanangement
tissues. high.
Microinjection Limitedusesinceonlyonecellcanbe microinjected at a
time.Technicalpersonnelshouldbe highlyskilled.
Liposome
fusion Confinedto protoplasts
thatcanbe regenerated intoviable
wholeplants.
fibres
Siliconcarbide Requiresregenerable Thefibres,however,
cellsuspensions.
require
careful
handling.
(B) Chemical methods
glycol(PEG)-mediated
Polyethylene to protoplasts.
Confined of fertileplantsis
Regeneration
problematical.
frequently
(DEAE)
Diethylaminoethyl dextran-mediated Doesnotresultin stabletransformants.

r Protection against viral infections

o Insecticidal activity
The gene transfer techniques in plant genetic
. lmp roved nu t r it ional qualit y
transformation are broadly grouped into two
o Altered flower pigmentation categories

. Tolerance to environmental stresses l. Vector-mediatedgene transfer

. Se lf in co mpat ibilit y
Griteria for commercial
genetically transformed
ll. Direct or vectorlessDNA transfer
The salient featuresof the commonly used gene
use of (DNA) transfer methods are siven in Table 49.1.
plants

For large-scale commercial application of

genetically engineered plants, the following
reouirementshave to be satisfied
Vector-mediated gene transfer is carried out
o ln trod uction of des ir able gene( s ) t o all p l a n t either by Agrobacterium-mediated transformation
cells. or by use of plant virusesas vectors.
Expression of cloned genes in the appropriate
AG R O B ACT E N I U M.MEDI AT ED
c ellsat t he ri g h tti me .
GE N E TR A N S FE B
Stablemaintenance of new gene(s)inserted.
Agrobacteriumtumefaciensis a soil-borne,
Transmissionof new senetic information to Cram-negativebacterium.lt is rod shaped and
subsequent generations. motile, and belongs to the bacterial family of
l.;
t'

574 B IOTECHNO LO CY

Wound
Crown
galltumor
f i.o<- Aorobacterium
"o" iumefaciens

Roots

Fig. 49.1 : Formation of a crown gall tumor in a plant infected n4th Agrobacterium tumefaciens.

Rhizobiaceae A. tumefaciens is a phytopathogen, gall disease is a naturally evolved genetic

and is treated as the nature's most effective plant
genetic engineer. Some workers consider this
bacterium as the natural expert of interkingdom
gene transfer In fact, the major credit for the
dev elopm ent of plant t r an s f o r m a t i o n t e c h n i q u e s
goes t o t he nat ur al uni q u e c a p a b i l i t y o f A .
tumefaciens. Thus, this bacterium is the most
belov ed by plant biot ec hno l o g i s t s .
There are mainly two speciesof Agrobacterium :
. A. tumefaciensthat induces crown gall disease.
. A rhizogenesthat induces hairy root disease.
CROWN GALL DISEASE AND
Ti PLASM'D
engineering process.
The T-DNA carries genes that code for protetns
involved in the biosynthesisof growth hormones
( a u x i n a n d c y t o k i n i n ) a n d n o v e l p l a nt m e ta b o l i te s
namely opfnes- amino acid derivatives and
agropines-sugar derivatives (Fig. 49.2). The
g r o w t h h o r m o n e s c a u s e p l a n t c e l l s to p r o l i fe r a te
a n d f o r m t h e g a l l w h i l e o p i n e s a n d ag r o p i n e s a r e
utilized lsy A. tumefaciensas sourcesof carbon and
energy. As such, opines and agropines are not
n o r m a l l y p a r t o f t h e p l a n t m e t a b o l i sm ( n e i th e r
produced nor metabolised) Thus, A tumefaciens
g e n e t i c a l l y t r a n s f o r m s p l a n t c e l l s a nd cr e a te s a
biosynthetic machinery to produce nutrients for its
own use.

Alm os t 100 y ear s ago ( 1 9 0 7 ) , S m i t h a n d A s t h e b a c t e r i a m u l t i p l y a n d c o n t i n u e i n fe cti o n ,

Townsend postulated that a bacterium was the
causative agent of crown gall tumors, although its
importance was recognized much later.
As A. tumefaciens infects wounded or damaged
plant t is s ues , in induc es t h e f o r m a t i o n o f a p l a n t
tumor called crown gall (Fig. 49.1) The entry of
the bacterium into the plant tissuesis facilitated by
t he r eleas e of c er t ain p h e n o l i c c o m p o u n o s
(acetosyringone, hydroxyacetosyringone) by the
wounded s it es .Cr own gall f o r m a t i o n o c c u r s w h e n
the bacterium releases its Ti olasmid (fumor-
induc ing plas m id) int o t he p l a n t c e l l c y t o p l a s m . A
fragment (segment) of Ti plasmid, referred to as
T-DNA. is actuallv transferredfrom the bacteriurrr
into the host where it gets integratedinto the plant
cell chromosome (i.e. host genome) Thus, crown
g r o w n g a l l d e v e l o p s w h i c h i s a v i s i b l e m a ss o f th e
a c c u m u l a t e d b a c t e r i a a n d p l a n t m a te r i a l . C r o w n
gall formation is the consequence of the transfer,
integrationand expressionof genes of T-DNA (or Ti
plasmid) of A. tumefaciens in the infected plant
The genetic transformation leads to the formation
of crown gall tumors, which interfere with the
normal growth of the plant. Several dicotyledonous
plants (dicots) are affected by crown gall disease
e g. grapes, roses, stone-fruit trees.
Organization of Ti plasmid
T h e T i p l a s m i d s ( a p p r o x i m a t es i z e 20 0 kb e a ch )
e x i s t a s i n d e p e n d e n t r e p l i c a t i n g c ir cu l a r D N A
m o l e c u l e s w i t h i n t h e A g r o b a c t e r i u m ce l l s: Th e
T - D N A ( t r a n s f e r r e dD N A ) i s v a r i a b l e i n l e n g th i n
 CF PL A N TS -ME TH OD OLOGY
Chaot er49 : C E N E T ICEN C IN EE R INO 575

NH a l s o t r a n s f e r r e dt o t h e p l a n t c e l l s . l t i s n o w c l e a r l y
il establishedthat the right border is more critical for
H2N-C-NH-(CHz)s -cH-cooH T - D N A t r a n s f e ra n d t u m o r i g e n e s i s .
I
NH 2. Virulence region : The genes responsiblefor
I the transferof T-DNA into the host olant are located
cH-cooH outside T-DNA and the rigion is referredto as viror
I virulence region. Vir region codes for proteins
CHs
OctoPine involved in T-DNA transfer.At least nine vir-gene
operons have been identified These include vir A,
NH
vir C, vir Br, vir C1, vir Dt,Dzand D4, and vir E,
I
H2N-C-NH-(CHz)s -cH-cooH and Et.
I 3. Opine catabolism region : This region codes
NH
for proteins involved in the uptake and metabolisms
I of opines.
CH-COOH
I
(CN)z Besides the above three, there is ori region rhal
i s r e s p o n s i b l ef o r t h e o r i g i n o f D N A r e p l i c a t i o n
I
COOH which permits the Ti plasmid to be stably
Nopaline maintained in A- tumefaciens.

T.DNA transfer and integration

The processof T-DNA transferand it integration

into the host plant genome is depicted in Fig. 49.4,
and is briefly described.

1. Signal induction to Agrobacterium : The

Fig. 49,2 : Structures of three opines-octopine,
nopaline, and agropine.
wounded plant cells release certain chemicars-
phenolic compounds and sugars which are
recognized as signals by Agrobacterium. The
s i g n a l si n d u c e d r e s u l t i n a s e q u e n c eo f b i o c h e m i c a l
events in Agrobacteriumthat ultimately helps in the
transfer of T-DNA of T-olasmid.

t he r angeof 12 to 2 4 k b , w h i c h d e p e n d so n the
bac t er ials t r ai n fro m w h i c h T i p l a s mi d sc o me.
Nopalines t r ai n so f T i p l a s mi dh a v e o n e T -D N A
with lengthof 20 kb while octopinestrainshave
two T-DNA regionsreferredto as T, and T* that
ar e r es pec t iv e l1ya k b a n d 7 k b i n l e n g th . (24 bp repeat)

A diagrammatic of a Ti plasmidis
representation
depicted tn Fig, 493. fhe Ti plasmid has three
importantregions. Virulence
regron
1. T-DNA region: This regionhasthe genesfor (y/rgenes)
the biosynthesisof auxin (aux), cytokinin (cyt) and
opine (ocs),and is flankedby leftand rightborders.
Thesethree genes-aux,cyto and ocs are referredto
as oncogenes,as they are the determinantsof the (ori gene)
t um or phenot y p e .
Fig. 49.3 : A dagrammatic representation of a
T-DNA borders- A set of 24 kb sequences Ti plasmid.
presenton eitherside (rightand left)of T-DNA are
576 B IOTE CHNO LO CY

Woundedplantcell

Phenoliccompounos
and sugars
Nuclear
,1

vir D1/D2
#

I OO
+r 09
virDllD2 virE2

Virulenceproteins
( Dr , Dz , E2,B e t c . )
Translation
+
I
Agrcbacteriumsp Attachment
Productionof
auxin,cytokinin
and opine

+
I
Autoetirnulation
,otceHdivision

Plant cell

Fig. 49,4 : A diagrammatic representation of T-DNA transfer and its integration into host plant cell genome
(pTi-Ti plasmid; RB-Right border; LB-Left border; ss-Single-stranded).

2. Attachment of Agrobacterium to plant production single-stranded T-DNA (ss DNA), its

celfs : The Agrobacteriumattachesto plant cells protection and export to plant cells.The ss T-DNA
th ro u g h p o l y s a c c h a ri d e sparti
, cul arl y cel l ul ose getsattachedto vir Dr.
fibres produced by the bacterium. Several
5. Transferof T-DNA out of Agrobacterium :
c h ro m o s o mavli ru l e n c e(c h v )genesresponsi blfor e
The ss T-D N A -vi r D , compl ex i n associat ion
th e a tta c h m e notf b a c te ri acl e l l sto ol antcel l shave
with vir C is exportedfrom the bacterialcell. Vir B
been identified.
\ productsform the transportapparatus.
3. Productionof virulence proteins : As the
6. Transfer of T-DNA into plant cells and
signalinductionoccursin the Agrobacterium cells
integration: The T-DNA-vir D, complex crosses
attachedto plantcells,a seriesof eventstakeplace
the pl ant pl asmamembrane.In the plant cells,
th a t re s u l ti n th e p ro d u c ti o nof vi rul enceprotei ns.
T-DNA gets covered with vir Er. This covering
T o s ta rt w i th , s i g n a l i n d ucti on by phenol i cs
protectsthe T-DNAfrom degradation by nucleases.
stimulates vir A which in turn activates (by
vir D, and vir E" interactwith a varietyof plant
phosphorylation) vir C. This inducesexpression of
protei nsw hi ch i nfl uencesT-D N A transpor tand
v i ru l e n c eg e n e s o f T i p l a smi d to produce the
i ntegrati on.The T-D N A -vi r D r-vi r E, - plant
c o rre s p o n d i nvgi ru l e n c ep rotei ns(D 1, D 2, E 2, B
protei ncompl exentersthe nucl eusthro ughnuclear
etc.).Certainsugars(e.g.glucose,galactose, xylose)
pore complex.Within the nucleus,the T-DNA gets
th a t i n d u c ev i ru l e n c eg e n e sh avebeen i ndenti fi ed.
integratedinto the plant chromosomethrough a
4. Productionof T-DNA strand: The right and processreferredto illegitimate recombination. Thrs
left bordersof T-DNA are recognizedby vir D,/vir is differentfrom the homologousrecombination, as
D2 proteins.These proteinsare involved in the it does not dependon the sequencesimilarity.
 C P L A N TS -ME TH OD OLOC Y
Chaot er49 : C E N E T ICEN C IN EE R INOF 577

HAIBY BOOT D'SEASE OF . The phytohormones (auxi n,cytoki ni n)produced

A . RHT Z O G E N ES- R 1 PL A SMT D S preventthe plant cells being regeneratedinto
plants.Thereforeauxin and cytokiningenesmust
Agrobacteriumrhizogenescan also infect plants.
be removed.
Butthis resultsin hairyrootsand not crowngallsas
is the casewith A. tumefaciens. The plasmids,of . Opine production in transformedplant cells
A. rhizogeneshavebeen isolatedandcharacterized. lowers the plant yield. Therefore opine
Theseplasmids,referredto as Ri plasmids,(Ri stands synthesizing geneswhich are of no useto plants
tor Rootinducing)areof differenttypes,Someof the should be removed.
Ri plasmidstrainspossess genesthatarehomologous
. Ti pl asmi ds cannotrepl i catei n E . col i .Thi sl i mi ts
t o T i plas m ide.g . a u x i nb i o s y n th e tigce n e s .
thei r uti l i ty as E . col i i s w i del y used i n
I ns t eadof v i ru l e n c eg e n e s ,R i p l a s mi d sc o n tai n recombi nant experi ments. A n al ternate
a seriesof open readingframeson the T-DNA.The arrangement is to add an origin of replicationto
products of these genes are involved in the Ii pl asmi dthat al l ow sthe pl asmi dto repl i catern
metabolismof plant growth regulatorswhich gets E. coli.
sensetized to auxin and leadsto root formation.
Consideringthe above limitations,fi plasmid-
Vectors of A. rhizogenes based vectors with suitable modifications have
been constructed. These vectors are mainly
As it is done with A tumefaciens, vectorscan be composedof the followingcomponents.
constructedby usingA. rhizogenes. Thesevectors
are alternatestrategies for genetransfer.However, 1. The ri ghtbordersequence of T-D N Aw hi ch
employmentof A. rhizogene-based vectorsfor plant is absolutelyrequiredfor T-DNA integrationinto
transformationis not common since more efficient plant cell DNA.
systemsof A. tumefaciens
have been developed. 2. A mul ti pl ecl oni ngsi te(pol yl i nker
D N A )that
promotesthe insertionof cloned gene into the
lmportance of hairy roots regionbetweenT-DNA borders.
Hairy roots can be cultured in vitro, and thus 3. A n ori gi nof D N A repl i cati on
that al l ow sthe
are importantin plant biotechnology.Hairy root pl asmi dsto mul ti pl yi n E . col i .
svstemsare usefulfor the productionof secondary
m et abolit esparti
, c u l a rl p
y h a rma c e u ti c pa ro
l te tns. 4. A selectablemarker gene (e.g. neomycin
phosphotransferase) for appropriate selectionof the
Ti P LA S M I D. D ER IVE D transformed cells.
VECTOR SYSTEMS
Two typesof Ti plasmid-derived vectorsare used
The ability of Ti plasmidof Agrobacterium to for genetic transformationof plants- cointegrate
geneticallytransformplantshas been described.lt vectors and binary vectors.
is possibleto inserta desiredDNA sequence(gene)
a n d th e nuse Cointegrate vectol
i nio t he T - DNAr e g i o n(o fT i p l a s m i d ),
A. tumefaciensto deliver this gene(s)into the
ln the cointegratevector system,the disarmed
genomeof plant cell. ln this process,Ti plasmids
and modi fi ed Ti pl asmi d combi nes w i th an
serveas naturalvectors.However,thereare several
intermediate cloning vector to produce a
limitations to use Ti plasmids directly as cloning
recombinantTi plasmid(Fig.a9.5).
vectors.
Production of disarmed Ti plasmid : The
. Ti plasmids are large in size (200-800 kb).
preferred T-DNA genes for hormone biosynthesisare
Smallervectors are for recombinant
removed(disarmed). In placeof the deletedDNA,
experiments.For this reason,large segmentsof
a bacteri alpl asmi d(pB R 322)D N A sequencei s
DNA of Ti plasmid, not essentialfor cloning,
incorporated. This disarmedplasmid,also referred
must be removed.
Io as receptor plasmid, has the basic structureof
. Absenceof uniquerestrictionenzymesiteson Ti T-DNA(rightand left borders,virulencegenesetc.)
olas m ids . necessary to transferthe plant cells.

Biotechnology
[37]
r-7

574 B IOTECHNO LO CY

Homologous
DNAsequence

Disarmed Ti plasmid Intermediatevector

Fig. 49.5 : Cointegrate vector system (vi-Ti plasmid virulence region; pBR322-Bacterial plasmid 322; LB-Left
border; RB-Right border; MCS-Multiple cloning site; PTM-Plant transformation marker; RES-Bacterial resistance
marker; col E,-Origin of a replication from col E, plasmid; orif -Origin of transfer site for
conjugative plasmid mobilization).

Construction of intermediate vector : The c e l l s a n d t h u s p e r m i t s t h e i r s e l e c ti ve i so l a ti o n

intermediate vector is constructed with tne
f ollowing c om ponent s .
o A pBR322 sequence DNA homologous to that
f ound in t he r ec ept or Ti p l a s m i d .
. A plant transformation marker (PTM) e g. a gene
coding for neomycin phosphotransferasell (npf
ll) . This gene c onf er s r es i s t a n c et o k a n a m y c i n i n
t he plant c ells and t hus p e r m i t s t h e i r i s o l a t i o n .
. A bacterial resistance marker e.g. a Bene coding
for spectinomycin resistance.This gene confers
spectinomycin resistance to recipient bacterial
. A multiple cloning site (MCS where foreign
g'enes can be inserted.
. A C o l E , o r i g i n o f r e p l i c a t i o n w h ich a l l o w s th e
replication of plasmid in E. coli but not in
Agrobacterium.
. An oriT sequence with basis of mobilization
(bom) site for the transfer of intermediate vector
from E. coli to Agrobacterium.
Production and use of cointegrate vectors : The
desired foreign gene (target-gene)is first cloned rn
t h e m u l t i p l e c l o n i n g s i t e o f t h e i n t e r me d i a teve cto r .
 Chapt er49 : C E N E T ICE N C IN E ER INO
GF P L A N TS -ME TH OD OLOC Y
Fig. 49.6 : Binary vector system (vir-Ti plasmid virulence region; LB-Left border; BB-Right border; MCS-Multiple
cloning s!!e; PTM:Planltransformatianmarker; RE9-Bacterialrcsistancemarker; oril.Origin of transfet site lar
The cloning process is carried out in E. coli, the
bacterium wl.rerethe cloning is most efficient. The
intermediatevector is mated with Agrobacteriumso
that the foreign gene is mobilised into the latter.
The transformed Agrobacterium cells with receptor
Ti plasmid and intermediate vector are selectively
iso late d wh en gr own on a m inim al m ed i u m
con tain ing spec t inom y c in. The s elec t ion pr o c e s s
becomes easy since E. coli does not grow on a
minimal medium in which Agrobacterium grows.
Within the Agrobacterium cells, intermediate
plasmid gets integratedinto the receptorTi plasmid
to p rod uce coint egr at e plas m id. This pla s m i d
containing plant transformationmarker (e.g. npt ll)
gene and cloned target gene between T-DNA
borders is transferredto plant cells. The transformed
pla nt ce lls can be s eleCt edon a m edium c ont ai n i n g
kanamycin when the plant and Agrobacterium cells
are incubated together.
Advantages of cointegrate vector
r Target genes can be easily cloned
. The pla smid is r elat iv ely s m all wit h a num be r o f
restriction sites.
o In terme dia teplas m id is c onv enient lyc loned i n E .
coli and transferred to Asrobacterium.
Binary vector
The binary vector system consists of an
Agrobacterium strain along with a disarmed Ti
plasmid called vir helper plasmid (the entire
T-DNA region including borders deleted while vir
579
of! ftom
gene is retained).lt may be noted that both of them
a r e n o t p h y s i c a l i y l i n k e d ( o r i n t e g r a t e d ) .A b i n a r y
vector with T-DNA can replicate in E. coll and
Agrobacterium.
A diagrammatic representation of a typical
binary vector system is depicted in Fig. 49.6 fhe
binary vector has the following components
1 Left and right borders that delimit the
T-DNA region.
2. A plant transformationmarker (PTM) e.g. npt
// that confers kanamycin resistance in plant
t r a n s f o r m e dc e l l s .
3 . A m u l t i p l e c l o n i n g s i t e ( M C S )f o r i n t r o d u c i n g
target/oreign genes.
4. A bacterial resistancemarker e.g. tetracycline
resistancegene for selecting binary vector colonres
in E. coli and Agrobacterium.
5. oriT sequence for conjugal mobilization of
the binary vector from E. coli to Agrobacterium.
6 . A b r o a d h o s t - r a n g eo r i g i n o f r e p l i c a t i o ns u c h
as RK, that allows the replication of binary vector
in Agrobacterium.
" Production and use of binary vector : The target
(foreign)gene of interestis insertedinto the multiple
cloning site of the binary vector. ln this way, the
target gene is placed between the right and left
border repeats and cloned in E. coli. By a mating
process,the binary vector is mobilised from E. coli
to Agrobacterium.Now, the virulence gene proteins
s80 B IOTECHNO LO G Y

of T-DNA facilitate the transfer of T-DNA of the

vector into plant cells.

Advantages of binary vectors

. The binary vector system involves only the

transfer of a binary plasmid to Agrobacterium
without any integration. This is in contrast to
cointegrate vector system wherein the
intermediate vector is transferredand integrated
wit h dis ar m ed Ti plas m i d . T-DNA integratedwith targetgene

r Due to convenience, binary vectors are more J

frequently used than cointegrate vectors.

PLANT
USING
TRANSFORMATION
AGROBACTEB'UM
TECHNIOUE @@
Agrobacterium-mediatedtechnique is the most Agrobacterium
widely used Ior the transformation of plants and tumafaciens
gener at ion of t r ans gen i c p l a n t s . T h e i m p o r t a n t
requirements for gene transfer in higher plants / ,rt et
\)
/1
\
/\{--'\\
i \-//-\ '
through Agrobacterium mediation are listed. L,K
. The ex plant sof t he pla n t m u s t p r o d u c e p h e n o l i c
compounds (e.g. autosyringone)for activation of
Normalplant
c Q9e
"Xo"
v ir ulenc e genes .

o Transformed cells/tissuesshould be capable to Co-cultureof leal disc

and A- tumefaciens
regenerateinto whole plants.

ln general, most of the Agrobactedum-mediated

plant t r ans f or m at ions h a v e t h e f o l l o w i n g b a s i c / rar)
l u- )
\
\ I
protocol (Fig. ag.V \
t:
/zo l ./

1. Development of Agrobacterium carrying the Cultureon selectivemedium

cointegrateor binary vector with the desired gene.
+
I
2. ldent if ic at ionof a s u i t a b l e e x p l a n t e . g . c e l l s , ----..----.--.\
protoplasts,tissues,calluses, organs. /v c-esr- M\
\.'\--t1}--__./ )
3. Co-culture of explants with Agrobacterium.
Shootinduction
medium
4. Killing of Agrobacterium with a suitable
ant ibiot ic wit hout har m in g t h e p l a n t t i s s u e . J
5. Selection of transformed plant cells.

6. Regenerationof whole plants.

Advantages ol Agrobactefium
mediated transformation
o This is a natural method of gene transfer.
c Agrobacterium can conveniently infect any Transformedplant
explant (cellsltissues/organs).
Fig. 49.7 : Transformationtechnique using
Even large fragments of DNA can be efficiently Agrobacterium -mediatedgene transfer.
'
transferred.
Chapt er49 : CE N ET ICEN C T N EE R T OF
N G PL A N TS -TV IE TH OD OLOC Y
. Stab ility o f tra ns f er r edDNA is r eas onably . go o d .
. Transformedplants can be regeneratedeffectively.
Limitations ol Agrobacterium-
mediated transformation
. There is a limitation of host plants for
Agrobacterium, since many crop plants
(monocotyledons e.g. cereals) are not infected
b y it. In rec ent y ear s , v ir ulent s t r ains o f
Agrobacterium that can infect a wide range of
plants have been developed.
. The cells that regenerate more efficiently are
ofte n d ifficu lt t o t r ans f or m . e. g. em br y onic c e l l s
lie in deep layers which are not easy targets for
Asrobacterium.
Plant viruses are considered as efficient gene
transfer agents as they can infect the intact plants
a nd amp lify th e t r ans f er r ed genes t hr ough v ir a l
genome replication. Viruses are natural vectors for
gen etic e ng ine er ing. They c an int r oduc e t h e
desirab le ge ne (s ) int o alm os t all t he plant c e l l s
since the viral infections are mostlv svstemic.
Plant viruses are
nonintegrative vectors
'Ihe
plant viruses do not integrate into the host
genome in contrast to the rrectorsbased on T-DNA
oI A. tumefacienswhich are integrative.The viral
gen ome s a re su it ably m odif ied by int r oduc i n g
desired foreign genes.
These recombinant viruses are transferred, c o d i n g r e g i o n s . T h e g e n e s l l a n d V l l a r e n o t
m ultip lied a nd e x pr es s edin plant c ells . They s pr e a d e s s e n t i a lf o r v i r a l i n f e c t i o n .
s ystemicallywith in t he hos t plant wher e t he ne w
genetic material is expressed.
Griteria for a plant virus vector
An ideal plant virus for its effective use in gene
transfer is expected to posses the following
characteristics.
celi
. The viru s mu s t be c apable of s pr eadingof r om
to ce ll thro ug h plas m odes m at a.
. The viral ge no m e s houle be able t o r eplic at e i n
the absenceof viral coat protein and spread from
581
c e l l t o c e l l . T h i s i s d e s i r a b l es i n c e t h e i n s e r t i o no f
foreign DNA l,r,illmake the viral genome too big
to be oacked.
. T h e r e c o m b i n a n tv i r a l g e n o m e m u s t e l i c i t l i t t l e o r
no disease symptoms in the infected plants.
. The virus should have a broad host range
. The virus with DNA genome is preferredsince
t h e g e n e t i c m a n i p u l a t i o n si n v o l v e p l a n t D N A .
The three groups of viruses - caulimoviruses,
geminiviruses and RNA viruses that are used as
vectors for gene transfer in plants are briefly
d e s cr ib e d .
CAULIMOVIRUSES AS VECTORS
The caulimoviruses contain circular doubre-
stranded DNA, anci are spherical in shape.
Caulimoviruses are widely distributed and are
r e s p o n s i b l ef o r a n u m b e r o f e c o n o m i c a l l y i m p o r t a n t
d i s e a s e si n v a r i o u s c r o p s . T h e c a u l i m o v i r u s g r o u p
has around 15 virusesand among Ihese cauliflower
mosaic virus (CaMV) is the most important for gene
transfer.l-he other cauI i movi rusesi ncl ude carnation
etched virus, dahlia mosaic virus, mirabilis mosaic
v i r u s a n d s t r a w b e r r yv e i n b a n d i n g v i r u s .
Gauliflower mosaic virus (GaMV|
CaMV infects many plants (e.9. members of
Cruciferae, Datura) and can be easily transmitted,
even mechanically. Another attractive feature of
CaMV is that the infection is systemic, and large
o u a n t i t i e so f v i r u s e s a r e f o u n d i n i n f e c t e d c e l l s
A diagrammatic view, of the CaMV genetic map
is depicted in Fig. 49.8. fhe genome of CaMV
consists of a B kb (8024 bp) relaxed but tightly
packedcircular DNA with six major and two minor
Use of GaMV in gene transfer
For appropriate transmission of CaMV, the
i o r e i g n D N A m u s t b e e n c a p s u l a t e di n v i r a l p r o t e i n .
Further,the newly inserted foreign DNA must not
interfere with the native assembly of the virus.
CaMV genome does not contain any rron-cociing
r e g i o n sw h e r e i n f o r e i g n D N A c a r r b e i n s e r t e d I t r s
fortunate that two genes narnely gene ll and gene
Vll have no essential functions for the virus it ts
therefore possible to replace one of them and
inserl the desired foreign gene
14

582 B IOTECHNO LO CY

T h e g e m i n i v i r u s e sc a n i n f e c t a w i d e r a n g e o f
crop plants (monocotyledons and dicotyledons)
which attract plant biotechnologists to employ
these virusesfor gene transfer.Curly top virus (CTV)
and maize streak virus (MSV) and bean golden
mosaic virus (BCMV) are among the important
gemtnrvrruses.

It has been observed that a large number of

Fig. 49.8 : A diagrammatic representation of the
genetic map of cauliflower mosaic virus genome
(1. .Vlll represent coding regions; lR land lR 2 are
intergeneric regions; The outside dotted circle
represerlts 305 transcript; The two circular lines
at the centre indicate viral DNA strands).
r e p l i c a t i v e f o r m s o ( a g e m i n i vi r u s Se n o m e
a c c u m u l a t e i n s i d e t h e n u c l e i o f i n f ecte d ce l l s. Th e
s i n g l e - s t r a n d e dg e n o m i c D N A r e p l i ca te s i n th e
nucleus to form a double-strandedintermediate
C e m i n i v i r u s v e c t o r s c a n b e u se d to d e l i ve r ,
amplify and expressforeign genes in several piants/
explants (protoplasts,cultured cells). However, the
s e r i o u s d r a w b a c k i n e m p l o y i n g g em i n i vi r u se s a s
vectors is that it is very difficult to introduce
p u r i f i e d v i r a l D N A i n t o t h e p l a n ts. An a l te r n a te
arrangement is to take the help oI Agrobacterium
and carry out gene transfer.
RNA PLANT VIRUSES AS VECTORS
There are mainly two types single-strandedRNA
v rr u s e s .

1 . Monopartite viruses : These viruses are

Cene ll of CaMV has been successfullvreplaced
wit h a bac t er ial gene e n c o d i n g d i h y d r o f o l a t e
reductasethat provides resistanceto methotrexate.
W hen t he c him er ic CaM V w a s t r a n s m i t t e dt o t u r n i p
plants, they were systemically infected and the
plants developed resistanceto methotrexate.
Limitations of GaMV as a vector
o CaMV vector has a limited caoacitv for insertion
of foreign genes.
o Infective capacity of CaMV is lost if more than a
few hundred nucleotides are introduced.
r Helper v ir us es c annot b e u s e d s i n c e t h e f o r e i g n
DNA get s ex pelled a n d w i l d - t y p e v i r u s e s a r e
oroduced.
u s u a l l y l a r g e a n d c o n t a i n u n d i v i d e d g e n o m e s fo r
a l l t h e g e n e t i c i n f o r m a t i o n e . g . t o b a cco m o sa i c
virus (TMV).
2. Multipartite viruses : The genome in these
v i r u s e si s d i v i d e d i n t o s m a l l R N A s w h i ch m a y b e i n
the same particle or different particles. e.g. brome
m o s a i c v i r u s ( B M V ) . H M V c o n t a i n s fo u r R N As
divided between three particles.
Plant RNA viruses, in general, are characterized
by high level of gene expression,good efficiency to
infect cells and spread to different tissues.But the
maior limitation to use them as vectors is the
d i f f i c u l t y o f j o i n i n g R N A m o l e c u l e s i n vi tr o .
Use of cDNA for gene transfer

C o m p l e m e n t a r y D N A ( c D N A ) co p i e s o f R N A
GEMINIVIRUSES AS VECTORS virusesare preparedin vitro. The cDNA so Senerated
The gem iniv ir us esar e s o n a m e d b e c a u s e t h e y can be used as a vector for gene transferin plants
hav e gem inat e ( Cem ini l i t e r a l l y m e a n s h e a v e n l y This approach is t.ediousand cumbersome.However,
t wins ) m or phologic al par t i c l e s i . e . t w i n a n d p a i r e d some successhas been reported.A gene sequence
capsid structures.These viruses are characterized e n c o d i n g c h l o r a m p h e n i c o l r e s i sta n ce ( e n zym e -
by pos s es s ingone or t w o s i n g l e - s t r a n d e dc i r c u l a r chloramphenicolacetyltransferase) has been inserted
DNAs ( s s DNA) . O n r epl i c a t i o n s ,s s D N A f o r m s a n into brome mosaic virus genome. This Sene
intermediate double-stranded DNA. expression,however,has been confinedto protoplasts.
 CF PLA N TS -ME TH OD OLOC Y
Chapt er49 : C E N E T ICEN C IN EE R INO 583

LIMITATIONS OF VIRAL VECTORS IN Plant

G E NE T RA N SF E R
+
I
The ultimate objectiveof gene transferis to Explants
transmit the desired genes to subsequent (leaf, embryo,merisiem,
generatians.With virus vectors,this is nof possible callus,cells,protoplasts)
unlessthe virus is seed-transmitted. However,in DNA------J PhYsicaland
caseof vegetativelypropagated plants,transmission d"lil;i
of desiredtraitscan be done e.B.potatoes.Evenin
Jcrremlcarmethods*
theseplants,thereis alwaysa riskfor the transferred
Seneto be lost anytime.
For the reasons referred above, plant
biotechnologists preferto insertthe desiredgenes
of interestinto a plant chromosome.

tI _ . .Testinofor
ptants**
I transgeniJ
+
T0 seeds
The term director vectorless transferof DNA is
+
I
used when the foreign DNA is directly introduced
Transformedplants
into the plant genome. Direct DNA transfer screenedin the
methodsrely on the deliveryof naked DNA into next generation
t he olant c e l l s . T h i s i s i n c o n tra s t to the
+
I
Agrobacteriumor vector-mediatedDNA transfer T1 seeds
which may be regardedas indirectmethods.
Majorityof the directDNA transfermethodsare Fig. 49.9 : An overview of the protocol for the
simpleand effective.And in fact,severaltransgenic
plantshave been developedby this approach.

Limitations of ditect DNA transfer

The majordisadvantage of directgenetransferis
that the frequency of "transgenerearrangementsis The salientfeaturesof the differentmethodsfor
high.This resultsin highertransgene copy number, direct DNA transferire given in Table49,1 (See
and high frequenciesof gene silencing (described p. 573). The commonly used methodsare briefly
on p. 593) . describedin the followingpages.

Types of direct DNA transfer PHYSICAL GENE TRANSFER METHODS

The direct DNA transfercan be brcadly divided An overview of the general scheme for the
into three categories. productionof transgenicplantsby employing physical
e details
1. physicat gene transrermethods-electro-i:::::H'"1*i]:l:,tl::".d]:':t:"11,:J:j"
o l t n e o l n e r e n tt e c n n l q u e sa r e o e s c r l o e c '
p ora tion . p artic le bom bar dm ent , m ic r oiniec ti o n ,
liposome fusion, silicon carbide fibres.
ELECT*O*ORATION
2. Chemical gene transfer methods-Poly-
Electroporation basically involves the use of
ethyleneglycol(PEG)-mediated,
diethylaminoethyl
high field strengthelectrical impulsesto reversibly
(DEAE) dextran-mediated,calcium phosphate
permeabilize the cell membranesfor the uptake of
precipitation.
DNA. This techniquecan be usedfor the delivery
3. DNA imbibitionby cells/tissues/organs. of DNA into intact plant cells and protoplasts.
 544 B IOTECHNO LO CY

Rupturedisc

Maclocarrier

Vector DNA-coated
particles(microcarriers)
Stoppingplate

Plant material

Microcarriersinserted Rupturedisc bursts, Macrocarrierstopped Microcarriershit

into the apparatus marcocarriermoves by the stoppingplate the plant material
oown

Fig. 49.10 : A diagrammatic representation of particle bombardment (biolistics) system

for gene transfer in plants.

The plant m at er ial is i n c u b a t e d i n a b u f f e r r E f f i c i e n c y o f e l e c t r o p o r a t i o n i s h i g h l y va r i a b l e

solution containing the desired foreign/targetDNA, d e p e n d i n g o n t h e p l a n t m a t e r i a l a n d th e
and s ubjec t ed t o high v ol t a g e e l e c t r i c a l i m p u l s e s . treatment conditions.
This resultsin the formation of pores in the plasma . Regeneration of plants is not very easv,
membrane thrbugh which DNA enters and gets
particularly when protoplastsare used.
integrated into the host cell genome. In the earlv
years, only protoplastswere used for gene transfer
PABT'CLE BOMBARDMENT
by electroporation.Now a days, intact cells, callus (BroLrsTtcs)
cultures anci immature embryos can be used with
suitable pre- and post-electroporationtreatments. Particle (or microprojectile) bombardment is
ElectrQporationhas been successfullyused for the the most effective method for gene transfer,and
production of transgenicplants of many cerealse.g. creation of transgenicplants. This method is
rice, wheat, maize. versatiledue to the fact that it can be successfully
usedfor the D N A transferi n mamm alian cellsand
Advantages of electroporation mtcroor8anrsms.

r This t ec hnique is s im pl e , c o n v e n i e n t a n d r a p i d , The microprojectilebombardmentmethodwas

besides being cost-effective.
. The transformed cells are at the
physiological state after electroporation.
. Efficiency of transformationcan be improved by
opt im is ing t he elec t r i c a l f i e l d s t r e n g t h , a n d
addit ion of s oer m idine.
Limitations of electroporation
. Under nor m al c ondit io n s , t h e a m o u n t o f D N A
deliv er ed int o plant c ell s i s v e r y l o w .
i ni ti al l ynamedas bi ol i sti csby i ts i n vent orSanf or d
(1988).Biolistics is a combinationof biologicaland
same bal/isfics. Thereare othernamesfor this technique-
particle gun, gene gun, bioblaster.
A diagrammatic representationof micro-
projectilebombardmentsystemfor the transferof
genesin plantsis depictedin Fig.49.10,and briefly
describedbelow.
Microcarriers(microprojectiles),
the tungstenor
gold particlescoated with DNA, are carried by
macrocarriers(macroprojectiles).These macro-
Chapt er49 : CE N E T ICE N C IN E ER INO
CF PL A N TS -ME TH OD OLOC Y 585

carriersare insertedinto the apparatus and pushed

downwardby rupturingthe disc.The stoppingplate Tlort 49.2 A selectedlist of transgenicplants
does not permit the movementof macrocarrier (along with cell sources)developedby
w hilet he m ic r o c a rri e (w
rs i thD N A)a rep ro p e l l e dat nicroproieetile bombardment
a high s peedint oth e a n tma te ri a lH
p l . e reth e D N A
Plant Cell source(s)
s egm entars e released w h i c h e n te rth e p l a n t c el l s
and integratewith the genome. Rice Embryonic
callus,
immature
zygotic
emDry0s
Plant material used in bombardment Wheat lmmature
zygoticembryos
Two typesof planttissueare commonlyusedfor Sorghum lmmature
zygoticembryos
particlebombardment. Corn Embryonic
cellsuspension,
immature
zygoticembryos
1. Primary explantswhich can be subjectedto
Barley immature
Cellsuspension, zygotic
bom bar dm entt h a t a re s u b s e q u e n tliyn d u c e d to
embryos
becomeembryogenicand regenerate.
Banana Ernbryonic
cellsuspension
2. Proliferating embryonic fissuesthat can be Sweetpotato Callus
cells
bombarded in crrltures and then allowed to
proliferateand regenerate. Cotton Zygotic
embryos
Grape Embryonic
callus
In order to protect plant tissuesfrom being
Peas Zygotic
embryos
damagedby bombardment, culturesare maintained
on high osmoticummedia or subjectedto linritecl Peanul Embryonic
callus
plas m oly s is . Tobacco Pollen
Alfalfa trmh^,^^i^
LI tur yuilru gailuJ
^^il,,^

Transgene integration in bombardment

It is believed(basedon the genetransferin rice A selected list of the transgenic plants
by biolistics)that the gene transfer in particle (developedby biolistics)along with the sourcesof
bombardmentis a two stageprocess. the plant materialsused is given in Table49.2.
1. ln the preintegrationphase,the vector DNA
molec ulesar e s p l i c e d to g e th e r.T h i s re s u l tsi n Factors affecting bombardment
fr agm entcsar r y i n gmu l ti p l eg e n ec o p i e s . Severalattemptsare made to studythe various
2. lntegrative phase is characterizedby the factors, and optimize the system of particle
insertionof genecopiesinto the hostplantgenome. bombardment for its mostefficientuse.Someof the
importantparameters are described.
The integrative phasefacilitates furthertransgene
integrationwhich may occur at the samepoint or Nature of microparticles: Inertmetalssuch as
tungsten, gold and platinum are used as
a point close to it. The net result is that particle
microparticles to carry DNA. Theseparticleswith
bombardmentis frequentlyassociatedwith high
relatively higher masswill have a better chanceto
c opy num ber a t a s i n g l e l o c u s . T h i s ty p e of
when bombarded and penetrate the
singlelocus may be beneficialfor regeneration of move fast
tissues.
olant s
Natureof tissues/cells : The targetcellsthat are
The success of bombardment capabl eof undergoi ngdi vi si on are sui tabl efor
transformati on. S omemoredetai l son the choi ceof
T he par t ic leb o m b a rd m e nte
t c h n i q u ew a s fi r st
pl ant materi alused i n bombardment are al ready
rtroducedin 1987. lt has been successfully used
grven.
'or the transformation of many cereals e.g. rice,
.rheat, maize. In fact, the first commercial Amount of DNA : The transformation may be
genetically modified (GM) crops such as maize low when too little DNA is used. On the other
:ontaining Bt-toxin gene were developedby this hand, too much D N A may resul t i s hi gh
: ppr oac h. copy number and rearrangement of transgenes.
586 B IO TECHNO LO CY

Holdingpipette

Therefore,the quantity ol DNA used should be one identifiedfrom intactcells,protoplasts, callus,

balanced.Recently,someworkershavestartedusing embryos,meristems etc. Microinjectionis usedfor
th e c h e m i c a l a mi n o si l oxane to coat the the transferof cellular organellesand for the
microparticles with low quantities of chromosomes.
of DNA adequate mani oul ati on
enoughto achievehighefficiencyof transformation. The techniqueof microinjectioninvolvesthe
Environmentalparameters : Many environ- transfer of the gene through a micropipette
mental variablesare known to influenceparticle (0.5-10.0pm ti p) i nto the cytopl asm / nucleus
of a
bombardment. These factors (temperature, plant cell or protoplast.While the genetransferis
humidity, photoperiod etc.) influence the done, the reci pi entcel l s are kept im m obilizedin
physiologyof the plant material,and consequently agaroseembedding,and held by a suctionholding
the gene transfer.lt is also observedthat some pipette(Fig.a9Jl).
explants,after bombardmentmay requirespecial As the processof microinjectionis complete,
regimesof light, humidity,temperature etc.
the transformedcell is cultured and grown to
The technologyof particle bombardmenthas developinto a transgenicplant. In fact,transgenic
been improvedin recent years,padicularlywith tobacco and Brassicanapus have been developed
regard to the use of equipment.A commercially by thi s approach.
produ ced parti cle bombardment apparatus namely The major limitationsof microinjectionare that
PDS-|000/HC'iswidely usedthesedays. it is slow, expensive,and has to be performedby
trai nedand ski l l edpersonnel .
Advantages of particle bombardment
. Gene transfer can be efficientlv done in L'POSOME.MED'ATED
organizedtissues. TBANSFOBMATION
r Differentspeciesof plantscan be usedto develop Liposomesare artificiallycreatedlipid vesicles
transgenicplants. contai ni nga phosphol i pi dmemb r ane.They ar e
successful l yused i n mammal i ancells f or t he
Limitations of particle bombardment deliveryof proteins,drugsetc. Liposomes carrying
. The majorcomplicationis the productionof high genescan be employedto fusewith protoplasts and
transgenecopy number. This may result in transferthe genes.The efficiencyof transformation
instabilityof transgeneexpressiondue to gene increaseswhen the process is carried out in
silencing. gl ycol ( PEC) .
conj uncti onw i th pol yethyl ene
o The targettissuemay often get damageddue tc
Lioosome-mediatedtransformation involves
lack of controlof bombardmentvelocity.
adhesionof liposomesto the protoplast surface, its
o Sometimes, chimericplantsmay be
undesirable fusion at the site of attachment and release of
regenerated, plasmids insidethe cell (Fig. 49.12).

M'CROTNJECT'ON Advantages of liposome fusion

Microinjection is a direct physical method r Being present in an encapsulatedform of
involving the mechanical insertion of the desirable liposomes, DNA is protectedfrom environmental
DNA into a target cell. The targetcell may be the i nsul tsand damage.
 49 : CE N E T ICEN C IN EE R INO
ChA P I CT CF PL A NTS
- ME TH OD OLOC Y 547

Liposomes
I o@@ o@o @ @c.r
9.99ie-
(?(U (9 ptalmids
@@@ @@o @@ 6
Plasmids

----+

Nucleus

Fig. 49.12 : A diagrammatic tepresentation of fusion of plasmid-filled liposomes with ptotoplasts

. DNA is stableand can be storedfor sometimein . The embryonicplant cellsare hard and compact
liposomesprior to transfer. and are resistantto SCFpenetration.
. A pplic ablet o a w i d e ra n g eo f p l a n t c e l l s In recentyears/some improvements have been
. Thereis good reproducibilityin the technique. made in SCF-mediated transformation.This has
helpedin the transformation of rice, wheat,maize
Limitations of liposome fusion and barl eyby usi ngthi s techni que.
T he m ajor pro b l e m w i th l i p o s o m e -m e d iated
transformation is the difficultyassociated with the C H E MIC A L GE N E TR A N S FE R ME TH OD S
regeneration of plantsfrom transformed protoplasts.
PO LYETHYLEN E G LYCO L.MED'AT ED
TNANSFORMATION
S'L'CON CABB'DE F'BBE.MED'ATED
TBATVSFOBMAT'ON Polyethyleneglycol (PEG),in the presenceof
divalent cations (using Ca2+) , destabilizesthe
T he s ilic on c a rb i d e fi b re s (S C D a re a b out
plasma membrane of protoplasts and renders it
0. 3- 0. 6pm in dia m e te a r n d 1 0 -10 0 p m i n l e n gth.
permeable to naked DNA. In this way, the DNA
Thesefibresarecapableof penetrating the cell wall
entersnucleusof the protoplasts and getsintegrated
and plas m am em b ra n ea, n d th u sc a n d e l i v e rD NA
w i th the genome.
i nt o t he c ells .
T he DNA c o a te d s i l i c o n c a rb i d e fi b re s a re The procedure i nvol ves the i sol ati on of
vortexedwith plant material(suspension culture, protopl astsand thei r suspensi on,addi ti on of
) . ingth e mi x i n g ,D N A a d h e ri n gto th e
c allus es Dur plasmid DNA, followedby a slow additionof 4O'/.
fibresentersthe cellsand getsstablyintegrated with P.EC-4000 (w/v)dissolvedin mannitoland calcium
the hostgenome.The siliconcarbidefibreswith the ni trate sol uti on.A s thi s mi xture i s i ncubated,
trade name Whiskersare availablein the marker. protoplasts get transformed.

Advantages of SGF-mediated Advantages of PEc.mediated

transformation transformation
. Direct deliveryof DNA into intactwalled cells. r A large number of protoplasts can be
T his av oidst he p ro to p l a sits o l a ti o n . simultaneouslytransformed.
. Procedureis simpleand does not involvecostly . Thi s techni quecan be successful lused
y for a
equipm ent . wide rangeof plant species.

Disadvantages of SGF-mediated Limitations, of PEG-mediated

transformation transformation
. S ilic on c ar bid e fi b re s a re c a n c i n o g e n i ca n d . The DNA is susceptiblefor degradationand
thereforehave to be carefullyhandled. rearranqement.
588
. Random int egr at ionof f or e i g n D N A i n t o g e n o m e
m ay r es ult in undes ir ablet r a i t s .
o Regeneration of plants from transformed
protoplasts is a difficult task
DEAE DEXTRAN-MEB'ATET' TRAAISFIiB
The des ir able DNA c an b e c o m o l e x e d w i t h a
high m olec ular weight poly m e r d i e t h y l a m i n o e t h y l
(DEAE) dextran and transferred. The major
lim it at ion of t his appr oac h is t h a t i t d o e s n o t y i e l d
stable transformants.
CALC'UM PHOSPHATE CO"
P R EC' P I T AT TO N. M E D TAFES TBAffSFEF
l- he DNA is allowed t o m i x w i t h c a l c i u m
c hlor ide s olut ion and is ot on i c p h o s p h a t e b u f f e r t o
form DNA-calcium phosphate precipitate. When
t he ac t iv ely div iding c ells in c u l t u r e a r e e x p o s e dt o
t his pr ec ipit at e f or s ev er al h o u r s , t h e c e l l s g e t
transformed. The success of this method is
dependent on t he high c onc e n t r a t i o no f D N A a n d
the protection of the complex precipitate.Addition
of dimethyl sulfoxide (DMSO) increases the
efficiency of transformation.
DhlA lMBlBl'rloru B'ir GE!-rs/TISSUES Neomycin
Someworkershaveseriously
triedto transform lnpt tI genef
c ells by inc ubat ing c ell s u s p e n s i o n s , t i s s u e s ,
embryos and even seeds with DNA. The belief is
t hat t he DNA get s im bibe d , a n d t h e c e l l s g e t
t r ans f or m ed. DNA im bibit io n a p p r o a c h h a s m e t
wit h lit t le or no s uc c es s .
In general, genetic transformation of plants is
a low-frequency event. Some methods for
selecting the transformd plant materials (cellsl
tissues) have been devised by using a set of
genes referred to as marker genes. These marker
genes are introduced into the plant material along
with the target gene The marker genes are of two
types.
I Selectable marker senes
Il. Reporter genes
B IOTECHNO LO CY
SELECTABLE MARKER GENES
T h e s e l e c t a b l e m a r k e r g e n e s a r e u su a l l y a n
integral part of plant transformation system. Ihey
are presentin the vector along with the target gene.
ln a maiority of cases,the selection is based on the
survival of the transformed cells when grown on a
m e d i u m c o n t a i n i n g a t o x i c s u b s t a n ce ( a n ti b i o ti c,
h e r b i c i d e , a n t i m e t a b o l i t e ) .T h i s i s d u e to th e fa ct
that the selectable marker gene confers resistance
t o t o x i c i t y i n t h e t r a n s f o r m e dc e l l s , w h i l e th e n o n -
t r a n s f o r m e dc e l l s g e t k i l l e d .
A large number of selectablemarker genes are
availableand they are groupedinto three categories-
antibiotic resistance genes, antimetabolite marker
genes,and herbicide resistancegenes(Table49.3).
ANr TB TOT IC FES'STA'VCE GEAIES
ln man1,plant transformationsystems,antibiotic
resistancegenes (particularlyof E. coli) are used as
s e l e c t a b l e m a r k e r s . D e s p i t e t h e p la n ts b e i n g
eukaryotic in nature, antibiotics can effectively
i n h i b i t t h e p r o t e i n b i o s y n t h e s i s i n th e ce l l u l a r
o r g a n e l l e s ,p a r t i c u l a r l y i n c h l o r o p l a s t s .So m e o f th e
antibiotic resistance selectable marker qenes are
briefly described.
phosphotransferase ll
The mosf widely used selectable marker is npt
// gene encoding the enzyme neomycin phospho-
transferasell (NPT ll). This marker gene confers
r e s i s t a n c e t o t h e a n t i b i o t i c k a n a myci n . Th e
transformantsand the plants derived from them can
b e c h e c k e d b y a p p l y i n g k a n a m y c i n so l u ti o n a n d
the resistantprogeny can be selected.
Hygromycin phosphotransferase
(hpt genel
T h e a n t i b i o t i c h y g r o m y c i n i s m o r e to xi c th a n
neomycin and therefore can kill non-transfornted
p l a n t c e l l s m u c h f a s t e r . H y g r o m y c in p h o sp h o -
transferase(hpf) gene thus provides resistanceto
transformed cells.
Aminoglycoside adenyltransferase
laadA gene|
Aminoglycoside3'-adenyltransferase (aadA)gene
confers resistanceto transformedplant cells against
t h e a n t i b i o t i c s s t r e p t o m y c i na n d s p e c t i o n o m yci n .
 Chaot er49 : C EN ET ICE N C IN E ER INOF
C P L AN TS -ME TH OD OLOC Y 589

Tmrs 49.3 A selected list of selectable marker genes used for gene transfer In plants,
' their source and substrates used for their selection

Selectable marker gene Abbreviation Source of scne Substrate(s)used

(encoded enzyme) for sclection
Antibiotic resistance
Neomycin phosphotransferase
ll nptll E coli geneticin
Kanamycin, (G418)
Neomycin phosphotransferase
lll nptlll Streptococcusfaecalis geneticin
Kanamycin, (G418)
Hygromycin phosphotransferase hp|hyg E coli Hygromycin
Bleomycin
resistance ble E. coli Bleomycin
Aminoglycoside erase
adenyltransf aadA Shtgellaflexneri Streptomycin,
spectinomycin
Antimetabolitemarkers
Dihydrofolate
reductase dhfr Mouse Methotrexate
Dihydropteroate
synthase dhps/sul E. coli Sulfonamides

Herbicideresistance
Phosphinothricin
acelyltransferaseuauPqr nyces hygroscopicusi Glufosinate,
Strepto L-phosphinothricin,
S viridochronogenes Bialophos
Enolpyruvyl
shikimatephosphate epsps/aroA Agrobacteriunspl
synthase Petuniahybrida Glyphosate
Acetolactase
synthase qle
Arabidopsi
s sp/maize/tobacco
Sulfonylureas
Glyphosate
oxidoreductase g0x Achromobacter LBAA Glyphosate
Bromoxynil
nitrilase bxn pneumoniae
Klebsiella Bromoxynil

Others
B-Glucuronidase gus/uidA E. coli glucuronide
Cytokinin
Xylose
isomerase xylA Thermoanaerobcterium Xylose
urogenes
thermosulf
Mannose isomerase pmi/manA
6-phosphate E. coli lvlannose
Betaine dehydrogenasebadh
aldehyde Spinach Betaine
aldehyde

ANTIMETABOLITE MABKER GE'VES Phosphinothricin acetytransferase

(pat/bar genef
Dihydrofolate reductase ldhfr gene|
B i a l o p h o s ,p h o s p h i n o t h r i c i na n d g l u f o s i n a t ea r e
The enzyme dihydrofolate reductase,produced commonly used herbicides.The pat/bar genes code
by dhfr gene is inhibited by the antinretabolite {or phosphinothricin acetyltransferase which
methotrexate. A mutant dhfr gene in mouse that converts these herbicides into acetylatedforms that
co de s for th is e nz y m e whic h has a low af f init y t o are non-herbicidal. Thus, pat/bar genes confer
-lhis
methotrexate has been identified. dhfr gene resistanceto the transformed plants.
fused with CaMV promoter results in a
methotrexate resistant marker which can be used Enolpyruvylshikirnate phosphate
for the selection of transformed plants. synthase (epspslaroA genesf

The herbicide glyphosateinhibits photosynthesis.

HERBIC|DE RESISTANCE MARKEBS
It blocks the activity of enolpyruvylshikimate
Cenes that confer resistanceto herbicides are tn phosphate (EPSP)synthase,a key enzyme involved
use as markersfor the selectionof transgenicplants. i n t h e b i o s y n t h e s i so f p h e n y l a l a n i n e ,t y r o s i n e a n d
590 B IOTECHNO LO CY

tryptophan.Mutant strains of Agrobacteriumand Avoiding selectable marker genes

Petuniahybridathat are resistantto glyphosatehave
Theoretically, it is possible to totally avoid
been identified. The genes epsps/aroA confer
marker genes and introduce only the transgeneof
resistanceto transgenicplants which can be
interest. The transformed palnts can then be
selected.
screened by an advanced technique like
polymerasechain reaction and the desirable plants
Bromoxynil nitrilase (Dxn genef
selected. This approach is not practicable due to
The herbicidebromoxynilinhibitsphotosynthesiscost factor.
(photosystem ll). Bromoxynilnitrilaseenzymecoded
by the genebxn inactivates this herbicide.The gene Gotransformation with two DNAs
bxn can be successfully usedas a selectable marker
for the selectionof transformed plants. The transgenicplants can be produced by
empl oyi ngtw o separate D N A s-one car r yingt he
PRODUCTION OF MARKER.FREE desiredtarget gene and the otherthe markergene.
TRANSGENIC PLANTS The transformed plantscontain both the genes,but
at different sites on the chromosomal DNA.
There is a growing concern among the public Traditionalbreedingtechniques(a few rounds)can
regarding the use of antibiotic ar herbicide be used to get rid of the transgenicplants with
resistance genes as selectable markers of plant selectablemarkers.
transformation
. The productsof somemarkergenesmay be toxic Removal of selectable markers
o r a l l e rg i c .
It is possibleto selectively
removethe selectable
. The antibioticresistance marker genes from the plant genome. For this
might be transferred
to
p a th o g e n i mi
c c ro o rg a n i sms purpose, site-specific recombinase sysferns are
i n the soi l .
utilized. Severalrecombinasesystemsare in fact
. There is a possibilityof creationof superweeds
availablewhich can be usedto selectivelyexcise
that are resistantto normallyused herbicides.
the markergenesfrom the plant genome.
. A transgenicplant with selectablemarkergenes
cannot be transformedagain by usingthe same Cloning of selectable markers between
selectablemarkers. transposable elements
In light of the apprehensions listed above,the A selectablemarkergenecan be clonedbetween
public is concernedabout the safetyof transgenic planttransposable elements(Ds elements) and then
technology,particularlyrelatedto the selectable inserted.The selectablemarkeris plankedby the
marker genes (antibiotic/herbicideresistance seouencesthat increase the intrachromosomat
genes). There are fears about the safety of recombi nati on. Thi s resul tsi n the excisionof t he
consumptionof foodstuffsderivedfrom genetically markergene.
engineeredplants.This is despitethe fact that so
far none of the marker geneshave been shown to
REPORTER GENES
adversely affect human, animal or environmental
nfety. A reporter gene may be regardedas the test
gene whose expression can be quantified. The
CLEAN GENE TECHNOLOGY plant transformationcan be assessedby the
expressionof reporter genes (also called as
The process of developing transgenic plants or scoreable genes).In general,an assay
screenable
without the presenceof selectablemarker genesor
for the reportergene is carriedout by estimating
by useof moreacceptable markergenesis regarded
the quantityof the proteinit producesor the final
a sc l e a ng e n ete c h n o l o g yA.n d thi sw i l l resul ti n the oroductsformed.
productionof many marker-free transgenicplants
that will be readilyacceptableby the public.Some A selectedlist of the reportergenesalong with
of the approachesfor clean gene technologyare the detectionassaysis given in Table49.4, spmeof
8 rv e n . the imoortantones are discussedbelow.
 CF PL A N TS -ME TH OD OLOC Y
Chaot er49 : G E N ET ICE N GIN EE R INO 59r

,genes used for gene transfer in

their sources, and detection assays

Reportergene Abbreviation Source of gene Detection assay

(enz,yme/proteinencoded)
Octopine
synthase 0cs um tumefaciens
Agrobacteri Electrophoresis,
chromatography
Nopaline
synthase n0s m tumefaciens
Agrobacteriu Flonlrnnhnrccic

chromatography
B-Glucuronidase gus/uidA E. coli Fluorometric
orhistochemical
or colorimetric
Greenlluorescentprotein stp victoria(jellytish)
Aequorea Fluorescence
(bacterial)
Luciterase luxNIuxB Vibrioharveyi Bioluminescence
(firefly)
Luciferase luc Photonus pyralis Bioluminescence
acetyltransferase v a t
Chloramphenicol E. coli Autoradiography

Op-ine synthase (ocs, nos genesl i n many i nstances, GFP has repl acedC U S si nce
assays of GFP are easier and non-destructive.Thus,
The common opinespresentin T-DNA of Ti or
screening of even the primarytransplants can be
Ri plasmidsof Agrobacteriumare octopine and
done by C FP w hi ch i s not possi bl ew i th other
nopaline,respectivelyproducedby the synthase
genesocs and nos. The transformedstatusof the reporterBenes.
plant cells can be easilydetectedby the presence C ene for C FP has been i sol atedfrom j el l y fi sh
of these opines. Opines can be separated by Aequoreavictoriawhich is a luminescent organism.
electrophoresisand identified. Alternately, the The original gfp gene has been significantly
enzymeactivitiesresponsible for the productionof modifiedto makeit more usefulas a reportergene.
opinescan also be assayed. C FP emi ts fl uorescence w hi ch can be detected
undera fl uorescent mi croscooe.
B.Glucuronidase lgusluidA gene)
genes|
p-Clucuronidase producinggene (gus/uidA)ts Bact'erial luciferase lluxNIuxB
the most commonly usedreporter genein assessing The bacterialluciferasegenes(/uxA and luxB\
plant transformation for the following reasons. have originatedfrom Vibrio harveyi.They can be
. p-Clucuronidase assaysare very sensitive. detectedin someplanttransformation vectors.The
detection assayof the enzyme is based on the
o Quantitative estimation of the enzyme principleof bioluminescence. Bacterialluciferase
can be done by fluorometric method (using catalyses the oxidation of long-chain fatty
substrate4-methylu mbelIiferryl B-D-glucuronide al dehydes of l i ghtw hi ch
thatresul tsi n the eri i i ssi on
which is hydrolysedto 4-methylumbelliferone). can be measured.
. Qualitativedataon the enzymecan be obtained
l e a n s (e n z y mel o c a l i z a ti on Fireffy luciferase Uuc genel
by his t oc he m i c am
can be detectedby chromogenicsubstance such The enzymefirefly luciferase,encodedby the
as substrate X-gluc). geneluc, catalyses the oxidationof D-luciferin(ATP
. No needto extractand identifvDNA. dependent)which resultsin the emissionof light
that can be detectedby sensitiveluminometers.
Green fluorescent {gfp genel
protein
The firefly luciferasegene, however, is not
Creen fluorescentprotein(CFP),coded by gfp widely usedas a markergenesincethe assayof the
qene,is beingwidely usedin recentyears.ln fact, enzymeis rathercumbersome.
592 B IOTECHNO LO C\

Ghloramphenicol acetyl transferase Promoters for monocots : Since 30S promoter ls

( c at genef
The c af gene pr oduc ing c h l o r a m p h e n i c o la c e t y l
transferase(CAT) is a widely used reporter gene in
m am m alian c ells . Due t o th e a v a i l a b i l i t y o f C U S
and CFP reporter systemsfor plant transformants,
CAT is not commonly used. However/ some workers
continue to use CAT by a sensitive radioactive
assay, for the detection of the reporter gene cat.
not very efficient for monocots, alternatesare used
Ubiquitin I promoter or the rice actin promoter
a r e f r e q u e n t l y u s e d f o r h i g h e xp r e ssi o n o t
transgenesIn monocots.
promoters
Inducible
I n r e c e n t y e a r s , c e r t a i n i n d u c i b l e p r o m o te r
systems for transgene expression have been
developed These promoters differ from 30S
oromoter lvhich is constitutive in nature. There are
m a i n l y t h r e e i n d u c i b l e e x p r e s s i o r rs y ste m s:

1 . Non-plant-derived systems: These inducible

The ultimate objectiveof gene transferis its promoters are independent of the normal plant
correctexpression to providethe desiredcharacter/ processes. A s p e c i f i c e x o g e n o us ch e m i ca l
trait. The appropriate expressionof genesis made w i l l i n d u c e t h e i r e x p r e s s i o n - e . g . te tr a cycl i n e ,
possible by the presence of promoters and ethanol, steroid, copper. Non-plant-derived
terminators. The DNA sequence upstream the inducible promoters are useful, but not
c o d i n gre g i o ni s th e p ro moterw hi l e the termi nator economically viable.
i s th e s e q u e n c e a t th e 3 ' termi nus.The promoters
2. Plant-derived systems based on responseto
are resoonsible for the commencement of
environmental signals : These systems are not
transcriptionwhereasthe terminatorsensurethe
i n d e p e n d e n t , s i n c e t h e y a r e p l a n t - d e r i ve d a n d
ceasationof transcriptionat the correctposition.
actually form a part of normal plant processes
Promoterspossesscertain inherentcharacters Plant-derived systems respond to a variety of
such as promoterstrength,tissuespecificityand e n v i r o n m e n t a l s i g n a l s e . g . - w o u n d , h ea t sh o ck.
d e v e l o p m e n tare
l g u l a ti o nw hi ch determi nethe
3. Plant-derived systems based on
efficiencyof promoterfunctionin geneexpression.
developmental control of gene expression: Certatn
Ag ro b a cte ri u m-detiv ed promoters genes that express at a particular stage of plant
and terminators development have been identified. The promoters
of some of these developmental genes are useful
The genescoding Ior napalinesynthase(nos)in for transgeneexpression.e.g
Ti plasmidof Agrobacterium arefrequentlyusedas
promotersand terminatorsin plant transformation . Senescence-specificgene expression
vectors.Originallvderivedfrom bacteria,the genes . A b s c i s i c a c i d i n d u c i b l e
B e n e e x p r e ssi o n
coding for opine synthesisare well adaptedto
functionin plants.In fact,nos promoteris regarded . A u x i n i n d u c i b l e g e n e e x p r e s s i o n
as constitutiveby many plant biotechnologists. Although plant-derivedsystems(2 and 3) are not
i n d e p e n d e n ta s n o n - p l a n t d e r i v e d o n e ( 1) , th e y a r e
The 3OS promoter of CaMV s t i l l u s e f u l i n p l a n t b i o t e c h n o l o gy si n ce n o
The cauliflow'er mosaic virus (CaMV) 30S exogenousinducer is required.
promoter is the mosf widely used as a promoter in
plants. This promoter, (a 30S RNA gene) is Tissue-specific promoters
e x p re s s eidn a l m o s ta l l th e t i ssues
of the transgenrc Effortsare on in recent yearsto isolatepromoters
plants. The driving strengthof 30S promoter is t h a t
c a n d r i v e g e n e e x p r e s s i o ni n a t issu e sp e ci fi c
mu c h h i g h e ri n d i c o tsc o mparedto monocots. m a n n e r . T h e a d v a n t a g e w i t h t issu e - sp e ci fi c
The 30S promoter is preferred for the promoters is that the expression (which may
appropriateexpressionof sclectablemarkergenes produce a harmful compound) is confined to
and reportergenes.ln recentyears,the efficiency selected tissuesthat are not consumed by humans
of 30S promoter has been further increasedby or animals. In fact, some tissue-specificpromoters
s u i ta b l em o d i fi c a ti o nisn th e enhancerresi on. h a v e b e e n i s o l a t e d ,a n d a r e i n u s e .
 C hapt er49 : CE N ET ICEN C IN EE R INO - ME TH OD OLOC Y
CF PL A N TS
The geneticallytransformedplant cells are grown
in vitro to finally regenerateto plants. At the initial
stagesof plant cell growth, some evidence for the
successof plant transformationcan be obtainer.le p
resistanceto antibiotics, herbicides etc.
The integration of transgenes with the host
plant genome can be confirmed by some molecular
techniques-Southern hybridization, polymerase
ch ain re action . This is us ually c ar r ied out b y
ana lysing the se eds at T1 gener at ion.
What is actually required for the success of
plant transformation is the efficient and stable
expressionof transgene,and not just its presencein
plan t g en ome . lt is jus t us eles st o hav e a des ir e d
ge ne witho ut ap pr opr iat e ex pr es s ion.
SCAFFOLD ATTACHM ENT REG I O NS
AND GENE STABI LI TY
Scaffold attachment regions (SARs;also known
as matrix attachment/issociated regions, MARs)
are the regions of DNA isolated based on their
ability to bind to the nuclear scaffold (protein
depleted chromosome forms a central scaffold
surrounded by DNA). SARs are known to stabilize
ge ne expre ssion in t r ans genic plant s . Dur ing t h e
course of transformation, SARs are ligated to the
flanking regions of foreign gene lt is observed that
the transformation efficiency by particle
bombardment is increased by use of SARs
fhe presence of SNRs along with transgene
stabilizes or normalizes gene expression. e.g. SARs
from yeast and tobacco have helped to stabilize
gene expression in plants. The degree of
stabilizationof genes by SARs is dependent on the
affinity of SARs towards the nuclear matrix of the
target plant cells. SARs are particularly useful for
sta bilizing the gene ex pr es s ion when t he c op y
numb er of DNA int r oduc ed is high. I n t he abs en c e
o f SARs,hig h DN A c opy num ber would r es ult r n a n
in sta ble a nd hig hly v ar iable gene ex pr es s ion.
I NTRONS AND G ENE EXPRESSI O N
The presence of introns between the promoter expression which may result in co-suppresion or
and cod ing reg ions of a gene will s ignif ic ant l y PTCS, and thus a lower level of gene expression.
influ en cethe g en e ex pr es s ion.The gene ex pr es s l o n For this reason, some workers prefer to use weak
is e nh an ce d w hen int r ons ar e us ed i n
Biotechnology [38]
593
monocotyledonous plant species. For instance,
introduction of introns between cauliflower mosarc
virus 35S promoter and B-glucuronidase
s i g n i f i c a n t l yi m p r o v e d g e n e e x p r e s s i o ni n m a i z e . I t
is believed that introns may stabilize mRNAs and
increase the protein biosynthesis.
There are conflicting reports on the affect of
i n t r o n s o n t h e g e n e e x p r e s s i o ni n d i c o t y l e d o n o u s
plants Some workers have reported stimulation
while others contradict this claim.
It is now accepted that the intron-containing
constructs will enhance gene expression in
transgenic monocots, although the picture is less
c l e a r a s r e g a r d sd i c o t s .
GENE SILENCING
There are in fact many instancesof gene transfer
that are not properly expressed.Ihe instability of
gene expression(or inadequategene expression)in
transgenic plants is referred to as gene silencing.
The mechanism of gene silencing is not well
understood although it is predominantly due to the
phenomenon of homology dependent gene
silencing (HDGg.
Two types of gene silencing are known in plant
biotechnology-transcriptional gene silencing and
post{ranscriptional gene silencing. The two differ
at the level of gene expression where silencing
occu rs.
Transcriptional gene silencing (TGSI
When the transgenesshare homology in their
promoter regions, TCS occurs. lt is due to altered
methylation patterns and altered chromatin
c o n f o r m a t i o n .C e n e s i l e n c i n g o c c u r s b y r e p r e s s t o n
of transcription.
Post-transcriptional gene
silencing (PTGSI
Sometimes, the expression of a homologous
transgene may inhibit the expression of the
transgene and endogenous gene. This primarily
occurs by decreasingthe stability of RNA, and thus
a reduced expression.This is often observed when
strong promoters are used to drive the transgene
promoters rather than strong promoters.
594 B IOTECHNO LO CY

' Pbst-transcriptionalgene silencing is due to the and exciting field in modern biotechnology as it
production of double-strandedRNA either in the offers the following advantages.
RNAis
T h e doubl e-stranded
n u c l e u so r c v to p l a s m. 1 . Chloroplasts are maternally inherited, hence
formedwhen antisense RNA is produceddue to the
there is no danger of gene transferthrough pollen
activityof RNA dependentRNA polymerase. to related weeds. This is because pollen does not
contain transgenes.
Strategies to avoid gene silencing
2 . M u l t i g e n e t r a n s f e r c a n b e co n ve n i e n tl y
T h e c o n tro lo f g e n es i l e nci ngi s not an easyj ob,
o u t i n c h l o r o p l a s t sw h i c h i s r a th e r d i ffi cu l t
s i n c ei t o c c u rsi n a n u n p redi ctablfashie on.S ome c a r r i e d
with nuclear genome.
generalrecommendations, are, however,made to
m i n i m i z eth e i mp a c to f g e nesi l enci ngi n transgeni c 3. Chloroplasts genome is fu n cti o n a l l y
plants comparable to prokaryotic Senome. A single
of of group
o Reductionin the numberof transgenes inserted promoter can control the expression
genes (transgenes). lt is therefore possible to
(i .e .re d u c e dc o p y n u m b er).
i n t r o d u c e d e s i r a b l e m u l t i p l e g e n e s w h i ch ca n
o Avoiding the use of promotersand transgenes
be expressed under the control of a single
with high degreeof homology. promorer.
. M i n i m i z i n g /a v o i d i nthg e use of mul ti pl ecopi es
4. High level of transgeneexpressionis possible
of the samepromoteror terminator.
. h e r e a r e a b o u t . l 0 0 ch l o r o p l a sts
w i t h c h l o r o p l a s t sT
Gene silencing is not observed in chloroplast p e r c e l l , e a c h c o n t a i n i n g a b o u t 1 0 0 co p i e s o f
(plastid) transformation,hence it is preferred in g e n o m e . T h u s , t h e r e i s p o s s i b i l i t yo f 1 0 ,0 0 0 co p i e s
recentyears. o f t r a n s g e n e sp e r c e l l ! T h i s i s a t r e m e nd o u sn u m b e r
of transgenescarried by transformed chloroplasts.
There is a tremendous potential for a very high
Ievel of gene expression and large scale productron
of active oroteins.
The chloroplasts (plastids) and mitochondriaare
5. Chloroplast transformation is nof associated
believedto have evolvedfrom prokaryotes during
with gene silencing which is a major problem with
the courseof evolution.Boththeseorganelles have
nuclear Benome transformation.
th e i r o w n g e n o me ,a l th oughi t i s much si mpl er
when comparedto nucleargenome.Further,many 6. Antibiotic resistancegenes need not be used
of the proteinsthat function in chloroplastsand as selectable markers. Even if used, thev can oe
mi to c h o n d ri a re e n c o d edby nucl eargenesand easily excised.
then transported to the organelle.
7. Toxicity associated with foreign protetn
p r o d u c t i o n i n c h l o r o p l a s t s i s m u ch l e ss w h e n
Ghloroplast genome
compared to nuclear-controlledforeign proteins.
M o s t o f th e h i g h e r pl ants have about- 100
p e r l e a fc e l l . Eachchl oropl ast
c h l o ro p l a s ts contai ns Design of vectors for chloroplast
a p p ro x i m a te l y1 0 0 c o p i e s of chl oropl astD N A transformation
genome.The chloroplastgenome(theplasfome)is
a circular double-stranded DNA molecule (or A diagrammaticrepresentation of two vector
chromosome)located in the stroma.Majority of constructsfor chloroplast is depicted
transformation
c h l o ro p l a sgte n o me a s re i n the si zeof 120-160 kbp in Fig. 49.13.
a n d c o n ta i n a b o u t 1 2 0 - 140 genes.A bout 100
1. A construct for expressionof a single
chloroplastgenesare known to code for proteins. gene : The vectorfor chloroplasttransformation is
The proteinsynthesis in chloroplasts resemblesthat
based on the selectablemarker gene aadA that
of prokaryotes. providesresistance The
to antibioticspectinomycin.
singleforeign(desirable) geneis fusedto regulatory
CHLOROPLAST ENGINEERING sequences (promoterand terminator)which in turn
Ceneticengineering DNA ( Cp
of chloroplastthat leadsto i s fl ankedon ei thersi de by chl oroplast
is an important DNA) (Fig.49.13A).
chloroplast(plastid)transformation
 49 : CE N E T ICE N C IN E ER INOF
C P L AN TS -ME TH OD OLOC Y

(A)
Foreign
gene 1

(B)

Foreigngenes

Fiq.49.13: A diagrammaticrepresentationof vectors for chloroplasttransformation(A) A constructdesigned

for a single loreign gene (B) A construct designed for multiple foreign genes (Cp DNA-Chloroplast DNA;
P-Promoter; aadA-A selectable marker gene that confers resistance to antibiotic spectinomycin; T-Terminator:
badh-A selectable marker gene encoding betaine aldehyde dehydrogenase; rbs-Ribasome binding site).

2. A construct for expressionof multiple DNA sequenceson the vector and those of on tne
genes: In this case,the selectablemarker is the g e n o m e . T h i s i s a s i t e - s p e c i f i ci n t e g r a t i o na n d t h u s
betaine-aldehydedehydrogenase (badh)gene. avoids the frequent problems associated with
randon insertion of foreign genes into nuclear
I t is f lank edb y a p ro m o te ra n d th e m u l ti p l e
transgenesare flanked by a terminator.At both S e n o m e .
e nds c hlor oplasD t N A s e q u e n c eas re p re s e n t.In The regenerated plants derived from the
betweenthe transgenes, theseare ribosome-binding m o d i f i e d p l a s t o m e ( c h l o r o p l a s t g e n o m e ) a r e
sites (one between two transgenes)to ensure regarded as transplastomic plants.
efficienttranslation(Fig.a9J 38).
The future of chloroplast
Introduction of foreign genes into transformation
chloroplast genome
T h e t e c h n o l o g y o f c h l o r o p l a s t t r a n s f o r m a t i o nr s
Most of the methodsused for introducingthe in the developing stages. In fact, it has not become
foreigngenesinto nucleargenomeare not useful as routine as transformationof nuclear genomes of
The mostsuccessful o l an t s .
for chloroplasttransformation.
methodfor insertingforeigngenesinto chloroplasts
Chloroplast engineering,however, holds a great
is particle gun bombardment.
promise in plant biotechnology being an efficient,
After the bombardment, homologous c l e a n a n d e n v i r o n m e n t a l - f r i e n d l ya p p r o a c h f o r t h e
re c om binat iono c c u rs b e tw e e n th e c h l o ro p l ast p r o d u c t i o n o f t r a n s g e n i cp l a n t s .
 genet ic m anipula t i o n sc a r r i e d o u t i n p l a n t s l ti / i, ABIOTIC
J he S T F r IISLS STRESSES
I f or t he pr oduc t ion o f t r a n s g e n i c p l a n t s h a v e
been des c r ibed ( Chapt er4 9 ) . T h e u l t i m a t e g o a l o f Insects-+ Herbicides
t r ans genic s( inv olv ing int r o d u c t i o n ,i n t e g r a t i o n ,a n d Viruses-+
expression of foreign genes) is to improve the Fungi-)
crops, with the desired traits. Some of the
-+
Bacteria
im por t ant ones ar e lis t e d .
-)
Wounds
r ft,esistanceto biotic stresses r.e resistance to
Ozone
dis eas es c aus ed by in s e c t s , v i r u s e s , f u n g i a n d
Intenselight
bac t er ia
. Res is t anc e t o abi o t i c stresses-herbicides,
t em per at ur e ( heat , c h i l l i n g , f r e e z i n g ) , d r o u g h t , Floods
s alinit y , oz one, int ens e l i g h t . Heavymetals
r lm pr ov em ent of c r op y i e l d , a n d q u a l i t y e . g .
storage, longer shelf life of fruits and flowers. Fig. 50.1 : Biotic and abiotic stresses that affect
plant growth, development and yield.
. Tr ans genicplant s wit h i m p r o v e d n u t r i t i o n
. Trans8enic plants as bioreactors for the
A l most al l the stresses,ei ther dir ect ly or
m anuf ac t ur e of c om m e r c i a l p r o d u c t s e . B .
pr ot eins , v ac c ines , and b i o d e g r a d a b l ep l a s t i c s .
indirectly,leadto the productionof reactiveoxygen
species(ROS)that createoxidativestressto plants
Thi s damagesthe cel l ul arconstit ut entofs plant s
Env ir onm ent aE s r t l' e s s e s t o p l a n t s
w i th a reductionin plantvield.
w hi ch i s associ ated
The different tvoes of external stresses that
The major objective of plant biotechnology is to
inf luenc e t he plant gr ow t h a n d d e v e l o p m e n t a r e
develop plants that are resistant to biotic and
depicted in Fig. 50.1, These stressesare grouped
abiotic stresses.
based on their characlers-biotic and abiotic
sfresses.The biotic stressesare caused by insects,
pat hogens ( v ir us es ,f ung i , b a c t e r i a ) , a n d w o u n d s RESISTANCETO BIOTICSTRESSES
The abiot ic s t r es s esar e d u e t o h e r b i c i d e s , w a t e r
def ic ienc y ( c aus ed by d r o u g h t , t e m p e r a t u r e , C e n e t i c e n g i n e e r i n g o f p l a n t s h a s l e d to th e
s alinit y ) ,oz one and int e n s e l i g h t development of crops with increased resistanceto

596
 Chapter50 : APPLICATIONS
OF PLANTTRANSFORMATION/|RANSCENIC
PLANTS 597

Tlrre 50.1 A selectedlist of common insect pests along with the major crops damagedby them

Common name of pest Botanical name Crop(s) damaged

Cotton
bollworm Helicoverpazea Cotton
Cotton
leafworm Spodopteru littiralis Rice,maize,cotton,tobacco
Tobaccohornworm Manduca sexta Tobacco, polato
tomato,
Europeancornborer Ostrinia
nubilalis Maize
Locust Locustamigratoria Grasses
Tobaccobudworm Heliothis
virescens Tobacco,
cotton
Tomatofruitworm Heliothis
armigera Tomato,cotton
Cowpea seedbeetle Callosobruchus maculatus Cowpea,
soybean
Coloradobeetle notarsa
Lepti dece n Iineata Potato
Brownplanthopper Nilaparvata
lugens Rice

biotic stresseswhich is described in three major . Chemicalpesticidesare not efficientlydegraded

categories
.l
. Insect resistance.
2. Virus resistance.
3 . Fu ng al an d bac t er ial dis eas e r es is t anc e.
lt is estimated that about 15"/, of the world's
crop yield is lost to insectsor pests.A selected list
of the common insects and the crops damaged is
given in Table 50.1.
The damage to crops is mainly caused by insect
larvae and to some extent adult insects. The
majority of the insectsthat damage crops belong to
the following orders (with examples).
o Lipidoptera (bollworms).
. Coleoptera (beetles).
r Orthoptera (grasshoppers).
o Homoptera (aphids).
Till sometime ago, chemical pesticides are the
only means of pest control. Scientistshave been
looking for alternatemethods of pest control for the
f ollo wing rea so ns( i. e. lim it at ionsof pes t ic ideus e ) . toxi
o About 95'/. of the pesticide sprayed is washed
away from the plant surface and accumulates In are amongthe endotoxinsproducedby sporul4ting
th e soil.
o lt is difficult to deliver oesticides to vulnerable
parts of plants such as roots, stems and fruits.
pol l uti on.
i n the soi l , causi ngenvi ronmental
. Pesticides,in general, are toxic to non-
target organisms, particularly humans and
ani mal s.
It is fortunatethat scientists
'havebeen able r<t
discover new biotechnologicalalternativesto
chemical pesticides thereby providing insect
resistance to crop plants.Transgenicplants with
insectresistance transgeneshave been developed.
About 40 genesobtainedfrom microorganisms of
higher plants and animals havd been used to
provide insectresistancein crop plants.Someof
the approachesfor the biocontrol of insects are
brieflydescribed.
R E S IS TA N C E GE N E S FR OM
MIC R OOB GA N IS MS
Bacillus thuringiensis fBU toxin
Bacillus thurin$iensiswas first discovered by
l shi w aki i n 1901, al though i ts commerci al
importance was ignoreduntil 1951. B. thuringiensis
i s a C ram negati ve,
soi l bacteri um. Thi sbacteri um
produces a parasporal crystalline proteinous
n w i th i nsecti ci dalacti vi ty. The protei n
produced by B.thuringiensisis referred to as
insecticidal uystalline protein (lCP). lCPs
bacteri a, and w ere ori gi nal l y cl assi fi ed as
6-endotoxins(to distinguishthem from other classes
of u-, p. and y-endotoxins)
598 B IOTECHNO LO Ci

Bt toxin genes
Severalstrainsof B. thuringiensrsproducing a
wide range ol crystal (cry) proteins have been
identified.Further,the structureof cry genesand
their correspondingtoxin (6-endotoxin)products
have been characterized.The cry genes are
c l a s s i fi e d(l a te s ti n 1 9 9 8) i nto a l argenumberof
distinct families (about 40) designatedas cry
1 ......c ry4 0 , b a s e coJ n t hei r si ze and sequence
s i mi l a ri ti e sAn. d w i th i n each fami l y, there may
be sub-families.Thus, the total number of Parasporalcrystal
genesproducingBt toxins (Cry proteins)is more
.l Io'nuti
than 00. +
Thereare differences in the structureof different o
250-kDasubunitprotoxin
Cry proteins,besidescertainsequencesimilarities.
The molecularweights of Cry proteinsmay be
e i th e rl a rg e(-1 3 0 K D a )o r smal l(-70K D a).D espi te
the differencesin the Cry proteins,they sharea
common activecore of three domains.

Mode of action of Gry proteins

Most of the Bf toxins (Cry proteins)are active
againstLepidopteranlarvae,while some of them
are specific against Dipteran and Coleopteran
insects.The protoxinof Cry I toxin group has a
m o l e c u l a rm a s s o f 1 3 0 ki l odal tons(130 K D a).
When this parasporalcrystal is ingestedby the
targetinsect,the protoxingetsactivatedwithin its
g u t b y a c o m b i n a ti o no f al kal i nepH (7.5 to 8.5)
and proteolytic enzymes. This results in the
conversionof protoxininto an activetoxin with a
molecufarweight of 58 KDa (Fig.50.2). 68-kDaactivetoxin
The active form of toxin protein gets itself
insertedinto the membraneof the gut epithelial Fig. 50.2 : A diagrammatic representation of the
cells of the insect.This resultin the formationof
i o n c h a n n e l sth ro u g h w hi ch there occurs an
excessiveloss of cellularATP.As a consequence,
cellular metabolismceases,insectstops feeding,
and becomesdehydratedand finally dies. Some Bt toxin as biopesticide
workers in the recent years suggestthat the Bt
Preparations of Bt spores or isolated crystals
toxin opens cation-selective pores in the
have been used as organic biopesticide for about
membranes,leadingto the inflow of cationsinto
50 years. This approach has not met with much
the cells that causesosmoticlysisand destruction
successfor the following reasons.
o f e p i th e l i a cl e l l s (a n d fi nal l y the deathof i nsect
larvae). . Low persistenceand stability (sunlight degrades
toxin) of the toxin on the surface of plants.
T h e B tto x i n i s n o t to xi cto humansand ani mal s
o The Bt toxin cannot effectively penetrate into
sincethe conversionof protoxinto toxin requires
(These various parts of plants, particularly roots.
alkaline pH and specific proteases are
a b s e n th u m a n sa n d a n i mal s). . Cost of production is high.
Chapter50 : APPLICATIONS
OF PLANTTRANSFORMATIONfRANSCENIC
PLANTS 599

Crop Trade name Bt protein Resistanceto insectk)

Cotton Bollgard Cry1Ac Cottonbollworm,
tobacco
budworm
Maize YieldGard
knockout Cry1Ab Europeancornborer
Maize Starlink Cry9C European
cornborer
Maize HerculexI C ry1F European
cornborer
Maize Bt-Xtra Cry1Ac European
cornborer
Potato New-leaf Cry3A Colarado
beetle

Bt based genetic transformation stems and fruits. This is not possible by any
of plants chemical pesticide.

It has been possibleto geneticallymodify (GM) . Toxic proteins are produced within the plants;
plants by inserting Bf genes and provide pest hence they are environmental-friendly.
resistanceto these transformedplants. For an
" Bt toxins are rapidly degraded in the
effective pest resistance,the bacterial gene in environment.
tra ns genic plan ts m u s t p o s s e s s h i g h l e v el
expression. Thisobviouslymeansthatthe transgene The problem of insect resistance
transcriptionshouldbe underthe effectivecontrol to Bt crops
of promoterand terminatorsequences.
The major limitation of Bt-gene possessing
The earlyattempts to expresscry 1A and cry 34 transgenicplantsis the developmentof Bt-resistant
oroteins under the control of CaMV 355 or insects.
The Bttoxin is a orotein.and the membrane
AgrobacteriumT-DNA promotersresultedin a very receptor (of the gut) through which the toxin
low expressionin tobacco,tomatoand potatoplants. mediates itsactionis alsoa protein.lt is possible
that
the appropriate mutationsin the insectgenecoding
Modificationol Bt uy 1A gene : The wild type
for receptorproteinmay reducethe toxin binding
transgene Bf cry 1A(b)wasfoundto express at a very
and renderit ineffective. This may happenwithin a
l o w lev els in t r a n s g e n i cp l a n ts .T h e n u c l e o ti de
few generations by repeated growingof BI crops.
sequence of this genewas modified(G + C content
altered,severalpolyadenylation signalsremoved, Several approachesare made to avoid the
ATTTA sequence deleted,etc). With appropriate developmentof resistance in insects.
sequence changes, an enormousincrease (about100
. lntroductionof two differentBt toxin genesfor
iold) in the Bt toxin oroductformationwasobserveo.
the same target insect.
The transgenicBt crops that were found to . Development of transgenicplantswith two types
provide effectiveprotectionagainstinsect damage of insect resistancegenes e.g. Bt gene and
were given approvalfor commercialplantingby protei nasei nhi bi torgene.
U S A in t he m i d -1 9 9 0 s (T a b l e 5 0 .A . So me
o RotatingBt crops with non-Bt crops may also
biotechnological companieswith their own trade
namesintroducedseveraltransgenic cropsinto the prevent the build-up of resistancein insect
Among these, maize and cotton Bt crops popul ati on.
fields. only
are currentlyin use in USA.The other genetically
modifiedolantsmet with failurefor variousreasons. The environmental impact oI Bt crops
The most serious impact of Bt crops on
Advantages of transgenic plants environment is the build-up of resistancein the
with Bt genes pest population.
r Bt genescould be expressedin all partsof the l n 1999, anotheri ssuew as broughtto l i ght
plants,includingthe rootsand internalregionsof aboutBf crops.lt was reportedthat the pollenfrom
600 BIOTECHNOLOC\

transgenlcplantswfth lnsectresistance

Plant gene Transgenicplant(s) Encodedprotein Resrctanceto insect(s)

Proteaseinhibitors
CpTi apple,rice,
Potato, Trypsin LePidoPtera
Coleoptera,
wheat,tomato
sun{lower,
CII Tobacco,potato protease
Serine LepidoPtera
Coleoptera,
PI-IV Potato,tobacco protease
Serine Lebidoptera
o Gl Tobacco, oilseedrape protease
Cysteine Homoptera
Coleoptera,
CMe Tobacco Trypsin !:-li-d'p':ll
a-Amylaseinhibitors
a-Al-Pv Pea,tobacco o-Amylase Coleoptera
WMAI-1 Tobacco o,-Amylase Lepidoptera

Lectins
GNA rice,sugarcane
Potato, Lectin Lepidoptera
Homoptera,
sweetpotato,tobacco
WGA Maize Agglutin _1. ptgi1 9_gl
p,d. f
9_.pt'
Others
BCH Potato Chitinase Lepidoptera
Homoptera,
TDC Tobacco decarboxylase Homoptera
Tryptophan

Bt maize might be toxic to the larvaeof Monarch Some of the importantones are
microorganisms.
butterfly.This generatedconsiderableoppositionto Iisted.
Bf crops by the public, since Monarchbutterflyis '1. Cholesterol oxidase of Strepotmycesculture
one of the mostcolorfulnativesin USA.It was later filtratewas found to be toxic to boll weevil larvae.
proved that the fearsabout the impact of 8t crops Cholesterol oxidasegenehas been introducedinto
on the monarchbutterflywere withoutthe required tobaccoto developa transgenicplant.
scientificevidence.
2. Isopentenyl transferase gene from Agro-
The lessonlearnt from the monarch butterfly bacterium tumefacienshas been introduced into
episodeis that the risks of CM crops should be tobacco and tomato. This gene codes for an
thoroughlyassessed before they are reported. importantenzymein the synthesis of cytokinin.The
transgenic plants with this transgene were found
Usageof 8f : The usageBt is commonlyusedfor to reduce the leaf consumption by tobacco
a transgeniccrop with a cry Senee.g. Bt cotton.In
hornworm and decreasethe survival of peach
the same way, Cry proteinsare also referredto as potatoaphid.
Bf proteins. lt may also be stated here that the
authorsusefour differentnamesfor the samegroup R E S IS TA N C E GE N E S FR OM
of proteins-6-endotoxin,insecticidalcrystalprotein
HIGHER PLANTS
(lCP),Cry and norv Bt.
Certaingenesfrom higherplantswerealsofound
to result irr the synthesisof productspossessing
Resistance genes from
insecticidalactivity.Someauthorsregardthem as
other microotganisms
non-Bt insecticidalproteins.A selectedlist of plant
The Bt toxin genes from B.thuringiensishave insecticidal(non-Bt)genes used for developing
beendescribedin the preceedingpages.Thereare transgenicplants with insect resistanceis given
certain other insect resistantgenes from other Table50,3.Someof them are brieflydescribed.
 PLANTS
OF PLANTTRANSFORMATIONIRANSCENIC
chapter 50 : APPLICATIONS 60r

PBOTE'NASE PNOTEASE) TNHTBITOFS of this enzymeby u-amylaseinhibitor,the larvae

can be starvedand killed. cr-Amylaseinhibitor
Proteinase inhibitorsarethe proteinsthat inhibit gene (o-Al-Pv) isolated from bean has been
the activityof proteinaseenzymes.Certainplants successfullytransferredand expressedin tobacco.
naturallyproduceproteinaseinhibitorsto provide It provides resistance against Coleoptera (e-g-
defenceagainstherbivorous insects. Thisis possible
Zabrotessubfasciatus).
since the inhibitors when ingested by insects
interfere with the digestive enzymes of the insect'
LECTINS
T his r es ult sin th e n u tri e n td e p ri v a ti o nc a u si ng
death of the insects. Lectinsare plantglycoproteinsand they provide
to insectsby actingas toxins.The lectin
resistance
It is possibleto control insectsby introducing gene (CNA) from snowdrop(Galanthusnivalis)has
proteinaseinhibitor genes into crop plants that been transferred and expressed in potato and
normallydo not producetheseproteins. tomato.The major limitationsof lectin are that it
acts only againstpiercingand suckinginsect,and
Cowpea trypsin inhibitor gene high dosesare required.
It was observedthat the wild speciesoi cowpea
plantsgrowing in Africa were resistantto attackbY RESISTANCE GENES FROM ANIMALS
a wide rangeoi insects.Research{indingsrevealed Proteinase inhibitorgenesfrom mammalshave
that insecticidal proteinwas a trypsininhibitorthat
also been transferredand expressedin plantsto
was capableof destroyinginsectsbelongingto the provide resistanceagainst insects,although the
orders Lepidoptera (e.8 Heliothis virescans), successi n thi s di recti oni s very l i mi ted.B ovi ne
Orthaptera(e.g.Locustam iI ratori a) and CoIeoptera pancreati c trypsi n i nhi bi tor (B P l ) and o(1 -
(e.g.Anthonous grandis). Cowpeatrypsin inhibitor genes appear to be promising to offer
antitrypsin
(CpfDhas no affecton mammaliantrypsins;herice insectresistance to transgenicplants.
it is non-toxic to mammals.
CpTi genewas introducedinto tobacco, potato INSECT RESISTANCE THROUGH COPY
and oilseedrape for developingtransgenicplants. NATURE STRATEGY
Survivalof insectsand damage to plants were Some of the limitations experienced by
much lower in plantspossessing CpTi gene.
transferring the insecticidalgenes(p4rticularly8t)
and developing transgenicplants have proinpted
Advantages of proteinase inhibitors scientiststo look for betteralternatives.The copy
o Many insects,not controlled by Bt, can be naturestrategy was introduced in 1993 (by Boulter)
effectivelvcontrolleo. with the objective oI insecl Pestcontrol which is
sustainableand environmentally friendly.
. Use of proteinaseg'enealong with Br gene will relatively
developmentin The copy naturestrategyfor the developmentof
helo to overcome8t resistance
insect-resistanttransgenicplantshasthe following
olants.
stages.
Limitations of proteinase inhibitors 1. ldentificationof leads:The first step in copy
plants (from
. Unlik e B f to x i n , h i g h l e v e l s o f p ro tei nase nature strategyis to identify the
worldover) that are naturally resistantto insect
inhibit or sar e re q u i re dto k i l l i n s e c ts .
damaEle.
. lt is necessary that the expression of proteinase
inhibitorsshould be very low in the plant parts 2. lsolation and purification of protein: The
c ons um edb y h u m a n s ,w h i l e th e e x p ressi on protein with insecticidal properties (from the
shouldbe high in the parts of plants utilizedby resistantplants) is isolated and purified. The
i nsects. sequence of the protein is determined,and the
gene responsible for its productionidentified.
' . A m y las e i n h i b i to rs 3. Bioassayof isolatedprotein: The activity of
The insect larvae secrete a gut enzyme the proteinagainstthe targetinsectsis determined
c- amylaseto digeststarch.By blockingthe activity by performinga bioassayin the laboratory.
 604 B IOTECHNO LO C\

3' Targetvirus RNA

3', AntisensemRNA

Fig. 50.3 : Hybridization of antisenEe nRNA with virus RNA to block replication.

to 5' -+ 3') and this is referredto as antisensegene. orientationand introducedinto

cloned in antisense
The mRNA produced by antisense gene rs tobacco plants. The transgenictobacco plants
complementaryto the mRNA synthesizedby expressed RNAof TCMV replicase.
antisense These
normal gene. As a result, both these mRNAs olantswere resistantto TGMV infection.
hybridizeand thusthe normaltranslation of mRNA
is blocked.The neteffectof employingan anfisense Ribozymes
gene into a cell is that it blocks a specificgene
R i bozymesare smal l R N A mol e culeswhich
expression.
promote the catalytic cleavage of RNA For
It is possibleto introducevirai antisensegenes providingvirusresistance, ribozymesin the form of
into plantsand producemRNAscomplementary to antisense RNAscapableof cleavingthe targetviral
v i ra l s e q u e n c eisn v o l v e di n vi rus repl i cati on.
The (sense)RNAshave been developed.The ribozyme
antisensemRNAs can block the replicationof (antisense RNA) bindsto a small sequenceof viral
viruses(Fig 50.3).Initially, antisense RNAapproach RNA and splits(Fig. 50.4).
was carriedout in single-stranded RNA viruses.The
successof this approach however,was limited In thisway, it is possibleto blockthe replication
probablydue to the following reasons. of viral RNA. However,the ribozymesapproach
has not been very successful in plants.
o High concentration of antisensemRNA may be
required.
. Proteinassociationwith mRNA interfereswith
hybridization (between sense mRNA and
antisensemRNA).
The plantsdo possesgeneraldefencesystems
AntisenseRNA approachmay be more useful againstinvadingpathogens.This is however,not
for DNA viruses.In fact, tomato goldqn mosaic truely comparablewith the immunesystemof the
virus (TGMV) replicase coding sequence was ani mal s.

Targetvirus RNA
Ribozyme(antisenseRNA)
$
,iI
i
I (*

Cleavedviral RNA

Fig. 50.4 : Action of ribozymes as antisense HNAs to block virus replication.

 PLANTS
OF PLANTTRANSFORMATIONf|RANSCENIC
Chaoter50 : APPLICATIONS 605

Wh en ever ther e is a c ellular dam age c aus ed b y

pathogens (fungi, bacteria) and plant pests, the TlsLS 50.5 types of pathogenesis-
general defence system of plants get geared up to rclated (PRl protelns produced in plants
provide some amount of protection to the plant.
Type Properties
Th is n atu ral d is eas e r es is t anc e of plant s i s
inadequate. However, knowledge on the natural P R .1 Antif
ungal
systems of plant resistance is useful for the PR-2 EndoB-1,3-glucanases
biotechnological approaches to develop disease PR-3 Endochitinases
reststance.
PR-4 Antif
ungal,endochitinase
Some of the defenses of plants and the PR.5 Antifungal,
thaumatin-like
biotechnological approachesare briefly described. proteins,
osmotins
PR-6 inhibitors
Protease
PATHOGENESI S. RELATED ( PRI
PR-7 Endoprotease
PROTEINS
PR-8 Chitinaseilysozyme
To defend themselves against the invading PR.9 Peroxidases
pathogens (fungi and bacteria), plants accumulate
P R -10 Ribonucleases
Iow molecular weight proteins which are
collectively regarded as pathogenesis-related(PR) P R -11 Endochitinase
acitivty
proteins. The different types of PR proteins and PR-12 Defensins
their properties are given in Table 50.5. Some of PR-13 Thionins
the most important ones are described. P R -14 proteins
Lipidtransfer
(non-specif
ic)
Chitinase

Chitin is a cons t it uentof f ungal c ell walls whi c h

approach, fungal resistant tobacco, tomato and
can be hydrolysedby the enzyme chitinase.Certain
carrot have been developed.
chitinase genes from plants have been isolated and
characterized.A bacterial chitinase gene obtained
from a soil bacterium (Serratia marcescens) was RIBOSOME.INAGTIVATING
introduced and expressedin tobacco leaves.Some PROTEINS (RlPsl
other workers isolated a chitinase gene from bean Ribosome-inactivatingproteins offer protection
(Phaseolus vulgaris) and developed transgenic against fungal infections. They act on the large
plants of tobacco and Brassica napus with this rRNA of eukaryote and prokaryote ribosomes
8ene. (remove an adenine residue from a specific site),
The transformed tobacco plants were found to and thus inhibit protein biosynthesisC. ertain RlPs
be resistant to infection of the pathogen that do not inhibit plant ribosomeswere identified
Rhizoctonia solani. In case of B.napus, the and the corresponding genes have been used to
protection however, was comparatively less. develop transgenic plants e.g. Type-l barley RIP is
used to provide resistanceto fungal infections.
Glucanase Some authors use the term antimicrobial
Clucanase is another enzyme that degradesthe proteins to RlPs. The other examples of
celt wall of many fungi. The most widely used antimicrobial proteins are lectins, defensins,
glucanase is p-1, 4-glucanase.The gene encoding lysozyme, thionins etc.
for p-1, 4-glucanasehas been isolated from barlev,
introduced, and expressed in transgenic tobacco Lysozyme
plants. This gene provided good protection against
Lysozyme degradeschitin and peptidoglycan of
soil-borne fungal pathogen Rhizoctonia solani.
cell wall, and in this way fungal infection can be
The resistance to fungal pathogens is much reduced. Transgenic potato plants with lysozyme
higher if both chitinase and glucanase producing gene providing resistance Io Eswinia carotovora
genes are present in transgenic plants. By this have been developed.
606 B IOTE CHNO LO CY

list along the genes

and the rcsistance provlded agalnst the pathogens (fungus/bacterluml

Crop Cene(s) transferred Resistanceagainst pathogen

(PR)proteins
Pathogenesis-related
Tobacco Chitinasefrombacterium Alternarialongipes
(Serratianarcescensl
Beanchitinase Rhizoctoniasolani,Phylophthora
parasitica
and1,3-Bgl ucanase Cercospora
C hi ti nase nicotinae
Rice Chitinase Rhizoctoniasolani
Carrot Chitinase and1, 3-Bglucanase Alternaria
dauci,A. radicina
Tomato Chitinase and1, 3-pglucanase Fusariumoxysporum
Brassica
napus Chitinase Rhizoctonia
solani
Antimicrobialproteins
Tobacco Barleyribosome proleinRhizoctonia
inactivating solani
Tobacco Defensinfromradish Alternaria
longipes
Tobacco genefrombarley
cr-Thionin Pseudomonas syringae
Potato Bacteriophage
T-4 lysozyme Erwiniacarotovora
Phytoalexins
Rice Stilbene
synthase Pyricularia
oryzae
Tobacco synthase
Stilbene Botrytis
cinerea

Defensins m o l e c u l a r w e i g h t a n d a n t i m i c r o b i a l i n n a tu r e . Th e
p h y t o a l e x i n su s u a l l y p r e s e n t i n s p e c i a l i ze dce l l s o r
Def ens ins ar e ant im ic r obi a l p e p t i d e s ( 2 6 - 5 0
o r g a n e l l e s a r e m o b i l i z e d w h e n i n f e c t i o n o ccu r s.
am ino ac id r es idues )f ound in a l l t h e p l a n t c e l l s .
F u r t h e r ,d u r i n g i n f e c t i o n t h e r e o c c u r s i n d u cti o n o f
They at t ac k t he m ic r obial p l a s m a m e m b r a n e ,
g e n e s f o r i n c r e a s e d p r o d u c t i o n o f p h yto a l e xi n s.
however this is not adequate to provide resistance
Stilbene synthaseis a key enzyme for the synthesis
to pathogens
o f a c o m m o n p h y t o a l e x i n .T h e g e n e c o d i ng sti l b e n e
In recent years, an artificial defensin gene has synthase has been isolated from peanut and
been developed and introduced into potatoes. introduced into tobacco, rice and Brassica napus.
These potatoes developed resistance to the The transgenicplantscarrying stilbenesynthasegene
bacterium Eswinia carotovora. were resistantagainst some fungi.
A selected list of transgenic plants developed,
Thionins along with the genes transferredand the controlled
Thionin proteins also offer protection against pathogens is given in Table 50.6.
bac t er ia. Thionin c oding g e n e s h a v e b e e n
introduced into tobacco and the transgenicplant so NEMATODE RESISTANCE
developeci showed resistance to Pseudomonas N e m a t o d e sa r e s i m o l e w o r m s f o u n d i n th e so i l .
syr!nqae. Thev possessa complete digestivetract. The annual
crop loss of the world due to nematode
PHYTOALEXINS ( r o u n d w o r m ) i n f e s t a t i o ni s v e r y h i g h .

Phytoalexinsare secondarymetabolitesproduced Some workers have identified and cloned a

in plants in responseto infection. They are low- nematode resistancegene from wild beet plants. lt
Chapt er50 : A P P L IC AT IONOSF PL A N TT R A N S FOR MA TION fi R A N S C EPNLA
ICN TS 607

is proposedthat this gene encodesa proteinthat
detects the pests (nematodes)and triggers a
r eac t io n si n th e p l a n t.l t i s b e l i e v e dth a t
d ef ens iv e
som ec hem ic alc o m p o u n d sth a t d e s tro yth e g u t o f
the nematodeare oroduced.
from crop plants. For this reason,the crops are also
affected by herbicides, hence the need to develop
herhicide-resistant plants.Thus, these plants provide
an opportunity to effectively kill the weeds (by
herbicides)without damaging the crop plants.

Attemots w'ere also made to transfer the

Strategies for engineering
nematoderesistance geneto sugarbeet.Not much
herbicide resistance
successwas reported,the major limitationbeing
the difficultyin cultivatingthe gene-altered
cellsof A number of biological manipulations
sugarbeet. particularly involving genetic engineering are rn
u s e t o d e v e l o p h e r b i c i d e - r e s i s t a npt l a n t s .

1 . Overexpression of the target protein : The

Plants are constantly being subjected to
environmentalstresses(SeeFig 50. 1) that may result
in deterioration of crop plants, and a very low or
even no yield. Plants are dependent orr the subtle
intern al mecha ni s m s f or t oler anc e of v ar iou s
stresses. The in situ tolerance of crop plants,
whenever present/ is inadequate and therefore
cannot give protection against the stresses.A wide
range of strategiesare required to engineer plants
against a particular type of stresstolerance.
Some of the abiotic stressesand the recombinant
strategies developed to overcome them are
describ ed .
HERBICIDE RE SI STANCE
target protein, being acted by the herbicide can be
produced in large quantities so that the affect of the
herbicide becomes insignificant. Overexpression
can be achieved by integrating multiple copies of
the genes and/or by using a strong promoter.
2, lmproved plant detoxification : The plants
do possess natural defense svstems against toxrc
compounds (herbicides) Detoxification involves
the conversion of toxic herbicide to non-toxic or
less toxic compound. By enhancing the plant
detoxification system, the impact of the herbicide
can be reduced.
3. Detoxification of herbicide by using a
foreign gene : By introducing a foreign gene into
the crop plant, the herbicide can be effectively
detoxified.

Weeds (wild herbs) are unwanted and useless 4. Mutation of the target protein: The target
plants that grow along with the crop plants. Weeds protein which is being affected by the herbicide
compete with crops for light and nutrients,besides can be suitably modified. The changed protein
harbouring various pathogens. lt is estimated that s h o u l d b e c a p a b l e o f d i s c h a r g i n gt h e f u n c t i o n s o f
t he wo rld's cro p yi eld is r educ ed by 10- 15% due t o the native protein but is resistantto inhibition by
the presence of weeds. To tackle the problem of the herbicide. Once the resistant target protein
weeds, modern agriculture has developed a wide gene is identified, it can be introduced into the
range of weedkillers which are collectively referred p l a n t g e n o m e s , a n d t h u s h e r b i c i d e - r e s i s t a npt l a n t s
to as herbicides. In general, majority of the can be developed.
herbicides are broad-spectrum as they can kill a For success in the development of herbicide
wide range of weeds. A good or an ideal herbicide resistant plants, good knowledge of the target
is expected to possess the following characteristics. protein and the action of herbicides is required.
r Capable of killing weeds without affecting crop Some of the developments made in the herbicide
o lan ts. resistanceof plant are briefly described.
. No t to xic to a ni m als and m ic r oor ganis m s .
GLYPHOSATE RESISTANCE
. Rapidly translocatedwithin the target plant.
Clyphosate, is a glycine derivative. lt acts as a
. Ra pid ly de gra de d in t he s oil.
broad-spectrumherbicide and is effectiveagainst76
, No ne of th e co m m er c ially av ailable her bic ide s of the world's worst 78 weeds. Clyphosate is less
f ulfils all th e a bo v e c r it er ia.The m ajor lim it at ion of t o x i c t o a n i m a l s a n d i s r a p i d l y d e g r a d e d b y
the herbicidesis that they cannot descriminateweeds microorganisms.In addition, it has a short half-life.
608 B IOTECHNO LO CY

Shikimate3-phosphate Phosphoenolpyruvate produced from tryptophan. The net result of

glyphosateis the deathof the plants.Clyphosateis
toxic to microorganismsas they also possess
shikimatepathway.
C l yphosatei s non-toxi cto anim als( including
humans),since they do not possessshikimate
acid 3-phosphate
5-Enoylpyruvylshikimic pathway. Of the three aromatic amino acids
+
I (synthesizedin this pathway), tryptophan and
Chorismate phenylalaninbare essentialand they have to be
suppl i edi n the di et,w hi l e tyrosi necan be f or m ed
+.--+ + l.t--------_=-_ from phenyl al ani ne.
Tryptophan Phenylalanine Naphthoquinone
Strategies for glyphosate resistance
+
..-'IIndoleacetic || || || There are three distinct strategiesto provide
lndoleac id+ l+
alkaloids Tyrosine glyphosphate to plants.
resistari'ce
JFlavanoids gene :
Phenols 1. Overexpression of crop plant EPSPS
An overexpressing gene of EPSPS was detectedin
Petunia.Thisexpression was foundto be due to gene
Fig. 50.5 : Shikimate pathway indicating the action
amplificationratherthan an increasedexpressionof
of the herbicide glyphosate (EPSP synthase-
the gene.EPSPS genefrom Petuniawas isolatedand
5 -Enolylpy ruvylshikimate s-ph asphate sy nthase)
introduced into otherplants.The increased synthesis
of EPSPS(by about 40 fold) in transgenicplants
providesresistance to glyphosate.Theseplantscan
T h eAme ri c a nc h e mi c acl omoanvMonsanto markets
tolerateglyphosate at a doseof 2-4 timeshigherthan
glyphosateas Round up
that requiredto kill wild+ypeplants.
Mechanism of action of glyphosate 2. U se of mutant E P S P Sgenes: An EPSPS
mutantgenethat conferredresistance to glyphosate
Clyphosateis rapidlytransported to the growing
was first detected in the bacterium Salmonella
p o i n tso f p l a n ts .l t i s c a p abl eof ki l l i ngthe pl ants
typhimurium. lt was found that a single base
even at a low concentration. Clyphosateacts as a
substitution(C to 1 resultedin the change of an
competitive inhibitor of the enzyme S-enoyl-
ami no aci d from prol i neto seri nein EPSPS. This
pyruvylshikimate 3-phosphate synthase (EPSPS).
modified enzyme cannot bind to glyphosate, and
This is a key enzyme in shikimic acid pathway that
thus orovidesresistance-
resultsin the formationof aromaticamino acids
(try p to p h a np,h e n y l a l a ni ne and tyrosi ne),phenol s The mutantEPSPS genewas introducedinto
and certainsecondarymetabolites(Fig.50.5). tobacco plants using AgrobacteriumTi plasmid
vectors.The transgene producedhigh quantitiesof
The enzyme EPSPScatalysesthe synthesisof
the enzyme EPSPS.However, the transformed
5 -e n o y l p y ru v y l s h i k i m ate 3-phosphate from
tobaccoplantsprovidedonly marginalresistance to
shikimate3-phosphate and phosphoenoylpyruvate.
Clyphosatehas some structuralsimilarilywith the
substrate phosphoenol pyruvate (Fig 50.6).
Consequently, glyphosatebinds more tightly with o?
to;/-oY\o* to.. ,o T ?
EP SP Sa n d b l o c k s th e normal shi ki mi c aci d
pathway.Thus, the herbicideglyphosateinhibits Ho' bH,
the biosynthesis of aromaticamino acidsand other "o,rP'.,,N'..Ao'.,
i mp o rta n tp ro d u c ts .T h i s resul tsi n i nhi bi ti onof
Phosphoenolpyruvate Glyphosate
proteinbiosynthesis (dueto lack of aromaticamino
Fig. 50.6 : Structures of phosphoenolpyruvate (the
a c i d s ).A s a c o n s e q u e nce, cel l di vi si onand pl ant
substrate) and the herbicide glyphosate (the
growth are blocked. Further,the plant growth competitive inhibitor).
re g u l a to ri n d o l e a c e ti c aci d (an auxi n) i s al so
 50 : A P P L IC A T IO NOSF PL A N TT R A N S FOR MA TION /TR A N S C E
ChA P t CT PNLA
ICN TS 609

Phosphinothricin

Alanine
I
Alanine
Bialaphos

I
Glutaminesvnthase
+ NHa-_#
L-Glutamate L-Glutamine

Fig. 50.7 : The formation, mode of action and detoxification of phosphinothricin

(PAT-Phosphinothricin acetyl transferase).

glyphosate. The reasonfor this was not immediately thricin is moreeffectiveagainstbroad-leafed weeds
identified.lt was later known that the shikimate but least effective against perennials.(Note :
pathway occurs in the chloroplastswhile the Phosphinothricin and glufosinate are two namesfor
glyphosate resistantEPSPS was producedonly in the the same herbicide.However,to avoid confusion
cytoplasm.This enzymewas not transported to the between glyphosateand glufosinate,phosphino-
chloroplasts,hence the problem to provide thricin is more commonlyused. BastaAventisand
resistance. Thiseoisodemadescientists to realizethe Libertyare the trade namesfor phosphinothricin).
importance of chloroplasts in geneticengineering.
Phosphinothricin-a natural herbicide
ln later years,the mutant EPSPSgene was
tagged with a chloroplast-specific transit peptide P hosphi nothri cii snan unusualherbi ci de, bei ng
sequence.By this approach,the glyphosate-resistant a derivative of a natural product namely bialaphos.
EPSPS enzymewas directedto freely enterchloroplast Certainspeciesof Streptomyces produce bialaphos
and confer resistanceagainst the herbicide w hi ch i s a combi nati on of phosphi nothri ciboundn
to tw o al ani neresi dues, formi nga tri pepti de.B y
3. Det ox if ic a ti o no f g l y p h o s a te:T h e s o i l the aciionof a peptidase, bialaphosis convertedto
microorganismspossessthe enzyme glyphosate activephosphinothricin (Fig 50.V.
oxidasethat convertsglyphosateto glyoxylateand
a m inom et hy lph o s p o naic i d . T h e g e n e e n c o d i ng Mechanism of action of
glyphosateoxidasehas been isolatedfrom a soil phosphinothricin
organism Ochrobactrum anthropi. With suitable
modifications, this gene was introducedinto crop P hosphi nothri ci n as a competi ti ve
acts i nhi bi tor
plantswere of the enzyme glutamine synthase (Fig5O.l.fhis ts
plantse.g.oilseedrape.The transgenic
possible since phosphinothricin has some structural
found to exhibitvery good glyphosate resistance in
the f ield. similarity with the substrateglutamate.As a
consequence of the inhibitionof glutaminesynthase,
Use of a combined strategy: More efficient ammoni aaccumul ates and ki l l s the ol ant cel l s.
resistanceof plants against glyphosatecan be Further,disturbancein glutaminesynthesisalso
providedby employinga combinedstrategy. Thus, inhibitsphotosynthesis. Thus,the herbicidalactivity
resistant(i.e. mutant)EPSPS gene in combination of phosphinothricin is due to the combinedeffectsof
with glyphosateoxidasegene are used. By this ammoniatoxicityand inhibitionof photosynthesis.
approach,there occursglyphosateresistance (due
to mutantEPSPS gene)as well as its detoxification Strategy for phosphinothricin
(due to glyphosateoxidasegene). resistance
The natural detoxifying mechanism of
PHOSPHINOTHRICIN RESISTANCE phosphinothricinobservedin Streptomyces sp has
(or
Phosphinothricin glufosinate)is also a broad prompted scientists to develop resistant
spectrum herbicide like glyphosate.Phosphino- pl ants agai nst thi s herbi ci de. The enzyme

Biotechnology
[39]
610 B IOTECHNO LO CY

phosphinothricinacetyl transferase(o[ Streptomyces It may however, be noted that some of the

sp) acetylatesphosphinothricin,and thus inactivates herbic ide-resistant transgenic pl ants are at f ield-trial
t he her bic ide. stage.Due to environmentalconcern, a few of these
plants are withdrawn e.g. atrazine- resistantcrops.
The Bene r es po n s i b l e for coding
phosphinothricin acetyl transferase(bar gene) has ENVIRONMENTAL IMPACT OF
been identified in Streptomyces hygroscopicus. HERBICIDE.BESISTANT CROPS
Some success has been reported in developing
T h e d e v e i o p m e n t g e n e t i c a l l y m o d i fi e d ( C M )
transgenic maize and oilseed rape by introducing
herbicide-resistant crops has undoubtedly
bar gene. These plants were found to provide
contributed to increase in the yield of crops For
r es is t anc et o phos phinot h r i c i n .
t h i s r e a s o n , f a r m e r s p a r t i c u l a r l y i n th e d e ve l o p e d
SULFO NYLUREAS AN D countries (e.g. USA) have started using these CM
IMIDAZOLINONES RESISTANCE crops. Thus, the proportion of herbicide resistant
soybean plants grown in USA increased from 17'h
The her bic ides nam e l v s u l f o n v l u r e a s a n d i n 1 9 9 7 t o 6 \ o h i n 2 0 0 1 . T h e f a r m e r i s i m m e n se l y
im idaz olinones inhibit t h e e n z y m e a c e t o l a c t a t e benefited as there is a reduction in the cost of
synthase (ALS), a key enzyme in the synthesis of herbicide usage.
br anc hed c hain am ino a c i d s n a m e l y i s o l e u c i n e ,
It is believedthat the impact of herbicide-resistant
leuc ine and v aline. M ut an t f o r m s o f t h i s e n z y m e
o l a n t s o n i h e e n v i r o n m e n t i s m u c h lo w e r th a n tn e
and the corresponding genes have been isolated,
d i r e c t u s e o f t h e h e r b i c i d e si n h u g e q ua n ti ti e s.Th e r e
identified and characterized.Transgenicplants with
are however, other environmentalconcerns.
the mutant genes of ALS were found to be resistant
t o s ulf ony lur eas and im id a z o l i n o n e s e . g . m a i z e , r D i s t u r b a n c ei n b i o d i v e r s i t yd u e t o e l i m i n a ti o n o f
tomato, sugarbeet. weeds.
o R a p i d d e v e l o p m e n t o f h e r b i ci d e - r e si sta n ce
Resistance to other herbicides weeds that may finally lead to the production of
Besides the above, some other herbicide super weeds.
resistant plants have also been developed e.g.
br om ox y nil, at r az ine, p h e n o c a r b o x y l i c a c i d s ,
TOLERANCETO WATER
cyanamide. A list of selected examples of gene
DEFICIT STRESSES
transferred herbicide resistant plants is given tn The e n v i r o n m e n t aI c o n d i t i o n s su ch a s
Table 50.7. temperature (heat, freezing, chilling), water

Tlcrr 50.7 Selectedexamplesof gene transferred

Herbicide Gene transfer/mechanismof resistance Transgeniccrop(s)

Glyphosate of EPSPS
lnhibition Soybean,
tomato
Glyphosate by glyphosate
Detoxification oxidase.
1 l ^ i -^
tvrqr4u, ouyu94rl
^a.,haan

Phosphinothricin bar genecodingphosphinothricin Maize,rice,wheat,cotton,potato,

acetyltransferase tomato,sugarbeet
midazoli
Sulfonylureas/i nones Mutantplantwithacetolactate
synthase maize,sugarbeet
Rice,tomato,
Bromoxynil Nitrilase ication
detoxif potato,
Cotton, tomato
Atrazine psb4 gene
Mutantplantwithchloroplast Soybean
Phenocarboxylic
acids Monooxygenase ication
detoxif Maizecotton
(e.9.?,4-Dand2,4,5-T)
Cyanamide gene
hydratase
Cyanamide Tobacco

(EPSPS-5-Enoylpyruvytshikinate
3-phosphate 2,4-D-2,4-Dichlorophenoxy
synthase; aceticacid)
aceticacid;2,4,5-T-2,4,S'Trichlorophenoxy
 S P L AN TT R A N S FOR MA TION | IR A N S GEPNLA
Chapt er50 : A P P L IC A T IO NOF
availability (shortage due to drought), and salinity
influe ncethe p lan t gr owt h, dev elopm entand y ield .
The abiotic stressesdue to temperature, drought
and sa linity are c ollec t iv ely r egar ded as wat e r
deficit stresses(See Fig 50.7).
Causes of water deficit
Water deficit may occur due to the following
ca uses.
. Reduced soil water ootential.
.In cre ased wate r ev apor at ion ( in dr y , hot and
wind y con ditio ns ) .
. Hig h salt con ce nt r at ionin t he s oil ( dec r eas es oi l
water ootential).
o Low temperatureresulting in the formation of ice
c rystaIs.
Effects of water deficit
. Resultsin osmotic stress.
. ln hib its oh oto sv nt hes is
. Increasesthe concentrationof toxic ions (reactive
oxyg en spe cie s )wit h, in t he c ells .
. Loss of water from the cell causing plasmolysis
a nd fin aliy cell deat h
Tolerance to osmotic stress
The plant cells are subjected to severe osmotic
stressdue to water deficit. They however, produce
certain compounds, collectively referred to as
osmoprotectants or osmolytes, to overcome the
osmotic stress. Osmoprotectants are non-toxic
comp ata blesolu tesand ar e div ided int o t wo gr oups .
1 . Sugar and sugar alcohols e.g. mannitol,
sorbitol, pinitol, ononitol, trehalose,fructans.
2. Zwitterionic compounds : These osmo-
: -c'lectantscarry positive and negativechargese.g.
:' ;iin e, glycin e b et aine.
Tne production of a given osmoprotectant rs
,ct<ies dependent. The formation of mannitol,
:- ,'ne a nd glycin e bet aine ar e m or e c los ely link ed
-- osmotic to lera nc e.
Strategies to develop water deficit
tolerance plants
As explained above osmoprotectantsoffer good
o'otection to plants against osmotic stress and
ICN TS 611
therefore water deficit. lt is therefore, logical to
think of genetic engineering strategies for the
increased production of osmoprotectants.
S o m e p r o g r e s sh a s b e e n n r a d e i n t h i s d i r e c t i o n .
The biosynthetic pathways for the production of
manv osmoprotectantshave been establishedand
genes coding key enzymes isolated. In fact, some
progress has been made in the development of
transgenic plants with high production of
osmoorotecta nts.
Transgenic plants with glycine
betaine production
Clycine betaine is a quaternary ammonium
compound and is electrically neutral. Besides
f u n c t i o n i n g a s a c e l l u l a r o s m o l y t e s ,g l y c i r r eb e t a r n e
stabilizes proteins and memebrane structures.
Some of the key enzymes for the production of
glycine betaine have been identified e.g. choline
m o n o o x y g e n a s e ,c h o l i n e d e h y d r o g e n a s e ,b e t a r n e
aldehyde dehydrogenase.The genes coding these
enzymes were transferred to develop transgenrc
Dlants
By using choline oxidase gene from Arthrobacter
sp, transgenic rice that produces higher glycine
betaine (which offerstolerance againstwater deficit
stress)has been developed.
RESISTANCE AGAINST
ICE.NUCLEATING BACTERIA
Formation of ice on the olant cells (outer
membrane) is a complex chemical process.The
i m p o r t a n c eo f i c e - n u c l e a t i n gb a c t e r i a i s r e c o g n i z e d
in recent years. The occurrence of these bacteria
has been reported in most of the plants- cereals,
fruits and vegetable crops. The ice-nucleating
bacteria synthesize proteins, rvhich coalesce with
water molecules to form ice crystalsat temperature
around 32oF. As the ice crystals grow, they can
pierce the plant cells and severely damage the
plants.
Chemical treatment of plants to
protect from ice formation
Plants can be treated with copper containing
c o m p o u n d s t o k i l l t h e b a c t e r i a .A n o t h e r a p p r o a c h
is to use urea solution so that the ice formation is
minimized.
612 B IOTECHNO LO CY

lce.minus bacteria to resist

plants from cold temperatures

The bacteriumPseudomonas syringaeis one of

th e h i g h l y p re v a l e n tice-formi ngorgani smsi n With the advances made in plant genetic
nature. With genetic manipulations,the gene engi neeri ng, i mprovement i n cropyieldand qualit y
that directsthe synthesisof ice-relatedbacterial have becomea reality.The crop yield is primarily
proteinsin P. syringaewas removed.Thesenewly dependent on the photosynthetic efficiencyand the
develooedbacteriaare referredto as ice-minus harvestindex (the fractionof the dry matterallocated
bacteria. to the harvested partof the crop).The qualityof tne
crop is dependenton a wide rangeof desirable
The researchers proposedto spraythe transgenic characters-nutritional compositionof edible parts,
ice-minus bacteria on to young plants. The flavour,processing quality,shelf-lifeetc.
intention was that these bacteria would give
frost tolerance to the plants; and thus increase GR E E N R E V OLU TION
the crop yield. The opponentsof DNA technology
The 'Green Revolution' led by Borlaug,
were against this approach-the main fear
Swaminathan and Khusenabledthe world's food
being that the bacterial mutants may create
supply to be tripled during the last three decades
s o m e h e a l th c o m p l i cati on i n humans. Tne
of 20th century. This was made possible by
researchersargued and justified that no new
adopting geneticallyimprovedvarietiesof crops,
genetic information is introduced into P. syringae
coupledwith advancesin crop management.
and it is closelyrelatedin all aspectto the parent
one which is already in the environment.After The devel opment of hi gh-yi e lding var iet iesof
prolongedcourt proceedingsin USA, clearance wheat and rice has enabled se"reraldeveloping
w a sg i v e nfo r s p ra y i n the
g i ce-mi nusbacteri ai n the countri es(a good exampl ebei ng lndia)t o m ove
fi e l d s . from a position of food scarcityto become net
exporterof these cereals.The Green Revolution
It was in 1987,ice-minusbacteriawere sprayed
became a reality as the farmers adopted to new
on to the field of potato plants and strawberry
cereal seeds, besi des empl oy ing high- input
plants.Anotherstraino( P. syringaecommercially
methodsof agriculture - useof nitrogenfertilizers,
labeled,as Frostbanwas laterdevelopedand used
herbi ci des,pesti ci des,modern equipm ent of
i n c ro p fi e d s .
agricultureetc.
lt may be noted here that ice-minushacteria of
P, syringae were the first transgenic bacteria that Selected examples of crops
were used outside the laboratory. Fortunately,the for quality and yield
experiments yieldedencouraging results,sincecrop Thereare a wide rangeof cropsthat havebeen
damagedue to frost formation was found to be manipulatedby scientistsfor improvedyield and
reduced. quality. Only selected examples are briefly
descri bed.
Arabidopsis with cold.
tolerant genes GE N E TIC E N GIN E E R IN G FO R
OF FRUI TS
Scientistswere successfulin developingcold- E X TE N D E D S H E LF.LIFE
tolerantgenes(around20) in Arabidopsls when this The geneti cmani pul ati on of fr uit r ipeninghas
plant was graduallyexposedto slowly declining becomean importantcommercialaspectin plant
temperatures.They also identified a coordinating geneti cengi neeri ng. D el ay i n fruit r ipeninghas
gene that encodes a protein, which acts as a many advantages.
transcription factorfor regulatingthe expression of o lt extendsthe shelf-life,keepingthe qualityof the
cold-tolerant genes. By introducing the fruit intact.
coordinating gene, expressionof cold-tolerant
o Long distancetransportbecomeseasy without
Beneswas triggered,and this protectedthe plants
to fruit.
againstcold temperatures. More work is in progress damage
i n th i s d i re c ti o n . . Slow ripeningimprovesthe flavour.
 Chapter50 : APPLICATIONS
OF PLANTTRANSFORMATTON/TRANSCEN|C
PLANTS 613

Chlorophyll
---\
\
\ Polygalacturonase
\ lz
\ / -/
\
,/ / ........"+.......,.,-"'
\
I \
\
E
- -t,ht -v. l-e n e

tl-.
. , / . . "j ." '*'

//r'
I
Lycopene

o \,,',\t"
c

t1
i ,,.1i\.
| /.r
'.j
\- .
\- r
I '.j \- - -

-_---

10

Fig, 50.8 : Biochemical changes during the process of tomato

Ceneticengineeringwork has been extensively (PC) and pectin methyl esferase. The
carried out in tomatoes, and some of the phytohormoneethylene production is intimately
developmentare described. linked to fruit ripeningas it triggersthe ripening
processof fruit. Addition of exogenousethylene
BIOCHEMICAL CHANGES DUR'NG promotesfruit ripening,while inhibitionof ethylene
TOMATO BIPEN'NG biosynthesisdrasticallyreducesripening.

Fruit ripening is an active process. lt is The breakdown of starch to sugars, and

characterized by increased respiration
accompaniedby a rapid increase in ethylene
synthesis.As the chlorophyllgets degraded,the
green colour of the fruit disappears,and a red
pigment,lycopeneis synthesized (Fig, 50.A. The
fruit getssoftenedas a resultof the activityof cell
wall degradingenzymesnamelypolygalacturonase
accumulationof a large number of secondary
productsimprovesthe flavour,tasteand smell of
the fruit.
Threedistinctgenesinvolvedin tomatoripening
have been isolated and cloned. The enzymes
encodedby thesegenesand their respectiverole in
fruit ripeningare given in Table 50.8.

Tnerr50.E Clonesof tonato ripening genes and their functions

Gene clone Enzvme svnthesized Function in ripening

pTOM5 Phytoene
synthase Lycopene
synthesis thatgivesredcolouration
pToM6 Polygalacturonase Degradation
of cellwall,resulting
in fruitsoftening
pTOM13 ACCoxidase formation
Ethylene fruitripening
thattriggers
ACC-1
-Amino-cyclopropane-1
-carboxylic
acid)
614 BIOTECHNOLOCY

Tomato plant

I *g#
DNA strand DNA
Complementary

\ovr r-E | | Inrrh, --r

Hybridi:
|
I
Y
+| '
r-T-T--r-T--r-T--r--I
' r l rrrr r.
n--
I ,-if\r
- / Sense mRNA neutra
/ \J
IJ J
(PG)
Polygalacturonase No polygalacturoni
produced
I
+J
No fruit ripening Transgenlc tomato plant
Fruit ripening

Fig. 50.9 : Genetic manipulation of the enzyme polygalacturonase (PG) by antisense RNA approach
(Production of Flavr Savr tomato plant)

GENET'Q MAN'PULAT'ONS OF 1. lsolationof the DNA from tomatoplant that

FRU'T R'PEN'NG encodesthe enzymepolygalacturonase (PC).

Scientistshave been trying to genetically of PC geneto a vectorbacteriaand

2. Transfer
manipulateand delay the fruit ripening prccess. productionof complementary DNA molecules.
Almost all the attemptsinvolve antisenseRNA
approach. 3. Introductionof complementaryDNA into
a fresh tomato plant to produce a transgenic
Manipulation of the enzyme pl ant.
polygalacturonase (development
Mechanismof PG antisenseRNA approach : In
of Flavr Savr tomatof
the normal tomato plant, PC gene encodes a
As alreadystated,softeningof the fruit is largely normal (sense)mRNA that producesthe enzyme
due to degradation of the cell wall (pectin)by the polygalacturonase that is activelyinvolvedin fruit
enzyme polygalacturonase(PG). The gene ripening.The complementary DNA of PC encodes
responsible for PG, the rottingenzyme,has been for antisensemRNA, which is complementaryto
cloned (pTOM 6). The genetic manipulationof normal (sense)mRNA. The hybridizationbetween
polygalacturonase by antisenseRNA approachfor the senseand antisensemRNAsrendersthe sense
the developmento( Flavr Savr tomato (by Calgene mRNA ineffective. Consequently, no poly-
company 'in USA) is depicted in Fig. 50.9, and galacturonase is produced,hencefruit ripeningis
m a i n l yi n v o l v e sth e fo l l o w i ngstages. del ayed.
 OF PLANTTRANSFORMATIONIRANSCENIC
Chapter50 : APPLICATIONS PLANTS 615

The rise and fall of Flavr Savr tomato Methionine

t^-
The genetically engineered tomato, known as VNP
I
Flavr Savr (pronouncedflavour saver)by employing lY PPi + Pi
Y
PC antisenseRNA was approved by U.5. Food and S-Adenosylmethionine
Drug Ad m inis t r at ion on lBt n M ay 1994. T h e F D A
ruled that Flavr Savr tomatoes are as safe as
I
S-MethylJ ACCsynthase
tomatoesthat are bred bv conventional means, ano adenosi ne' I
Y
therefore no special labeling is required. The new 1-Amino-cyclopropane
tomato could be shipped without refrigeration to 1-carboxylicacid (ACC)
far off places, as it was capable of resistingrot for
more than three weeks (double the time of a
conventional tomato).

Although Flavr Savr was launched with a great

fa nfa re in 1995, it did not f ulf ill t he ex pec t a t i o nf o r Ethylene
th e fo llow ing r eas ons .
Fig. 50.10 : Biosynthesis of ethylene.
. Transgenictomatoescould not be grown properly
in different parts of U.S.A.
geneof ACC synthase,and the
insertingarrtisense
. The yield of tomatoes was low.
tomato ripeningwas markedlydelayed.
. The cost of Flavr Savr was high.
3. lnsertion of ACC deaminasegene : ACC
it is argued that the company that developed deaminase is a bacterial enzyme. lt acts on
Flavr Savr, in its overenthusiasmto become the first ACC (removesamino group), and consequently
Biotech Company to market a bioengineeredfood, the substrateavailabilityfor ethylenebiosynthesis
had not taken adequate care in developing the is reduced. The bacterial gene encoding ACC
transgenic plant. And unfortunately,within a year deaminasehas been transferredand expressed
after its entry, Flavr Savr was withdrawn, and it is in tomato plants. These transgenic plants
now almost forgotten! i nhi bi ted about 90% of ethyl ene bi osynthesi s.
The fruit ripening was delayed by about six
Manipulation of ethylene biosynthesis weeKs.
It has been clearly established that ethylene The strategies1 and 2 may be referredto as
plays a key role in the ripening of fruits. The antisenseethylene technolagy.
biosynthetic pathway of ethylene is depicted in
Fig 50.10. Ethylene is synthesizedfrom S-adenosyl LONGEB SHELF-L'FE OF FNU'TS
methonine via tlie formation of an intermediate, AND VEGETABLES
namely 1-aminocyclopropane-| carboxylic acid
The spoilageof fruits,vegetablesand senescence
(ACA, catalysed by the enzi-me ACC synthase. The
of picked flowers, collectively referredto as posf-
next step is the conversion of ACC to ethylene by
harvestspoilage is major concern in agriculture.
ACC oxidase.
This hampersthe distributionsystemparticularly
Three different strategies have been developed when the transportis done to far off places.
to block ethylene biosynthesis,and thereby reduce
The successful to del ayri peni ng,
mani pul ati ons
fruit rip en ing.
senescenceand spoilageof various foods will
'l . Antisense gene of ACC oxidase : Transgenic significantlycontribute to the appropriatefood
plants with antisense gene of ACC oxidase have distributionand thus good economicpracticesin
been developed. In these plants, production of agri cul ture.
ethylene was reduced by about 97"/" with a
Suppressing of ethyleneappears
the biosynthesis
sign ifica ntdelay in f r uit r ipening.
to be a promisingarea to reducethe spoilageof
2. Antisense gene of ACC synthase : Ethylene fruits, vegetables and senescence of flowers.The
biosvnthesiswas inhibited to an extent of 99.5'h by to block ethylenesynthesis
threedifferentstrategies
616 BIOTECHNOLOCY

Phenylalamine

+
i
4-Coumaryl-CoA
lrz 3X Malonyln
CoA
I Cnalconcsynthase
Niringen
in-chalcone

Naringenin
I
I Ravone&hydroxylase
+
Dihydrokaempferol

Dihydroquercetin Dihydromyricetin
I
I D.Ffi I ot*
| 3cr J3Gr
Pelagonidin-3-glucoside Cyanidin3-glucoside o"toni il'ilr Iucoside
(brickred/orange) (red) "
"ll
Fig. 50.11 : Biosynthesis of anthocyanins.

in tomato have been described. The same GE N E TIC E N GIN E E R IN G FOR FLO WER
approachesin fact can be successfullyused for PIGMENTATION
other fruits, vegetablesetc., to achievelonger shelf-
Thereare continuousattemptsin flower industry
Iife
to make the ornamentalflowersmore attractive(by
improving or creating new colours), besides
G E N E T IC EN G IN EE R IN G FOR prolonging post-harvestlifetime. The cut flower
PREVENTING DISCOLORATION
industryis mostly (about 70'/") dominatedby four
Discoloration is a major plants-roses,tulips,chrysanthemums
of fruitsand vegetables and carnations.
postharvestproblem encounteredin food industry.
The most common type of flower pigmentsare
Certain food additives are added to prevent
anthocyanins,a Broup of flavonoids.They are
discoloration.However,theseadditivesmay cause
synthesizedby a seriesof reactions,startingfrom
h e a l thc o mp l i c a ti o nisn h u mans.
the amino acid phenylalanine(Fig. 50.11).The
Biochemically, discoloration of fruits and colour of the flower is dependenton the chemical
vegetables is mainlydue to the oxidationof phenols natureof the anthocyaninproduced.
(mono-and diphenols)to quinones,catalysedby a . Pelagonidin3-glucoside - brick red/orange.
group of enzymes namely polyphenol oxidases.
Theseenzymesare localizedin the membranes of . C yani di n3-gl ucosi de - red.
mitochondriaand chloroplasts. . D el phi ni di n3-gl ucosi de-bl ue to pur ple.
Ceneticmanipulations usingantisenseapproach
Manipulation of anthocyanin pathway
of polyphenoloxidasehas
to inhibit the synthesis
enzymes
been carried.Somesuccesshas been reportedin
preventing the discoloration of potatoes by this for differentreactions,
The enzymesresponsible
strategy. in the anthocyaninpathwayhave been identified.
 OF PLANTTRANSFORMATIONIRANSCENIC
Chapter50 : APPLICATIONS PLANTS 617

B y genet ic m a n i p u l a ti o n sa n d m u ta ti o n s,i t i s t r a n s g e n i cp l a n t s c o n t a i n i n g r i b o n u c l e a s ei n h i b i t o r
possible
to developflowerswith the desiredcolours.
Most of the flowers (roses, carnations
c hr v s ant he m u m sl a) c k b l u e c o l o u r d u e to the
absenseof the key enzyme flavonoids 3', 5'-
hy dr ox y las(F
e 3 ' 5 ' H ) th a tp ro d u c eds e l p h i ni di ne
3
gluc os ide. O n e c o m p a n yb , y th e n a m e F l o ri gene,
has geneticallymanipulatedand introducedthe Cenetic manipulations for improving the
geneencodingthe enzymeF 3' 5' H (fromPetunia nutritional quality of plant products are of great
hybrida)into the following plants. importance in plant biotechnology. Some success
has been achieved in this direction through
The world's first genetically modified (GM)
conventional cross-breeding of plants. However,
flower was introducedin '1996.lt was a mauve
this approach is very slow and difficult, and many
( bluis h)c olo u re d c a rn a ti o nw i th a tra d e name
a times will not give the traits with the desired
Moondus{'. Subsequently,many other flowers
improvements in the nutritional quality. Selected
have been oroducedand marketed.
examples of genetic engineering with improved
Gan GM-flowers be eaten? nutritional contents are described.

In majority of countries,flowers are used for A M I N O A C I D S O F S E E D

ornamental purposes and not usually eaten. STORAGE PROTEINS
However, in some countries like Japan,flower
petals are used for decoration of foods, and . l O f t h e 2 0 a m i n o a c i d s p r e s e n t i n t h e h u m a n s ,
frequentlyeaten also. This raises an important 0 a r e e s s e n t i a l w h i l e t h e o t h e r 1 0 c a n D e
questionaboutthe safetyof CM flowers,sincethey synthesized by the body. The 10 essential amino
are not thoroughly screened for human acids (EAAs)have to be supplied through the diet.
consumption.But the presentbelief is that the Cereals (rice, wheat, maize, corn) are the
s e c o l o u ri n gc h e m i c a lm o l ecul es) predominant suppliers of EAAs. However, cereals
ant hoc y anin(th
are naturalplant materials,and their consumption do not contain adequate quantity of the essential
may be in fact beneficialto health. amino acid lysine. On the other hand, pulses
(Bengal gram, red gram, soybean)are rich in lysine
G E NE T I C E N G IN EE R IN G F O R a n d l i m i t e d i n s u l f u r - c o n t a i n i n ga m i n o a c i d s ( t h e
M A LE S T ER IL IT Y e s s e n t i a lo n e b e i n g m e t h i o n i n e ) .

The plantsmay inheritmale sterilityeitherfrom Transgenic routes have been developed to

the nucleus or cytoplasm. cytoplasmic male improve the essential amino acid contents in the
sterility (cms) is due to the defects in the seed storage proteins of various crop plants.
mitochondiralgenome.lt is possibleto introduce
m ale s t er ilityth ro u g hg e n e ti cma n i p u l a ti o nwshi l e Overproduction of lysine
the femaleplantsmaintainfertility. by deregulation

In tobaccoplants,male sterilitywas introduced The four essential amino acids namely lysine,
by using a mitochondrialmutatedgene encoding m e t h i o n i n e ,t h r e o n i n e a n d i s o l e u c i n ea r e p r o d u c e d
t he enz y m e ri b o n u c l e a s eT. h e g e n e e n c odi ng from a non-essential amino acid aspartic acid
ribonucleasenamely barnasegene from Bacillus (Fig. 50.12).The formation of lysine is regulatedby
amyloliquefaciens was transferred to tobacco feedback inhibition of the enzymes aspartokinase
' plant s . T he ri b o n u c l e a sies to x i c to ta p e ta lcel l s, (AK) and dihydrodipicolinate synthase (DHDPS).
and thus preventsthe developmentof pollen, Theoretically, it is possible to overproduce lysine
ult im at ely le a d i n g to ma l e s te ri l i ty . By thi s by abolishing the feedback regulation. This is what
approach,transgenic plantsof tobacco,cauliflower, has been accomplished.
cotton,tomato,corn,lettuceetc.,with malesterility The lysine feedback-insensitivegenes encoding
have been developed. the enzymes AK and DHPDS have been
It is possibleto restoremale sterility in the respectivelv isolated from E.coli ano
aboveplantsby crossingthem with a secondsetof Cornynebacterium with appropriate genetic
618 B IOTECHNO LO CY

Asparticacid Construction of artificial genes to

produce proteins rich in EAAs
fnsUartot<inase Atternptsare being made to constructartificial
B-Aspartylphosphate genesthatcodefor proteinscontainingthe essential

Aspartic
l
B-
ami no aci ds i n the desi red propor t ion.Som e
successhasbeenreportedin the productionof one
syntheti c protei n contai ni ng 13% m et hionine
,semialdehYder Dihvdrodioico-
tinatesynfhase residues.
,/ \
{)
Homoserine 2, 3-Dihydropicolinate GE N E TIC E N GIN E E R IN G FOR
/\
/\
J\
II IMPROVING PALATABILITY OF FOODS
More than the nutritivevalue,tasteof the food
Methionine Threonine
I I
Lysine
is importantfor attractinghumans.lt is customary
+ to make food palataHe by adding salt, sugar,
lsoleucine fl avorsand many other i ngradi ents. lt would be
ni ce i f a food has an i ntri nsi ca llyappet izing
Fig. 50.12 : An overview of the biosynthetic pathway character.
ol some essentia! amino acids derived from aspartic
acid (Note : Lysine regulates its own synthesis by A orotein monellin isolatecifrom an African
feedback inhibition) plant (Dioscorephyllum cumminsil is about
100,000 sweeter than sucroseon molar basis.
Monel l i n gene has been i ntroduce dint o t om at o
manipulations, these genes were introduced into and lettuceplants.Somesuccesshasbeenreported
s oy bean and c anola plant s . T h e t r a n s g e n i c p l a n t s i n the producti onof monel l i n i n t hese plant s,
so developed produced high quantities of lysine. i mprovi ngthe pal atabi l i ty.

Transfer of genes encoding GOLDEN RICE - THE PROVITAMIN A

methionine-rich proteins
Severalgenes encoding methonine-rich proteins
have been identified.
. I n m aiz e, 21 KDa z ein w i t h 2 8 % m e t h i o n i n e .
o I n r ic e, 10 KDa pr olam in w i t h 2 0 % m e t h i o n i n e . B-carotene,
. I n s unf lower ,s eed album i n w i t h 1 6 % m e t h i o n i n e .
These genes have been introduced into some peopl esubsi sti ng
crops such as soybean, maize and canola.
The transgenic plants produced proteins with
high contents of sulfur-containingamino acids.
Production of lysine-rich
glycinin in rice
Cly c inin is a ly s ine- r ic hp r o t e i n o f s o y b e a n .T h e
gene enc oding gly c inin ha s b e e n i n t r o d u c e d i n t o
rice and successfullyexpressed.The transgenicrrce
plant s pr oduc ed gly c inin w i t h h i g h c o n t e n t s o f
iysine. Another added advantage of glycinin s
t hat it s c ons um pt ion in h u m a n s i s a s s o c i a t e d
with a reduction in serum cholesterol (hypo-
cholesterolaemiceffect).
E N R IC H E D R IC E
About one-third of the world's population is
dependenton rice as staplefood. The milled rice
that i s usual l y consumed i s al most def icit in
the provitaminA. As such, vitamin A
defi ci ency (causi ng ni ght bl i ndness)is m ajor
nutritional disorder worldover, particularly in
on ri ce.
To overcome vitamin A deficiency, it was
proposedto geneticallymanipulaterice to produce
B-carotene,in the rice endosperm.The presence
of p-carotene in the rice gives a characteristic
yellow/orange colour, hence the provitamin
A-enrichedrice are appropriately consideredas
C ol den R i ce.
ln Fig. 50.13 an outline of the biosynthetic
pathwayfor the formationof p-caroteneis given.
to produc eColden Rice
The geneti cmani pul ati on
requiredthe introductionof three genesencoding
the enzymes phytoene synthase, carotene
desaturase and lycopeneB-cyclase. lt took about 7
yearsto insertthree genesfor developingColden
R i ce.
Chapte r5 0 : AP PL IC AT IONOSF P L AN TTR A N S FOR MA TION IR A N S C E N
P LA
IC N TS 619

lsoprenoid units Golden Rice has met almost all the objections
i raised by the opponents of GM foods. However,
i Sequential
addition many people are still against the large scale
v
p r o d u c t i o n o f C o l d e n R i c e , a s t h i s w i l l o p e n d o or
Geranylgeranyldiphosphate
to the entrv of manv other CM foods. Another
IPhytoenesynthase argument put forth against the consumption of
I
Colden Rice is that it can supply only aboul 2O"h
J of daily requirement of vitamin A. But the
Phytoene
proponents justify that since rice is a part of a
I
I Carotenedesaturase m i x e d d i e t c o n s u m e d ( a l o n g w i t h m a n y o t h er
Y
I foods), the contribution of provitamin A through
Lycopene Colden Rice is quite substantial.
I
I
Recently (in 2004), a group of British scientists
I Lycopenep-cyclase
J have developed an improved version of Colden
p-Carotene R i c e . T h e n e w s t r a i n ,C o l d e n R i c e 2 c o n t a i n s m o re
(provitamin
A) t h a n 2 0 t i m e s t h e a m o u n t o f p r o v i t a m i n A t h a n i ts
p r e d e c e s s o rl.t i s c l a i m e d t h a t a d a i l y c o n s u m p t i on
Fig. 50.13 : An outline of pathway for the
of 70 g rice can meet the recommended dietary
biosynthesis of provitamin A (B-carotene).
aliowance for vitamin A.

Tlrlr 50.9 A selectedlist of transgeniccrop plants (GM cyopsapprovedin USA)for commercialuse

Crop plant Genetically altered trait Product name

Cotton lnsectresistance Bollguard
Glyphosateresistance Roundupready
Bromoxynilresislance BXN
resistance
Sulfonylurea
Maize lnsectresistance YieldGuard
lnsectresistance Maximizer
Glyphosateresistance Roundup Ready
ferietqrgg
9lqlsqirils LibertyLink
Ric e A enrichment
Vitamin Rice
Golden
Tomato Delayedripening FlavrSavr
Delayedripening EndlessSummer
Virusresislance
Ready
Roundup
ig!ill9l99
_9lrylqlgP
lnsectresistance Newleaf
Modifiedstarch
0ilseedrape(canola) resistance
Glufosinate lnn0val0r
Glyphosateresistance RoundupReady
Highlauricacid Laurical
hybrid
Malesterility
Squash Virusresistance ll
Freedom
Tobacco Virusresistance
Capsicum Virusresistance
Carnation llowercolour
Modified
620 B IOTE C HNO LO CY

Tlsle 50.10 Some examplesof transgeniccrop plants (( plantsl at the developmental stages

PIanl Gene transfer Trait t ra nsfer red/app Ii cati on (s)

For improvinghuman health
Tomato Phytoene
desaturase ProvitaminA (p-carotene)supplement
Canola y-Tocopherol erase
methyltransf VitaminE supplement
Sugarbeet fructosyl
Sucrose-sucrose erase
transf Fructans-lowcalorie to
alternatives
sucr0se
Rice Fenitin lronsupplement
Potato Antisensethreoninesynthase lncreasedmethionine levels
Potato Seedalbumin withallessential
Protein aminoacids
Tomato decarboxylase Increased
S-Adenosylmethionine lycopene levels
Tomato Chalcone isomerase Flavanols-act
asantioxidants,
reducerisk'ofcancer, heart
diseases
Arabidopsis lsoflavonesynthase lsoflavones-reduce
serum cholesterol,
andreduce osteooorosis
Canola Modified canierprotein
acyl-acyl theriskof heart
cr's-Stearateslower
thioesterase diseases
For increasedcrop yield
Rice Phosphoenolpyruvate
carboxylase lncreased of photosynthesis
efficiency
Tobacco PhytochromeA Avoidsshades
Lettuce acid(GA)oxidase
Gibberellic Inhibits
GAaccumulation andstem
growth(dwarfing)
Potato Phytochrome
B Increasedphotosynthdsis
andlonger
lifesoan

Others
Tobacco
andsoybean Pouo
Cytochrome Synthesis of epoxyfattyacidsfor
manufacture oJadhesivesandpaints
Rice Nicotianamine
aminotransferase Tolerance to lowironavailability
Tobacco Nitroreductase Reduces landcontaminationbv
trinitrotoluene

G E N E T IC E N GIN EE R IN G TO IN C R E A S E was activated by insertinga regulatorygene from a

VIT A M IN S A N D MIN ER AL S bacterium. This resulted in an efficient production
' The transgenicrice (Colden Rice) developed o f v i t a m i n E .
with high provitaminA contentis describedabove. Some workers are trying to increasethe mineral
Transgenic crop plantsare alsobeingdevelopedfor contentsof edible plantsby enhancingtheir ability to
increased production of other vitamins and absorbfrom the soil. Some successhas been reporteo
mi n e ra l s . w.ith reeard to increasedconcentrationof iron.

A transgenic Arabidopsis thaliana that can

produce ten-fold higher vitamin E (u-tocopherol)
thanthe nativeplant hasbeendeveloped.Thiswas
done by a novel approach.A. thalianapossesses
the biochemical machinery to produce a
compound close in structureto cr-tocopherol. A The very purposeof productionof transgenic
genethat can finally producec-tocopherolis also plantsis for theircommercialimportancewith high
present,but is not expressed.This dormantgene productivity.lt was in 1995-96,transgenicplants
 OF PLANTTRANSFORMATION/TRANSCENIC
Chapter50 : APPLICATIONS PLANTS 621

(potato and cotton) were, for the first time, made o Modified sensory attributes e.g. increased
availableto farmersin USA. By the Vear1998-99, sweetness as in thaumatin.
five major transgenic crops (cotton, maize,
soybean,canola and potato)were in widespread Concerns about transgenic plants
use. They accountedfor about 75"/oof the total The fearsabout the harmfulenvironmental ano
area plantedby crops in USA. hazardoushealth affectsof transgenicplantsstill
A selected list of transgenic crop plants exists,despitethe fact that there have been no
(approvedin USA)for the commercialuse is given reportsso far in this regard.
in Table50.9. Someexamplesof transgeniccrop The transferof almostall the transgenicplants
plants,which are at the developmental stagesare from the laboratoryto the crop fields is invariably
given in Table 50.10. These plants are carefully associatedwith legal and regulatory hurdles,
designedto give rise to products,which will besi desthe soci aland economi cconcerns.
im pr ov ehum a nh e a l tha n d i n c re a s eo f c ro p yi el d.
The major concern expressedby public (also
Goals of biotechnological acknowledged by biotechnologists) is the
improvements in crops development of resistancegenes in insects,
generationof super weeds etc. Severalremedial
There are about 30-40 crops that have been measures are advocated to overcome these
genet ic allym o d i fi e d ,a n d m a n y m o re a re b ei ng oroblems.
added. However, very few of them have got the
The farmersi n devel opi ngcountri esare much
c lear anc ef or c o m m e rc i a ul s e . A s e l e c te dl i st i s
worried about the seed terminator technology
alreadygiven in Table50.9.
which forcesthemto buy seedsfor everynew crop.
The ultimategoalsof geneticallymodified(CM) Thesefarmersare traditionallyhabituatedto use
crop plantsare listedbelow. the seedsfrom the previouscrop which is now not
o Resistance to diseases (insect,microorganisms). possibledue to seedtermintortechnology.
o lmprovednitrogenfixing ability.
o Highery iel d i n gc a p a c i ty .
e Resistance
to droughtand soil salinity.
o Betternutritionalproperties.
o lmprovedstoragequalities. A nother i mportantappl i cati onof geneti cal l y
transformedplantsis their utility as bioreactors
to
a Production of pharmaceutically important producea wide rangeof metabolicand industrial
c om pound s . products.Theseaspectsare describedin the next
Absenceof allergens. chaDter.
 f enet ic ally m odif ied pla n t s c a n b e m a n i p u l a t e d STARCH
\J to act as bioreactorsto produce a wide range
Starchis a polymerof glucose,and is composed
of biologic ally im por t an t c o m p o u n d s . T h e s e
of amyl oseand amyl opecti n. S tarchis used as a
inc lude c ar bohy dr at es ,lipid s a n d p r o t e i n s , b e s i d e s
food, feed and for industrialpurposes.Cenetic
t he s ec ondar y pr oduc t s . The c o m m e r c i a l p r o d u c t s
engi neeri ng
techni ques can be usedto m anipulat e
of plant s ar e us ef ul f or indu s t r i e so r f o r i m p r o v i n g
the quanti tyand qual i tyof starch.
t he hum an or anim al health .
The terms molecular farming/pharming or Increasing the production of starch
metabolic engineering of plants are also used to
B i osynthesiofs starch i n pl ants is a highly
c ollec t iv ely r epr es ent t he m o i e c u l a r b i o l o g i c a l
regulated process involvingthe key enzymeADP-
techniquesfor the synthesisof commercial products
glucose pyrophosphorylase. This enzyme rs
in plant s . M et abolic engi n e e r i n g o f t r a n s g e n i c
al l osteri cal l control
y l ed(feedbacki nhibit ion)by
plant s is gaining im por t ance i n r e c e n t y e a r t i r u n
attractive alternative to animals and micro-
metabolites such as phosphate.
or ganis m s f or t he m anufa c t u r e o f b i o l o g i c a l l y ADP-glucosepyrophosphorylase in E.coli was
important products. mutatedto alterits allostericproperties. Thismutant
gene was transferredand expressedin potato
plants.The transgenicpotato plants produced high
quantities of starch.

Synthesis of amylopectin starch

An outline of the biosynthetic pathways for the
Starchnormallycontains20-30% amyloseand
production of different carbohydrates is given rn
70-B0Toamylopectin.The relativeproportionof
Fig 51.1. As is evident, the important carbohydrates
amyl oseand amyl opecti ndetermi net he physico-
ar e s y nt hes iz ed and s t or e d i n d i f f e i 'e n t c e l l u l a r
chemi calproperti es gelsar e
of starch.A myl op ect in
compartments preferred
more stable and thereforeare in fooo
o Starch and its derivatives are synthesized in the processi
ng i ndustri es.
plas t ids .
The enzyme namely granule-bound starch
. Sugar sand s ugar der iv at i v e sa r e p r o d u c e d i n t h e
synthase(GBSS)is responsible for the synthesis
of
c y t os ol and ac c um ulat e t h r o u g h o u t t h e c e l l . amylose r,r,hiiesfarcf branching enzyme (SBE)
o Fr uc t anss y nt hes iz edand s t o r e d i n t h e v a c u o l e s . producesamyl opecti n(Fi g 51.1). By using an

622
Chaot er51 : T RA N S C E N IC
P L AN T SA S T | OR EA C TOR S 62a.

Cytosol

Sucrose

UDP-Glucose

1
Glucose1-phosphate
TREHALOSE
1
Glucose6-phosphate MANNITOL

1 myalnositol
Fructose6-phosphate
t +
I
I
I P IN ITOL
Triose-phosphate

,
GLYCOLYSIS

Fiq.51.1 : An outline of the metabolic pathway for the biosynthesis of carbohydrates and
theit imoorlant derivatives in a plant cell.

antisense approach, the enzyme C BSS was gene coding for this enzyme was transferredto
inhibitedin potatoplants Consequently, amylose- potatoplants.However,the transgenic potatoplants
free starchi.e. starchcontainingonly amylopectin did not show any enhancement in the synthesis
of
was produced. cycl odextri ns
due to vari ousreasons.

Synthesis of high-amylose starch FRUCTANS

High- am y losseta rc hw i th l i mi te d b ra n c h i n gi s Fructans,also referredto as polyfructans,are
useful as a food, feed and for certain industrial solublepolymersof fructose.They are synthesized
purposes.lt is possibleto produce high-amylose and storedin cellularvacuoles.Short-chain fructans
starchin potatoesby antisenseinhibitionof starch (oligofructans) are almostas sweetas sucrose. Thus,
b r anc hingenz y m eA a n d B (S BEA a n d SB EB). they can be usedas sugarsubstitutes in food and
soft drink industries.Another advantageis that
CYCLODEXTRINS fructans are not digestedin the gut and therefore,
they serveas low-caloriesweeteners.
Cyclodextrins are cone-shaped ringsformed by
6 - 8 gluc opy r ano sseu b u n i tsT. h e y a re h y d ro p h i li c Transgenic potato plant with fructosyl
in natureand can pockethydrophobiccompounds. transferase gene (from Streptomyced has been
Due to this property,cyclodextrinsare used as developed. Fructosyl transferase,being a key
therapeutic agents to solubilise hydrophobic enzymeenhancedthe synthesis of fructans.
pharmaceuticals such as steroids. ' In recent years, oligofructansof sugarbeet
A key enzyme responsible for the synthesisof (fructanbeet) with suitablegenetic manipulation
cyclodextrins from starch namely cyclodextrin have been produced.They serve as low calorie
hasbeen identified.A bacterial sweeteners.
glyaosyl transferase
624 B IOTECHNO LO CY

Commerciallyimportant
nolecular fannlng (metabollc eEglncerlngl

Compound Transgenicplant(s) Application(s)

Carbohydrates
Amylose{ree
starch Potato Food,
industrial
(amylopectin
starch)
Highamylose
starch Potato Food,
industrial
Cyclodextrins Potato pharmaceutical
Food,
Fructans potato,
maize,
Tobacco, sugarbeet Industrial,
food
Trehalose Tobacco Foodstabilizer
tipids
Mediumchainfattyacids Oil seedrape Food,industrial,
detergent
fatty
Mono-unsaturated acids Tobacco Food
fattyacids
Saturated Oil seedrape Food,confectioneries
(polyhydroxybutyrale)Cotton,oil seedrape
Bioplastics plastics
Biodegradable

TREHALOSE with differentcarbonsatoms.Someexamolesare

l i sted.
Trehaloseis a disaccharideproducedin plants
and microorganisms in response to osmoticstress. o Laurate - C 12
C e n e ti c ma n i p u l a ti o n sh ave been successful l y.
- Ct+
Myristate
carriedout for enhancedsvnthesisof trehaloseto
withstandabiotic stress(thatcausewater deficit). . Palmitate - C''u
Trehalose is usefulas a food preservative. Some . Stearate- Ct a
workersare attempting to producetrehalosein bulk . Ol eate-C ra,
r
quantitiesfor its utility in food industries.
By the actionof acyl-ACPthioesterases, the fatty
acids are releasedand exportedto the cytoplasm.
Here, they may undergocertain modifications-
elongation, desaturation (insertion of double
bonds), hydroxylationetc. These modified fatty
Plantoils are usefulas foods(abouttwo thirds), acids react with glycerol 3-phosphate(in the
and for industrialpurposes (about one third). The endopl asmi creti cul um)to fi nal l y f or m t r iacyl-
s f p l ant oi l s i ncl udethei r glycerols(TC). TC are transportedto lipid oil
i n d u s tri aal p p l i c a ti o n o
use in the manufacture of soaps, detergents, bodies and stored.The oil bodies of seedsalso
l u b ri c a n ts
a n d b i o fu e l s . contain proteins called oleosins,in the lipid
monol ayers (fordi scussi on
on ol eosi n s, Seep. 631) .
A n o u tl i n eo f th e s y n thesiof s oi l s (chemi cal l y
triacylglycerols) is depictedin Fig 51.2.Thereis an Someof the approaches of geneticengineering
involvement of plastids, cytoplasm and for the manipulation of plant oil biosynthesis are
endoplasmicreticulumfor the production of oils. briefly described.
Aftertheir synthesis, oils are storedin lipid bodies.
PRODUCTION OF SHORTER CHAIN
Acetatefrom the cytoplasm is taken up by the
FATTY ACIDS
plastidsand convertedto acetylCoA and malonyl
CoA. These two moleculesundergo a seriesof Majority of the plant oils contain more than
reactions,catalysedby the enzyme fatty acid ' I6-carbonse.g. pal mi ti c,steari c,ol e ic acids.O ils
synthase(FAS),to producefatty acyl carrierproteins with shorterchains (C6-C1a) arg more useful in
 Chapter51 : TRANSCENIC
PLANTSAS BIOREACTORS 625

Acetate
Plastid

Acetyl CoA ------* Malonyl CoA

I PalmitoylACP (C16)
(c1s)
i--+StearoVlACP Endoplasmic
OleoylACP (C16: 1) reticulum
Triacylglycerol
\ioesterase
"/
lipids Free fatty acid i
Diacylglycerol

1
Phosphatidicacid
Fattyacid modified
(elongation,desaturation,
hydroxylationetc.) Glycerol3-phosphate

Cytoplasm

Fig. 51.2 : An outline of the biosynthesis of triacylglycerols in plants

(FAS-Fatty acid synthase; ACP-Acyl carrier protein).

many industries - for the production of soaps, Some attemptsare being made to genetically
detergents,cosmeticsetc. manipulatecrops to yield oils with high contents
eruci c aci d. The approachesi ncl ude to
It is possibleto terminatethe hydrolysisof acyl- of
overexpress genesencodingthe enzymeselongases
ACP by specificthioesterases and produce high
proportionof selectedfatty acids. One acyl-ACP and transfer of genesto produceenzymesthat can
preferentially incorporate erucic acid into
thioesterase that specifical ly hydrolyses lauroyl-ACP
tri acyl gl ycerol s.
has been isolated from California bay tree
(lJmbellulariacalifornica).The gene encodingthis
enzvmehasbeenclonedand transferred to oilseed PRdDUCTION OF FATTY AcIDs wITH
MOD IFIE D D E GR E E OF S A TU R A TION
rape.The transgenicplantswere found to produce
oils with high proportionof lauric acid (12-carbon A fatty acid is said to be saturatedif it has no
fatty acid). double bonds.Saturatedand unsaturated (with on
or more double bonds)fatty acidsoccur in nature.
P RO DUCT I O N O F L ON G E R C H A IN It is possibleto geneticallymanipulatethe degree
FATTY ACIDS of saturationin fatty acids.
F or t he a p p l i c a ti o n a s i n d u s tri a l o i l s,
triacylglycerolswith longer chain fatty acids Production of unsaturated fatty acids
(> Cre) are preferred.Thus, erucic acid (22C)
Ol ei c aci d (C ,u:1)ri ch oi l s are usefulas food,
containingoils are usefulin industries.However, feed and in some industries. Therehas been some
e r h u ma nc o n s u mp ti on. successin the transferof antisense
e r uc icac id is un s u i ta b l fo gene encoding
By conventionalplantbreedingtwo distinctcrops the enzyme desaturase in oilseedrapeand soybean
of oilseedrape have been developed- high-erucic plants. These transgenicplants were found to
acid rape (HEAR)and low-erucic acid rape (LEAR). produceoils with very highproportionof oleicacid.

Biotechnology
[40]
626 B IOTE CHNO LO CY

Production of saturated fatty acids Several experimental studies are in progressto

p r o d u c e b u l k q u a n t i t i e s o f b i o p l a s t i c s i n p l a n ts.
Som et im es ,it is des ir able t o p r o d u c e o i l s w i t h
Two approaches are described hereunder
high contents of saturatedfatty acids. Successhas
(Fig 51.3).
been reported by use of antisenseRNA approach.
The objective wes to reduce the conversion of
PHB production in cytoPlasm
stearate(C',u : 0) to unsaturatedfatty acids such as
oleic ac id ( C.u, : 1) , linoleic a c i d ( C , u . , u : 2 ) a n d Startingfrom acetyl CoA, polyhydroxybutyrateis
linolenic acid (C.'u : 3). The enzyme stearoyl-ACP a three-stage pathway, involving the following
desaturase catalysesthe conversion of stearoyl-ACP enzymes (with corresponding gene).
to oleoyl-ACP. The gene encoding the enzyme o 3-Ketothiolase (phaA).
stearoyl-ACP desaturase has been isolated from
o Acetoacetyl CoA reductase(phaB).
Brassica rapa and its complementary sequence
c loned. The t r ans genic plan t s d e v e l o p e d b y t h i s o PHB synthase(phaC).
approach were found to contain high contents of
The three genes coding the respectiveenzymes
saturated fatty acid nameiy stearic acid, and low
have been isolated {rom Alcalig,eneseutrophus and
c onc ent r at ionof oleic ac id.
c l o n e d . T h e c y t o p l a s m o f p l a n t ce l i ( e .8 .
Arabidopsis\ contains 3-ketothiolase.Therefore, rn
PRODUCTION OF RARE FATTY ACIDS
t h e i n i t i a l e x p e r i m e n t so n l y t w o g e n e s ( p h a B a n d
There are some rare fatty acids, which are phaC) coding acetoacetyl CoA reductase and PHB
indus t r iallyim por t ant ,but not n o r m a l l y s y n t h e s i z e d synthase were transferred to develop Arabidopsts
in the plants. Some p.lantsproducing the rare fatty (Fig 51.3A). By this approach, the quantity of BHP
acids have been identified and the genestransferred prodr-rcedwas very low. Another limitation was that
f or t heir ov er or oduc t ion. the plants had stunted growth.

One good example is the production of

petroselenic acid that is found in coriancler
Tobacco olants were transformed with coriander
acyl-ACP desaturase that led to a high production
of petroselenicacid in tobacco plants. Petroselenic
ac id is c om m er c ially im por t a n t . O n o x i d a t i o n b y
ozone, it forms lauric acid (for use in soap and
detergent production) and adipic acid (for the
manufacture of nylon).
Some success has also been reported in the
increased production of several unsaturated fatty
ac ids t hr ough genet ic manipulation by
incorporating genes coding special. enzymes
namely front-end desaturasese.g. 1-linolenic acid,
ar ac hidonic ac id, r ic olenic a c i d .
BIODEGRADABLE PLASTICS
(BToPLASTTCSI
B i o d e g ra d a b l ep l a s ti c s (or bi opl asti cs)are
chemically polyhydroxy alkanoates(PHAs).They
are currentlybeingproducedin largequantitiesby
microbial fermentation.The structuresand the
biosyntheticpathwaysfor the productionof PHAs
a re g i v e n i n C h a p te r 3 0 . A mong the P H A s,
polyhydroxy butyrate (PHB) is the most important
one.
PHB production in plastids
T h e o r o b l e m s e n c o u n t e r e d i n th e a b o ve
approach were overcome by transferringall three
genes (phaA, phaB, phaQ of PHB synthesis and
targeting them to chloroplast (Fig 5I .3D. fo
achieve this, each gene was separatelyfused with
a coding sequence of transit peptid bound to
N{erminal fragment of Rubisco (ribulose 1,5,
b i s p h o s p h a t e c a r b o x y l a s e ) s u b u n i t p ro te i n . Th e
genes expression was carried out by CaMV 35S
promoter. TransgenicArabidopsis plants with each
gene construct were first developed. Then a series
of sexual crossingswere carried out between the
i n d i v i d u a l t r a n s f o r m a n t s . T h e t r a n s ge n i c p l a n ts
developed by this approach yielded good quantity
of bioplastics (about 14"h dry weight of the plant),
and in addition there was no observable adverse
affect on the growth or fertility of these plants.
Production of bioplastics in
cotton fibres
Cotton fibres containsthe enzyme p-ketothiolase.
Therefore,the genes for the other two enzymes of
PHB pathway (pha7 and phaQ from Alcaligenes
eutrophus were transferred into meristems of cotton
C hapt er51 : T RAN SC EN IC
PL A N T SAS B IO R EA C TOR S 627

(A)

, CoA reducrase
pnaal
+I
: 3-Hydroxy
I butyrylCoA

I PHuryntnu""
(phaQ
.f
J
Polyhydroxy- Flavonoids
butyrate

(B)
Plastid
Cytoplasm
AcetylCoA---- AcetylCoA
./
| ,-*"ro,n,o,u." ./
(PhaA) / \
|
AcetoacetylCoA MalonylCoA
AcetoacetylCoA
.
I RcetoacetylCoA
I reductase
@haB)
J
3-Hydroxy
butyrylCoA +
j PHnryntn"." lsoprenoids rlavoloios
j (Phac)
Polyhydroxybutyrate

Fig. 51.3 : Development of transgenic Arabidopsis for the biosynthesis of polyhydroxybutyrate (PHB)
(A) Cytoplasmic synthesis of PHB (B) Chloroplast (plastid) synthesis of PHB (Note : phaA, phaB and phaC
represent genes for the corresponding enzymes)

plan t by pa rticle b om bar dm ent .Adequat e s y nt hes r s e n g i n e e r i n g a p p r o a c h e s f o r i t s m a n u f a c t u r e i n

of PHB wa s re po r t ed in t he f ibr es of t r ans geni c p l a n t s a r e d e s c r i b e d a b o v e B u t t h e d i s a d v a n t a g e
cotto n Dla nts. with PHB production is that it is found as stiff and
bristle plastics, since it forms highly crystallrne
Production of polyhydroxyalkanoate polymers.
co-polymers
The other bioplastic, composed of
Th e most impo r t ant bioplas t ic of c om m er c ia l polylydroxyalkanoate (PHA) co-polymer is a
im p orta nce is po ly hy dr ox y but y r at e The genet i c polymer made up of longer monomers. lt is less
628 B IO TECHNO LO CY

crystallineand more flexible comparedto PHB. 2. C hoi ceof ti ssue.

The PHAsare producedfrom the intermediates of
3. Expression
strategies.
B-oxidationof fatty acids-notably3-hydroxyacyl
CoA. Some successhas been reported in the processing.
4. Post-translations
productionof PHAsthroughgeneticmanipulations 5. Recoverystrategies.
o{ peroxisomes and glyoxisomes.
Selection of crop species
The mostimportantconsideration for choosinga
plant as a bioreactoris that it should be readily
manipulatedto producea stabletransgenicline,
with stableexpression.
Molecular farming or metabolic engineeringof
proteinsin plantsis a novel approachthat makes Tobacco-the most preferred plant as a
plantsto serveas living factors (bioreactors)for the transgenicbioreactor : Tobaccois thb most widely
p ro d u c ti o no f p ro te i n sUse
. of ani mal s,ani malor used plant for the bioproductionof proteinsby
microbial cell culturesfor the bioproductionof researchers as well as biotechcompanies,for the
proteinshas beendescribed(Chapters15, 33, 41). following reasons.
Productionof humanor animal proteinsby plants
1. lt can be easilyengineered
and transformed.
is a more recentand novel approach.
2. Tobaccois an excellentbiomassproducer
The transgenic plants have the following
(about40 tonsof freshleaf/acreland against4 tons
advantagesas bioreactors,
by rice).
r Low cost of production.
3. Seed production is very high (about one
. Eukaryoticproteinprocessing.
mi l l i on seeds/pl ant).
. U n l i mi te ds u p p l y .
4. Tobaccocan be harvested severaltimes in a
o No spreadof animal bornediseases.
year.
. Reducedtime to market.
. Safeand environmental The major limitationsof tobaccoplant are- it is
friendly.
a regionalcrop and relativelylabourintensive, and
. No need for skilledworKers. it is not suitableto be fed to humansor animals.
Ceneticallyengineeredplantscan manufacture Other crop species as bioreactors : For the
a wide range of proteins. Theseinclude : developmentof edible products (e.9. vaccines)
. En z y mefo purposes. plantssuch as potato,tomato,banana,maizeand
s r i n d u s tri aal nd agri cul tural
. Lysosomal enzymes. lettuce are used. Some workers prefer to produce
proteins in the seeds of plants such canola,
o An ti b o d i e s(p l a n ti b o d i es).
soybean,corn, rice and barley. This is mainly
. Su b u n i vt a c c i n e s . becauseseeds can accumulateand store good
. O th e r b i o p h a rm a c e uti cal ors medi cal l yrel ated quantitiesof proteins.
oroteins.
Choice of tissue
The generalapproachesfor the productionof
desired proteins in plants are first described, The preferredtissuesfor protein production by
followed by the differentproductsof commercial plants include leaves,storageorgans(e.g.tubers)
importance. and seeds.Thischoiceof tissuemainlydependson
the followingfactors.
APPROACHES FOR PROTETN o Compatibilitywith the desiredprotein.
PRODUCTION IN PLANTS
. Correctprocessing.
The generalmethodologyfor the productionof
proteinsin plantsinvolvesthe followingaspects . S tabl eaccumul ati on.
1. Selectionof crop species. . Efficiencyof recovery.
ChA P I CT
51 : T R A N S C E N IC
P L AN T SA S B IO R E A C TOR S 629

Many proteinshave been producedin leaves w i th di sul fi debondsand sui tabl ymodi fi edami no
( par t ic ular ly
u s i n g to b a c c o p l a n t) e .g . l y s o somal aci dsi s i mportantfor [hi s purpose.
enz y m es ,m a mma l i a na n ti b o d i e s h , u ma n s erum
The changesthat occurafter(or sometimes even
album in.
during)the protein is synthesized are collectively
In recentyears,seedshavebecomean attractive referredto as post-translational modificationse.g.
high pr ot einp ro d u c ti o n(e .g .h i ru d i n )v e h i c l esdue phosphoryl ati on,hydroxyl ati on, carboxyl ati on,
to good storagecapacity and stability.lt is no glycosylation, proteolyticprocessing etc. Variations
surprisethat someof the seedproteinsretaintheir in post-translational changesare often limitingthe
biologic alac t i v i tyfo r s e v e rayl e a rs O . n e l i mi t ati on successfulprroductionand use of recombinanr
<lf seedsis that it is sometimesdifficultto recover proteinseither in plantsor even in other systems
the protein. (transgenianic mal sor cel l cul tures).
'fhe major limitations plants with regardto
Expression strategies of
post-translational modificationsof proteins are
There are two major approachesfor the
Iisted.
transgene expression in plantsto produceproteins-
stableintegration approachand useof plantviruses . Clycosylation (incorporationof carbohydrate
as transientvectors. moiety)is differentin plants.
1. Stableintegrationapproach: This strategyis . Plant glycosylationresultsin the productionof
most suitablefor the bulk productionof proteins complex glycanscontainingfucose or xylose,
par t ic ular lyin l e a v e s . l n th i s a p p ro a c h , the w hi ch do not occur i n humans.
transgeneis regulatedby a strong constitutive
promotersuchas 35Spromoter.Anotheralternative r Plantscannotperformspecializedcarboxylation
is to usetissue-specific promoter.By restricting the necessary for certainclottingfactors(ll, Vll, lX,
transgeneexpressionto a particulartissue (e.g. and X) and enzymes(proteinC).
seed),the yield of the protein can be substantially Due to abovelimitations,many a timesproteins
increased.In addition,the protein is stablefor a of transgenicplantsare inefficientor lessefficient
long time from weeksto years. i n thei r functi on, besi desexhi bi ti ng i mmuno-
A diagrammaticrepresentation of stablegene genecity.
expressionapproach for protein production is In recent years,cloning of genes responsible
depictedin Fig 51.4. for post-translationalmodificationsof proteins(e.g.
2. Transientexpressionby using plant viruses: glycation,carboxylation)have met with successto
Bioproduction strategiesinvolvingvirusesare useful producemore bi ol ogi cal l yacti veprotei ns.
for the protein productionat the desiredperiod
(discreteperiod).By genetic manipulations,it is Recovery strategies
possibleto insertthe desiredgene(for protein)into (purif ication strategiesf
the coat proteingeneof the virus genome.The so
A rational approach for a cost-effective
develooedinfectiousRNA virusesare introduced purificationstrategyis desirablefor the recoveryof
into the plantsfor the productionof proteins.
the proteins(downstream processing) producedin
Transient
expression systemby viruseshas been transgenic plants.The degreeof purityis dependent
used in tobaccoplantsby employing
successfully on the purposefor which the proteinis produced.
tobaccomosaicvirus (TMV).Tobaccoplantsat an For instance,industrialenzymesin general,do not
appropriateage were inoculatedwith genetically require high degree of purity. Sometimes,
modified TMV. Recombinantorotein could be purificationmay not be necessary at all. A good
extractedwithin 2-3 weeksof harvesting. exampleis the productionof the enzymephytasein
seeds.Phytaseacts on phytateand increases the
Post-translational processing avai l abi l i tyof phosphateto ani mal s. For thi s
The ultimate purposeof the use of plants as purpose,milledtransgenic seedscontainingplrytase
bioreactorsis to produce proteins with native can be directly added to the feed. This totally
conformation, good biological activity and avoids the purification/downstream processing
The specificfoldingof the protein
biocompatibility. costs.
630 B IOTECHNO LO G Y

plstsin ----+t...::F..r- {- Transgene (for desired protein)

argeting ) \
to vectors

lissue specific
promoter Terminator

Transgenicplant plant
Transgenic

Extractionof Extractionof desired

desiredprotein proteinfrom targettissues

Fig. 51.4 : Stable gene expression strctegy for the

For.a great majority of proteins,particularly
pharmaceuticalproteins, purificationstrategyis
absolutelyrequired.lt is thereforenecessaryto
develop efficient methods for downstream
processing.Some of the recoverystrategiesare
brieflydescribed.
Affinity tag-based purification : The use of
affinity tags for the purificationof recombinant
proteinshas been describedelsewhere(Chapter
worksin plantsystems
1l), and a similarstrategy as
well. The techniqueinvolvesthe fusionof desired
proteinwith anotherproteinor peptide(tag),which
can bi nd to l i gand,and thus the prot eincan be
i sol ated.Fol l ow i ng puri fi cati onof t he desir ed
protein, the affinity tag can be removed.Some
workers have been successfulin purifying the
enzyme glucocerebrosidase by this approach.
Purification through compartmentalization: lt
is possibleto directthe accumufationof proteinsin
a specificsub-cellularorganelle.This is achieved
by usingsignalpeptidesor fusion peptides.Once
the protein is localized,the technique of sub-
cellularfractionationcan be used for preliminary
isolationof proteinfollowed by its purification.
 : TRANSGENIC
PLANTSAS BIOREACTORS

The human neuropeptideleu-eukephalinhas referred to as oleosin partition technorogy

been compartmentalizedin the endoplasmic (Fig 51.5). The oil bodies along with the
oleosin_
reticulumand vacuolesby this strategy.Then by fusion proteins are quite stable within the seed and
appropriatepurificationtechniques,it is isolated. during the course of processing. The
Protein purification by production in oil pharmaceutical protein of interest remains stable
bodies : O i l b o d i e s a re n a tu ra l s u b -c e l l ul ar r e t a i n i n g i t s b i o l o g i c a l a c t i v i t y w i t h i n t h e s e e d sf o r
organellesthat store triacylglycerols(TC). Oil years. Further, the purification oleosin_fusion
bodiesare composedof triacylglycerols proteins is relatively easier.Hence oleosin partition
enveloped
by a phospholipidmonolayer. technology is preferredfor the purification of many
A groupof proterns,
referred lo as oleosins are associatedwith the pharmaceutically important proteins. Two
surfaceof the oil bodies. functional proteins namely hirudin ancr
p-glucuronidase have been recovered
by oleosin
Oil bodies are found in the oil/seedsthat partition technology.
representthe storageform of energy (as TC). Oil
seedscontain oleosin at a concentrationin the
Production of hirudin in
rangeof 2-1O% of the total seedproteins.
Brassica napus
Oil bodiescan be used for the productionof
Hirudin is a natural anticoagulant protein
foreign proteins in plants. The developmentof
produced in the salivary glands of leeches (Hirudo
transgenic plantsand the purificationof the desired
medicinalis). lt is an effective therapeutic agent as
protein by employing oil bodies are shown in
it specifically inhibits thrombin and blocks blood
Fig. 5t.5.
coagulation.
A transgenewith oleosin and desiredprotein
Till sometime ago, hirudin was produced by
can be constructed.By introducingthis gene,
employing bacteria and yeasts. The annual
transgenicplantsare deveioped.Oleosinstolerate
r e q u i r e m e n to f h i r u d i n i s v e r y h i g h , a n d i t i s a g o o d
foreign proteins;hence it is possibleto proouce
c a n d i d a t e f o r p r o d u c i n g i n t r a n s g e n i cp l a n t s .
oleosin-fusionproteinsthat accumulatein the oil
bodies. A synthetic gene encoding hirudin was fused
with Arabidopsrs
oleosin gene. An endopeptidase
T he plantm a te ri a l(m
s a i n l ys e e d so i l b o d i e s)are
factor Xa gene was inserted between hirujin and
crushed and centrifugedto separateinto three
oleosin genes.(The endopeptidasegene servesas a
fractions.
recognition site for the protease action). The
. A pelletof insolubleplant materialat the bottom transgene construct was introduced into Brassica
of the tube. napus.The so developed transgenicplants produce
oil bodies
containing oleosin-hirudin fusion
. An aqueo u s p h a s e c o n ta i n i n g s o l ubl e
protein. Hirudin can be purified by the procedure
componentsof the seedsat the middle.
described already and depicted in Fig 51.5.
. A floatinglayer at the top with oil bodiesand
Oleosin partition technology holds a great
their associated
oleosinfusionproteins. promise for the successfulcommercial produition
The top layer of oil bodies is isolated, of several pharmaceutically important proteins.
resuspended in a buffer and re-centrifuged.
The
concentratedoil bodies are treated with the PRODUCTION OF INDUSTRIAL
proteolytic enzyme protease that cleaVes the ENZYMES IN PLANTS
desired protein from oleosin. On further
centrifugation,the pharmaceuticallyimportant There is an ever-growing demand for indusirial
protein (aqueousphase)can be separatedfrom the enzymes. Existingsources may not be able to meet
oil bodies . the requirements. Large-scaleproduction of plant_
derived transgenic enzymes for commercial ano
OLEOSIN PARTITION TECHNOLOGY industrial purposes are preferred. Besides several
advantages of plants as bioreactors (See p. 628),
The purification of foreign proteins through there is no fear of spread of animal pathogens(lixe
their production in oil bodies (descr.ibedabove) is BSE)as is the case with animal production.
 B IOTECHNO LO CY
632

ttlr
Oleosinpromoter Oleosin Desiredprotein Terminator
Structure of a transgene
I
I Genetransfer
+

TransgenicPlant
I
Plantcrushed
I
+

Centrifuged
I Desired
YI
oleosin Protein
n
-F-E R n nfnTnffitriacylglycerol
{ (I (] {I
{\ i | \ l ] i I ll(|| i
\ \\--- lr -rl ,/
( Phospholipid
'monolayer
btdy-
I transterred
+
-;il

I Treatedwith
Protease
J
)
An oil bodY
 Chapter51 : TRANSCENIC
PLANTSAS BIOREACTORS 633

undigested phytate gets excreted and accumulates

in the soil and water, leading to eutrophication.
enzymes ploduced In plants and their
Transgenic plants capable of synthesizing
phytase in their seeds have been developed. These
Enzyme Application(s) seeds(no need to isolatethe enzyme) can be mixed
with the feed of animals, and fed. The phytase
cr-Amylase Foodprocessing
enzyme has successfully solved nutritionar
Avidin In diagnostic
kits (phosphate) and environmental (euirophication)
Cellulase Production
of alcohol
lrom problems.
wastes(cellulose)
B-Glucanase In brewing
industry Cellulase and xylanase

B-Glucuronidase ln diagnostic
kits T h e t w o e n z y m e s n a m e l y c e l l u l a s ea n d x y l a n a s e
Ligninperoxidase In papermanufacture are used in several industries- bioethanol, textile,
paper and pulp. In all these processes,they are
Phytase lmproved phosphate
basically involved in the degradation of plant
(byphytate
utilization
m a t e r i a l s ( p r e d o m i n a n t l yc e l l u l o s e ) .
breakdown)
By a novel approach, it is possible to produce
Trypsin Pharmaceutical
cellulaseand xylanasein the plants for which the
Xylanase Biomassprocessing,paper target itself
is the digestion of plant cell. The risk of
andtextileindustries autodigestionof the plant cells (by these enzymes)
is overcome by genetic manipulation to produce
thermostable enzymes. Thus, cellulase and
A selectedlistof industrialenzymesproducedin xylanase, produced
by transgenic plants, are
plantsand their importantapplications
transgenic is inactive at the temperatures
at which plants
given in Table 5I .2. Some of the important normally grow.
The activity of these enzymes is
enzymesare described. restored on heating the plant extracts. And these
extracts are very successfully used in many
Avidin and B.glucuronidase i n du s t r i e s .
The first proteins/enzymes that were produced While the recovery of enzymes from transgenic
in t r ans ge n i cp l a n ts (ma i z e ) a re a v i d i n and plants is often required, there are some instances
p- gluc ur on i d a sTeh. e y a re u s e di n d i a g n o s ti cki ts. where the crude extracts
can be directly used. The
industrial applications of cellulase, xylanase and
Trypsin phytase(describedabove) are some good examples.
Trypsinis an imptrrtantproteolyticenzyme and
its production by conventional recombinant PRODUCTION OF LYSOSOMAL
approachesis rather difficult. Trypsin is now ENZYMES IN PLANTS
produced in plants. lt has a wide range of Lysosomes are the cellular organelles
the productionof insulin,vaccines,
applications-for responsiblefor the degradationof macromolecules.
wound care etc. The degradative enzymes present in lysosomes
include proteases,nucleases,lipases,glycosidases,
Phytase phosphatases, phospolipases and sulfatases
Deficiency of a specific lysosomal enzyme results
Phytaseis a hydrolyticenzymethatcatalyses
the
in the accumulation of undegraded substrate
hydrolysisof phytate(inositolhexaphosphate) to
causing clinical manifestations.Some examples of
inositoland inorganicphosphate.
lysosomaldisordersand the correspondingenzyme
Phytateis presentin high quantitiesin many deficiencies are listed below.
plant seedsused as a feed to pigs and poultry
r Tay-Sachsdisease- c-hexosaminidase.
( m onogas t ri ca n i ma l s ).T h e s e a n i m a l s d o not
possessthe enzyme phytase;hence they cannot . Caucher's disease- glucocerebrosidase.
derive the nutrient phosphatefrom phytate.The . H u r l e r s y n d r o m e- i d u r o n i d a s e .
 634 B IOTECHNO LO CY

o Mucopolysaccharidoses - Several enzyme of PRODUCTION OF ANTIBODIES

(a group of diseases) glycosaminoglycan (PLANTIBODIESI lN PLANTS
degradation. A n t i b o d i e s o r i m m u n o g l o b u l i n s a r e th e d e fe n se
proteins produced in mammals. For details on the
A couple of lysosomal enzymes produced in
structure and functions of different immuno-
transgenic tobacco plants are described belorv.
globulins, refer Chapter 68.

Glucocerebrosidase The use of plants for commercial production of

antibodies, referred to as plantibodies, is a novel
Clucocerebrosidaseis a lysosomal enzyme that approach in biotechnology. The first successful
degrades glucocerebroside (predominantly
production of a functional antibody, namely a
' produced when erythrocytes are destroyed due to
m o u s e i m m u n o g l o b u l i n l g C l i n p l a n ts, w a s
aging or dam age) t o gluco s e a n d c e r a m i d e . T h e reported in 1989. This was achieved by developing
deficiency of this enzyme causes Caucher's two transgenic tobacco plants-one synthesizing
disease,characterized by swelling of spleen, liver, h e a v y 1 - c h a i n a n d t h e o t h e r l i g h t r - ch a i n , a n d
and bone dam age c aus ing e x t r e m e p a i n . crossingthem to generateprogeny that can produce
Gaucher's diseasecan be effectively treated by an assembledfunctional antibody.
administering the enzyme glucocerebrosidase. At
Production of secretory lgA (slgAl
present, this enzyme is obtained from human
plac ent a or f r om m am m a l i a n c e l l c u l t u r e s , b o t h SecretorylgA is an immunoglobulin that protects
being very costly. lt is estimated that a single against dental caries produced by Streptococcus
pat ient r equir es 10- 12 t o n s o f h u m a n p l a c e n t a mutans. For the production of slgA, transgenic
annually c os t ing $100, 000 t o 4 0 0 , 0 0 0 ! plants were developed to synthesize different
subunitsand then crossedto produce the functional
Glucocerebroside was the first lysosomal antibody (Fig 51.6).
enzyme produced in plants (Nicotiana tobacum). Four separatetransgenicplants synthesizingfour
The production process of this enzyme has been d i s t i n c t p i e c e s o f a n t i b o d y ( H - , L - , J- ch a i n s a n d
patented, and FDA approved it for use in humans.
secretory component) were developed. The plants
It is hoped that through the plant-basedproduction 'l
expressingH- and L-chains were crossed (cross )
of glucocerebrosidasq (marketed as Cerezyme), the giving plants that produce lgA. When these plants
Datientsof Caucher's disease can be treated in a were crossed (cross 2) with plants expressing
cost-effectivemanner.
J - c h a i n , t h e p r o g e n y p r o d u c e d d i m er i c l g A. Th e
dimeric lgA synthesizingplants were then crossed
u-L-lduronidase (cioss 3) with the plants expressing secretory
The deficiency of the enzyme iduronidasecauses component, the functional slgA could be produced.
Hurler's svndrome, the most common disorder of Secretory antibodies have many advantages.
defective mucopolysaccharidedegradation. They are resistantto proteolytic degradation;hence
t h e i r y i e l d i n p l a n t s i s s u b s t a n t i a l l yh i g h e r . sl g A a r e
Tobacco plants have been genetically
the predominant antibodies that protect against
engineered to produce cr-L-iduronidase.
mucosal infectionsof microorganisms.They bind to
antigensmore effectivelyand offer good protection
Limitations for the production of
The secretoryantibodies produced in transgenic
lysosomal enzymes in plants
plants have been tested on humans. When slgA
Both the enzymes referred above are was topically applied to teeth, it could prevent the
glycoproteins(glucocerebrosidase and iduronidase). colonization l:y Streptococcus mutans up to 4
As described elsewhere (See p. 629), appropriate months. This is comparable to the protection
glycation of animal proteins is not feasible in plants. offered by immunoglobulins produced through
This poses a major problem for the production of hybridoma technology. This was possible despite
lysosomal enzymes in plants. Through a series of some structural di{ferences between olantibodies
genetic manipulations, biotechnologists were and monoclonal antibodies. lt appearsthat there is
successfulin achieving the desired glycation for the no difference in the binding propertiesbetween the
production glucocerebrosidaseand iduronidase. two tvpes of antibodies.
 P L AN T SA S B I OR E A C TOR S
Chaot er51 : T R A N S C E N IC 635

Heavy

Cross3

Fig.51.6: Production of secretory l9A (slgA) molecule in transgenic plants'

636 BIOTECHNOLOCY

IN

Tmrr 51.3 A selectedlist of antibodies(plantibodies)producedin transgenicplants

Antibody Targetedagainst lransgenic plant Application

lmmunoglobulins (lg)
slgA(hybrid) mutans
Streptococcus Tobacco Dentalcaries
lgG(guy's-13) mutans
Streptoccus Tobacco Dentalcaries
lgG(Co17-14) Surfaceantigen Tobacco Coloncancer
lgG(antiHSV-2) Viralantigen Soybean Herpes virus
simplex

Single-chainFv (scFv)
scFv(T84.66) Viralantigens Cereals cancer
Carcinoembryonic
scFv(38C13) Viralantigens Tobacco Lymphoma

Production of other antibodies Transgenicplants provide an alternatesystemfor

Thr ough genet ic m anipu l a t i o n s , i t h a s n o w
become possible to produce a wide range of
ant ibodies in t r ans genic pla n t s - w h o l e a n t i b o d i e s ,
ant igen- binding f r agm ent s , s i n g l e - c h a i n v a r i a b l e
f r agm ent ant ibodies . So m e examples of
plant ibodies and t heir appl i c a t i o n s a r e g i v e n t n
Table 51.3.
It is striking to note that despite certain (described on p. 629) can be used to produce
differencesin glycosylation of antibodies produced v a c c i n e s i n p l a n t s . S o m e e x a m p l e s o f va cci n e s
in plant s and anim als , t he pl a n t i b o d i e sa r e b y a n d produced in plants are given in Table 51 .4.
lar ge c om par able in t heir f u n c t i o n w i t h a n i m a l
antibodies.
PRODUCTION OF VACCINES IN
PLANTS
Vaccination is an effective approach for proper
healt hc ar e of m illions of p e o p l e i n t h e w o r l d .
Sometimes, the cost of the vaccines is coming in
the way, and consequently,thousands of children The need for use of edible vaccines arose from the
become susceptibleto preventable diseases.
Vaccinesare designedto elicit immune response
wit hout c aus ing t he dis ease . I n t h e c o n v e n t i o n a l
appr oac h, v ac c ines c om p o s e d o f k i l l e d o r
attenuated disease-causing organisms are
adm inis t er ed.This m et hod is s o m e t i m e sa s s o c i a t e d
with side effects. With the advances in molecurar
biology, recomhinant subunit vaccines that are
effective in preventing the disease,without causirt5l
side effects have been identified.
There are many production systems for the
synthesis of recombinant vaccines. These include
mammalian cell cultures, yeast, bacteria and insect
c ell c ult ur es , and t r ans genica n i m a l s .
the production of recombinant vaccines.The major
advantage of vaccine production in plants is the
direct use of edible plants tissues for oral
administration. By use of edible vaccines or veggie
vaccines (vegetable vaccines), the problems
associated with purification of vaccines can be
totally avoided.
The stable or transient expression system
The procedures adopted for the production of
vaccines by plants, and the use of plants as edible
subunit vaccinesare given in Chapter 16.
Edible vaccine
Plant-basedproduction systemsare designed to
p r o v i d e l o c a l l y a v a i l a b l e e d i b l e v a c c in e s, a t l o w -
cost, for easy administration to millions of people.
fact that a large number of people in the developing
world are the victims of enteric diseases. Edible
vaccines provide mucosal immunity against
infectious agents.
Choice of plants for edible vaccines : Most of
work on the production of vaccines in plants was
i n i t i a l l y c a r r i e d o u t i n t o b a c c o p l a n t th a t i s n o t
edible. These vaccines are now being produced in
e d i b l e p l a n t s s u c h a s b a n a n a , t o m a t o , an d to so m e
extent potato. For use in animals, the common
fodder crops or food crops can be considered.
Some workers consider banana as an ideal
system for the production of edible vaccines. This
is mainly because it is grown in most parts of the
Chapter51 : TRANSCENIC
PLANTSAS BIOREACTORS 637

Tnerr51.4 Some examplesof vaccinesproducedin transgenicplants

Recombinantprotein (vaccine) Transgenicplant Application (protection against)

Hum an
Heatlabile B
enterotoxin Tobacco E. coli
CtoxA andCtoxB subunits
Cholera Potato Vibriocholerae
Envelope
surfaceprotein Tobacco/potato Hepatitis
B
Capsidprotein Tobacco/pctato Morwalkvirus
Rabiesvirusglycoprotein Tomato Rabiesvirus
Animal
Virusepitope
VP, A.ltalfalArabidopsis Footandmouthvirus
ViralepilopeVP, Blackeyed
bean Milkenteritis
virus
Viralglycoprotein Maize/tobacco Porcine
coronavirus
lromVP, capsidprotein
Peptide Arabidopsis Canineparvovirus

world, and eaten raw. Another advantage is that
child ren (to whom v ac c ines ar e m os t needed) l i k e
e atin g b an an a.
The procedures adopted for the production of
vaccines by plants, and the use of plants as edible
su bu nit vaccin es ar e giv en in Chapt er 16.
The future of edible vaccines : Despite several
limitations, biotechnologistssee a great hope for
the new wave of transgenic veggie vaccines. But it
may take severalyears before the banana or tomato
replaces the needle as a vehicle for vaccrne
delivery!

Delivery of vaccine to the gut : Vaccines,being PRODUCTION OF THERAPEUTIC

proteins, are likely to be degraded in the stomach.
This is true to some extent. However, it is now
known that the orally administered plant materials
(e dib le vaccin e) c an induc e im m une r es pons e.T h e
particulate materialsof plants are more effective in
th is reg ard .
Food-based tablets to replace edible vaccines :
It is true th at the edible v ac c ines m ay wor k wel l i n
the laboratoryor under controlled conditions. There
is a d ifficu lty of dos e adjus t m ent when ed i b l e
vaccines are consumed as a part of foodstuff. Some
workers prefer to discontinue the direct use of
plant materials, and prefer to opt for food-based
tablets containing a known dose of vaccine. This
a pp roa ch is b ei ng applied t o v ac c ines pr oduc ed i n
tomatoes.
Limitations of edible vaccines : Direct
consumption of transgenic fruit or vegetable or
food-based tablets may create some problems.
PROTEINS IN PLANTS
The general approaches,and the production of
industrial enzymes, lysosomal enzymes, antibodies
and vaccines in transgenic plants have been
described in the proceeding pages Many of these
products actually representtherapeutic proteins or
biopharmaceuticals e.g. lysosomal antibodies,
vaccines.Therefore,they must also be referredagain.
ln Table 51 .5, a selected list of therapeutic
proteins, and the plants producing them, along
with the applications is given.
The major limitations of protein production by
transgenic plants are the low product yield andthe
difficulties associated with recovery. With
improvements in genetic engineering and recovery
processes, it is hoped that the production of
t h e r a p e u t i c p r o t e i n s w i l l s u b s t a n t i a l l yi n c r e a s e r n
the coming years.

The risk of loss of vaccines by the action of Ghloroplasts in the production of

enzymes in stomach and intestine. therapeutic proteins

The possibility of allergic reactions as they enter Chloroplasts are capable of folding the foreign
circulatio n. proteins, besides bringing out most of the post-
638 B IOTECHNO LO CY

Trsrr 51.5 Selectedexamplesof therapeutic plotcins producedin transgenicplants

Therapeuticprotein Transgenicplant Application(s)

Hernoglobin Tobacco Bloodsubstitute
Serum albumin Tobacco Burns/fluid bloodextender
replacement,
Prolein
C Tobacco Anticoagulant
Hirudin Canola Anticoagulant
u.,-lnterferon Rice Viralprotection,
anlicancer
B-lnterferon Rice/tobacco/turnip for hepatitis
Treatment B+C
T-lnterferon Tobacco Phagocyteactivalor
Collagen Tobacco As collagen
Somatotrophin Tobacco As growthhormcme
Trypsin
inhibitor Maize Transplant
surgery
c,-Antitrypsin Rice Cysticfibrosis
Erythropoietin Tobacco As mitogen
Lactoferrin Potato Antimicrobial
growth
Tissue factor Tobacco As mitogen
Enkephalin CanolalArabidopsis Opiate
Angiotension
converting
enzyme Tobacco/tomato Hypertension
Glucocerebrosidase Tobacco Gaucher's
disease
u-Galactosidase Tobacco Fabrydisease
Trichosanthin-cr Tobacco HIVtherapy,
cancer

tra n s l a ti o n aml o d i fi c a ti o nA. s the chl orool asts

are a lnterferon s
inheritedmaternally, they will not spreadvia pollen a Serum albumin
to non-transgenic systems. This type of o Hemoglobin
biotechnological approach will help to get
a Proinsulin
clearance from the regulatory bodies of
biotechnology.Some of the therapeuticproteins a Monoclonal antibodies
p ro d u c e di n c h l o ro p l a s ts a re l i sted. a Protective antigen of Baciilus anthracis.
f crta in be ne f ic ial m ic r oor ganis m s , pr es enl i n
\- the so il, ar e k nown t o inf luenc e t he pla n t
growth, development and yield. These bacteria and
fungi may provide growth-promoting products to
plants or inhibit the growth of soil pathogenic
microorganisms (phytopathogens), which hinder
the plant growth. The former is the direct effect
while the latter is the indirect effect of growth-
pro motin g ba cte r ia in plant s .
The growth-promoting activity of micro-
orga nismsa nd th e biot ec hnologic alappr oac hesa r e
describ ed b riefl y wit h r es pec t t o t he f ollowi n g
aspects.
1 Biolo gical nit r ogen f ix at ion.
2 Biocontrol of phytopathogens.
J. Brote rtrltz er s .
N itro ge n is an es s ent ial elem ent of m a n y
oro mole cu les, th e m os t im por t ant beir r g nuc le i c
:cid s an d a mino ac ids . Alt hough nit r ogen is t h e
-rost abundant gas (about B0%) in the atmosphere,
-e ithe r a nima ls n or plant s c an us e t his nit r ogen t o
.,nth esize b iolo gic al c om pounds . Howev er , t he r e
=-e ce rtain micr oor ganis m s on whic h t he liv i n g needed by the plant comes from nitrogen fixing
: an ts (an d a nim als ) ar e dependent t o br i n g bacteria. These are two types of nitrogen fixrng
-'i.og en into th eir biologic al s y s t em s .
The phenomenon of fixation of atmospheric
nitrogen by microorganisms is known as
diazotrophy and these organisms are collectively
referred to as diazotrophs Diazotrophs are
biological nitrogen fixers, and are prokaryotic in
nature.
NITROGEN CYCLE
An outline of the nitrogen cycle is depicted in
Fig. 52.1. Nitrogen enters the soil with the deposits
of dead animals and plants, and urea of urine.
These waste materials (proteins, urea) are
decomposed by soil bacteria into ammonia and
other products. The ammonia is converted ro
nitrite (N02) and then nitrate (NOr) by certarn
bacteria belonging to the genera Nitrosomonas
and Nitrobacter. The nitrate is degraded by
various microorganisms to release nitrogen
t h a t e n t e r s a t m o s p h e r e .T h i s a t m o s p h e r i c n i t r o g e n
is taken up by the nitrogen fixing bacteria
(present on the roots of leguminous plants)
and us'ed for the synthesis of biomolecules (e.g.
amino acids) As the animals consume the
leguminous plants as food, the nitrogen cycle rs
complete.
NITROGEN FIXING BACTERIA
lt is estimated that about 5O"k of the nitrogen
m i c r o o r g a n i s m s - a s y m b i o t i ac n d s y m b i o t i c .
639
640 B IOTECHNO LO CY

Soil

Nitrosomonassp
Nitrobactersp

Fig. 52.1 : An outline of the nitrogen cycle.

Asymbiotic nitrogen fixing bacteriado not interactwith plantsother than the

microorganisms
The gaseous nitrogen of the atmosphere is
dir ec t ly and independent ly u t i l i z e d t o p r o d u c e
nit r ogen- r ic h c om pounds . Wh e n t h e s e n o n -
symbiotic organisms die, they enrich the soil with
nitrogenouscompounds. Severalspeciesof bacteria
and fungi can do this job e.g. Clostridium
pasturianum, Azatobacter chrooccum.
The mechanism of nitrogen fixation by
asymbiotic bacteria is not clearly understood. lt is
believed that nitrogen is first converted to
hy dr ox y lam ine or am m oniu m n i t r a t e , a n d t h e n
incorporated into biomolecules.
Symbiotic nitrogen fixing
microorganisms
naturalhosts.
The relationshipbetweenthe symbioticbacteria
and the legumesis well recognized. On the rootsof
l egumes, thereare a numberof nodul es( swellings)
in which Rhizobiumsp thrive.Thesebacteriatrap
atmosphericnitrogenand synthesizenitrogen-rich
compounds(aminoacids,proteinsetc) usedby the
l egumes.A t the same ti me, the l egum essupply
importantnitrogencompoundsfor the metabolism
of R hi zobi a.
The growth of legumeshas been known to
enrichthe soil fertility.This is due to the fact that
the concentrationof nitrogencompoundsin the
soil increasesas a result of the presenceof
svmbiotic bacteria. For this reason normally,

These microorganisnrs live together with the

plant s in a m ut ually be n e f i c i a l r e l a t i o n s h i p ,
phenomenon referred to as symhiosl's. The most
important microorganisms involved in symbiosis
belong to two relatedgeneranamely Rhizobium and Bacterial species Host plant(s)
Bradyrhizobium. These symbiotic bacteria also Rhizobiumleguminosarun Pea,broadbean
referred to as nodule bacteria are Cram negative, Rhizobiummelilati Alfalfa
flagellated and rod-shaped. The host plants
Rhizobiumfredii Soybean
harbouringthese bacteriaare known as legumese.g.
Rhizobiumloti Lotus
soybean, peas, beans, alfalfa, peanuts,and clover.
Rhizobiumelti Mungbean,kidneybean
Each one of the species of Rhizobium and Rhizobiumciceri Chickpea
Bradyrizobium are specific for a limited number japonicun
Bradyrhizobium Soybean
of plants, which survive as the natural hosts
Bradyrhizobiu
m elkanii Soybean
(Table 52.1). lt is norn, clearly known that these
 Chapt er52 : C R OW T H -PR O N 4 O T | N
BAG C T E R TA
tN P LA N TS 641

nitrogen fertilizers are not needed in the fields o2

cultivated lesumes.
l,-tro
M E CHA NI S M OF N IT R O GE N F IXA T IO N J
OxyLHb
Insidethe root nodulesof leguminousplants,
the bacteriaproliferate.Thesebacteriaexist in a
f or m t hat has n o c e l l w a l l .
J
The bacteriaof the nodulesare capableof fixing
nitrogenby meansof the specificenzymenamely
nitrogenase.

Nitrogenase
+H 2
Nitrogenaseis a complex enzyme containing
two oxygen sensitivecomponents.ComponentI
has two o-protein subunits and two B-protein n
subunits,24 moleculesof iron, two rnoleculesof
molybdenum and an iron molybdenum cofactor NITROGENASE
COMPLEX
(FeMoCo).Component ll possesses t\,vo o-protein
subunits(differentfrom that of componentl) ano a
Fig. 52.2 : Nitrogen fixation in symbiotic bacteria.
lar genum berof i ro n m o l e c u l e s .
ComponentI of nitrogenase catalyses the actual
conversionof N, to ammoniawhile componentll
Hydrogenase
donateselectronsto component I (Fig. 52.2).
During the course of nitrogen fixation by
Leghemoglobin nitrogenase, an undesirablereactionalso occurs.
A proteincomparable of hemoglobinin animals That is reduction of H+ to H2 (hydrogengas).For
has been identified in the nodules of leguminous the production of hydrogen,ATP is utilized,rather
planfs.Leghemoglobin (tHb) containsiron and is red wasted. Consequentlythe efficiencyof nitrogen
in colour.lt is an oxygenbindingprotein.The heme fixation is drastically lowered. lt is possible
partof leghemoglobin is synthesizedby the bacterium theoreticallyto reduce the energy wastage by
while the protein(globin)portionis producedby the recyclingH, to form H+.
hostplant.Leghemoglobin is absolutelynecessaryfor ln f act, some strains of Bradyrhizobium
nitrogenfixation.The noduiesthat lack LHb are not japonicum in soybean plants were found to use
capableof fixingnitrogen. hydrogenas the energysource.Thesestrainswere
It is LHb that facilitatesthe appropriatetransfer found to possessan enzyme namely hydrogenase.
of oxygen(by formingoxylHb) to the bacteriafor Recyclingof the hydrogengas that is formed as
respiration to produceATP.And energyin the form a byproduct in nitrogen fixation is shown in
of ATP is absolutelyrequiredfor nitrogenfixation. Fig. 52.3.
Another imoortantfunction of LHb is that it It is advantageousfor nitrogenfixation if the
preventsthe damagingeffectsof direct exposureof symbiotic bacteria possess the enzyme
O, on nitrogenase. hydrogenase.However, the naturally occurring
strainsof Rhizobium and Bradhyrhizobiumdo not
ln Fig.52.2,the fixationof nitrogenby symbiotic
normallypossess the gene encodinghydrogenase.
bacteriais depicted.As the oxylHb supplies02,
bacterialrespirationoccurs.The ATP generatedis
usedfor fixing nitrogento produceammonia.The GE N E TIC MA N IP U LA TION S FOR
complexreactionis summarizedbelow. NITROGEN FIXATION
N, + B H+ + Be - + 1 6 A T P The nitrogen-fixingbacteria that are closely
associatedwith the world'sfood supplyare among
2NH-
JZ
+ H " t + 1 6 AD P + 1 6 P i the favouredorganismsfor geneticmanipulations.

a':technology [41]
642 B IOTECHNO LO CY

procedure basically involves the identification and

characterizationof clones fronr a wild-type library
to restorenitrogen fixation in the various mutants of
the original organism.

The nif genes were first isolated from the clone

banks of the diazotroph Klebsiellapneumonia.This
organism is found in soil, water and human
i n t e s t i n e ,a n d i t s m o l e c u l a r b i o l o g y i s w e l l kn o w n .
The isolation of nif genes by genetic
Fig. 52.3 : Action of the enzyme hydrogenase for complementation from K. pneumonia is depicted rn
recycling of hydrogen gas. Fig, 52.a, and involves the following steps.

1. The wild-type DNA of K. pneumonla (Nif+)

was cut with restriction endonucleases.
Biot ec hnologis t sc ons ider t h e f o l l o w i n g p o s s i b i t i e s
for effective utilization of diazotrophs (or the A clone bank was constructed with a vector
process of diazotrophy) as natural biological a n d m a i n t a i n e d i n E . c o l i .
fertilizers. 2. K. pneumonia cells are exposed to a
. Cene alterations in Rhizobium sp to improve mutagenic agent. This may result in the mutation of
nitrogen fixing efficiency,and bacteria-hostplant nif genes to form Nif- cells.
interactions
3. The Nif- K. pneumonia cells are then
. Cenetic engineering of Rhizobium sp so that it conjugated wilh E.coli cells, carrying the clone
can form a symbiotic relationship with non- bank on a vector.
legum inous plant s s uc h a s w h e a t , r i c e a n d c o r n .
4. The transformantsof K. pneumonia possessing
. Transfer of genes for nitrogen fixation from Nif+ phenotype can be selected by growing the
Rhizobium sp to other bacteria such as c e l l s o n a m i n i m a l m e d i u m t h a t d o es n o t co n ta r n
Agrobacterium tumefaciens. The so developed fixed nitrogen.
A. tumefaciens can infect several imoortant
5 . T h e D N A f r a g m e n t i n t h e p l a sm i d th a t
crops (e.9. tomato, tobacco, petunia) and fix
c o n t a i n s n i f g e n e s , w h i c h c o m p l e m e n ts N i f
nitrogen.
mutation in K. pneumonia can be isolated.
Some other considerationsinclude the insertion
of nitrogen-fixinggenes into plants or even animals The isolation of nif genes will be more effective
inc luding hum ans . This ap p r o a c h , w h i c h e n a b l e s if a series of independently derived Nif mutants
the plants, animals and humans to directly trap and are employed in genetic complementation
utilize nitrogen from the atmosphere is rather experiments. This approach increasesthe likelihood
theoretical. Life would be certainly different if of different nif genes isolation. Further, by using
introduction of nitrogen-fixing genes in man can DNA hybridization probes of nlf genes, DNA clone
dispense with the habit of eating daily! bank of K. aneumonia can be screened.This allows
the identification of additional genes that are
Despite several possibilities and optimistic involved in nitrogen fixation.
predictions, the success in the genetic engineering
in relation to nitrogen-fixation is very limited, and
Nitrogenase gene cluster
briefly described below.
The entire set of nitrogenase genes from
GENETTC ENG,NEEBING OF K, pneumonia has been identified. Some highlights
N'TBOGENASE GEVE are listed.

It is absolutely necessaryto identify, isolate and r The nitrogenaes(nifl genes are located as a single
characterize the nftrogen-fixing genes, nifgenes to cluster, occupying approximately 24kb of the
undertake any kind of genetic manipulations. bacterial genome.

Genetic complementation is the common . There are seven distinct operons that encode 20
technique used for the isolation nif genes. The different oroteins.
l ra p t er 52 : CRO W T H -P R OMOT INBA l N P LA N TS
C C T E R IA
Fig. 52,4 : An outline of the method for the isolation of nit genes from K. pneumonia
by genetic complementation.
o :, the nif Benes transcribe and translate in a
. el I-coordinatedfashion.
. -'e nif genes are under the regulatorycontrol of
-.., qenes namely nifA and nifL.
-r
: :1ough the nifgenes have been characterized
: -" iru tion of this or ganis mf or biologic al nit r ogen
" ,- ,,r is almost insignificant. However, the nif
-
-: : j. of K. pneumonia have been used as
' --: za tion p rob es t o ident if y nif genes f r om t he
- \ . : clon e ba nks of diaz ot r ophic or ganis m s
-,- --;larly Rh izo bium s p) . lt is now k nown t hat
, -- .: all the nitrogen-fixing bacteria possess
. - a: type o f n ifge ne c lus t er s .
Hanipulation of nif genes
Sonre workers have been successful In
-'-rciucing extra copies of nitrogenaseregulatory
For
-=-es namely nifA and nifL into diazotrophs.
643
K. pneumonia
(Nif*cells)
K. pneumonia
(Nif- cells)
Conjugated,grown on
mediumlackingnitrogen
K. pneumonia (Nit-)
transformants
I
+
,iffi"
isolated
instance, insertion of more nifA genes into
Rhizobium meliloti resulted in an increased
b i o m a s s p r o d u c t i o n i n a l f a l f a p l a n t s .T h i s i s d u e t o
the fact that nitrogen fixation in these plants is
m u c h i n c r e a s e d .B u t t h e m a j o r l i m i t a t i o n o f t h i s
approach is that the geneticallyengineeredbacteria
have a diminished growth rate. As a result, these
newly developed bacteria loss their efficiency as
plant-growth promoting agents.
Despite continuous efforts by several groups of
workers, no significant success has been reported
in the transfer of nif genes into plants. The major
limitationsare listed.
. Transferof 24 kb nif gene cluster has not been
effective since the normal cellular concentration
of oxygen would inactivate nitrogenaseenzyme.
A n y r e d u c t i o n i n O , r e s u l t si n c e l l d e a t h .
. Transferof one or two genes of nlf gene cluster is
usetess.
 644 B IOTECHNO LO CY

. Thereare no plantpromotersthat can respondto genes (about 20 nodA- nodX have been
nifA regulatoryprotein. Consequently,it is not identified in diazotrophs. The nod genes are
possibleto turn on the nlf genesin transgenic broadly divided into three groups.
plants. . Common genes
. The plant cells cannot process nif gene r Host-specificgenes
which are multigenicin nature.
transcripts,
o Regulatorygenes.
For the variousreasonsgiven above,it has not
been so far possible to introduce functional The functions of each one of the nod genes in
nitrogenfixationcapabilityinto plants. nodulation have not been clearly identified. Further,
many more new nod genes are being discovered
every year.
GENETIC ENGINEER'NG OF
HYDBOGENASE GENE
Manipulation of nod genes?
The role of the enzyme hydrogenasein
The process of nodulation is complex thlough
promoting nitrogen fixation has already been
the participation of a large number of nod genes,
described(Seep. 641).Hydrogenase is synthesized
besides various other factors-concentration of
by hup (fiydrogen uptake)genes, which are not
nutrients,soil temperature,light, CO, concentration
p re s e nitn th e n a tu ra l l yo ccurri ngR hi zobi alstrai ns.
etc.
Considerable variationshave been identifiedin
Despite attempts by severalworkers, no success
hydrogenases from differentorganisms.There are
has been reported to enhance the ability oi
different types of hydrogenases, which usually
Rhizobium sp for nodulation through genetic
contain subunits. Different eenes code these
manioulations.
subun its.

lsolation of hydrogenase genes

T h e te c h n i q u e o f g eneti c compl ementati on
(similarto that describedfor nif geneson p. 642)
Phytopathogens can drasticallyreducethe crop
can be successfully employedfor this isolationof
yield, which may be in the range of 25-100"/o.
hydrogenase (hup)genes.In fact, hup geneswere
Chemical agentsare commonly used to control
first isolatedfrom E.coli by this approach.Later,
with ill-healthaffectson
them. This is associated
hup genes from B.japonicum have also been
humans,besi desenvi ronmental pollut ion.
isolated.
There are certain bacteriathat can lessenor
Manipulation of hup genes preventthe deleteriouseffectsof plant pathogens-
fungi or bacteria.And thus, they promote plant
The hydrogenase(hup7 genes have been
growth by indirectmeans.
introduced into R.leguminosarum.These Hup+
strainsof bacteria,when inoculatedinto legumes, Plantgrowth-promoting bacteriaare capableof
resultedin highernitrogenfixation. producinga wide range of substancesthat can
restrictthe damagecausedby phytopathogens to
G E N E T IC E N GIN EE RIN G OF plants. The important plant growth-promoting
N OD U L A T IO N G E N E S antibioticsand certain
substancesare siderophores,
enzymes.
Establishmentof nodules on the roots of
leguminousplants is a prerequisitefor nitrogen
SIDEROPHORES
fixation. Certain genes involved in nodulation
namely nod genes have been identified in There are some bacteriain the soil that can
Rhizobium melitoti. The technique of genetic synthesizea low molecular weight (400-1,000
complementation (similarto that describedlor nif Daltons)iron-bindingpeptides,collectivelyreferred
geneson p. 642) has been used to isolatenod to as siderophores. Siderophores have high affini\
genes from R. melitoti. A large number of nod to bind to iron in the soil ancitransportit to the
Chapter52 : CROWTH-PROMOTING lN PLANTS
BACTERIA 6,45

m ic r obial c el l . T h i s i s re o u i re d s i n c e i ron i s
s i n c e 1 9 8 9 . T h i s t r a n s g e n i co r g a n i s m p r o d u c e st h e
essential, and cannotdirectlyenterthe bacteriadue
antibiotic agrocin 84 that is toxic to crown gall
to a very low solubility. disease-causingAgrobacterium tumefaciens. By this
r
biocontrol approach, the crop yield of almond trees
The growth-promoting bacteria, through
and peach trees can be improved by preventing
can take up largequantitiesof iron
siderophores
crown gall disease.
from the soil, and make it unavailablefor the
growth and existenceof fungal pathogens.
This is
ENZYMES
possiblesincethe siderophoresproducedby fungi
have a very low affinity when compared to Certain plant growth-promoting bacteria are
sideroohoresof bacteria. capable of synthesizing some enzymes that can
T her eis no h a rm to th e p l a n tss i n c eth e y can degrade fungal cell walls and lyse them. These
grow at a much lower concentration of iron in the enzymes include chitinase, p-l,3-glucanase,
s oil. protease and lipase.

Genetic manipulations for siderophores Genetic manipulation for enzymes

It is now clearly established that siderophores The genes responsiblefor encoding the enzyme
can preventthe proliferationof phytopathogens. lt n a m e l y c h i t i n a s e a n d B - g l u c a n a s e h a v e b e e n
is thereforelogicalto think of siderophoregenesfor isolated and characterized. Chitinase gene has
more effectivebiocontrolof plant pathogens. been isolated from Serralia mercescens ano
introduced into Rhizobium meliloti and
Pseudobactinis a siderophoresynthesizedby Trichoderma harzianum. The genetically
plant growth-promotingbacteria Pseudomonas engineered organisms displayed an increased
putida. By genetic complementationand other antifungal activity. By this biocontrol approach, the
techniquesof molecular biology, at least five phytopathogen Rhizoctonia solani has been
separategene clustersinvolved in pseudobactin effectively control led.
oroductionhave been identified.
Ceneticmanipulationfor improvedsynthesis of
siderophore is not an easyjob, sinceit is a complex
pr oc es sinv olvi n ga l a rg en u m b e ro f g e n e s .S ome
success, however,hasbeenreportedin cloningthe It i s esti mated thatmorethan 100 mi l l i ontonsof
genesfor iron siderophorereceptorsfrom certain fixed nitrogen are needed for global food
plant growth-promoting bacteria,and introducing production.The useof chemical/synthetic fertilizers
them into other bacteria. is the common practiceto increasecrop yields.
Besidesthe cost factor, the use of fertilizersis
A NT I B I O T I C S associ ated w i th envi ronmental
ool l uti on.
Certainantibioticsproducedby plant growth- Scientists are on a constantlook for alternate,
promotingbacteria,can preventthe growth and cheap and envi ronmental -fri endlsources y of
proliferationof plant pathogens.Some of the nitrogenand other nutrientsfor plants.The term
antibiotics synthesizedby pseudomonadsare blofertilizersis used to refer to the nutrient inputs
agrocin 84, agrocin434, herbicolin,phenazine, of biological origin to support plant growth. This
oom y c in,py r ro l i n i tri n . can be achieved by the addition of microbial
inoculantsas a sourceof biofertilizers.
Genetic manipulations for antibiotics
B i oferti l i zersbroadl y i ncl udesthe fol l ow i ng
It is possibleto enhancethe growth-promoting tategories.
activityof the bacteriaby insertinggenesencoding o Symbioticnitrogenfixers
the synthesis of antibiotics.
o Asymbioticnitrogenfixers
ln f act, a geneticallyengineeredbiocontrol
. Phosphate solubilisingbacteria
bacteriumof Agrobacteriumradiobacferhas been
dev eloped,an d i s b e i n g ma rk e d c o m m e r ci al l y o Organi cferti l i zers.
646 B IOTECHNO LO CY

The most important microorganisms used as

biofertilizersalong with the crops are listed in
Table 52.2. rsed as blofertilizers along wlth the crops

Some of the important features of these Category/microorganism Useful for crops

biofertilizers
are brieflydescribed.
(pulses,
Symbioticnitrogenfixers Legumes oilseeds)
S YMB IO T IG N IT R O GE N FIX E R S um Ieguminos
Rhizobi arum,
R. meliloti.
R. ciceri.
The diazotrophic microorganisms are the Bradyrhizobiumjaponicun
symbioticnitrogenfixersthat serveas biofertilizers.
e.g. Rhizobium sp and Bradyrhizobiumsp. The Asymbiotic nitrogenfixers Wheat,rice,sugarcane,
Azobacte
r, Azospiillum jower,
vegetables
detailson these bacteria.arithsoecial referenceto
nitrogen fixation must be referred now. Many Bluegreenalgae(Anabaena, Rice
attemptsare beingmade (althoughthe successhas Plectonemia)
Nostic.
been limited)to geneticallymodify the symbiotic Phosphate solubilizing
bacteriafor improvingnitrogenfixation. bacteria Pulses
Thiobacillus,
Bacillus
Green manuring (Glomus)
Mycorrhiza Pulses
It i s a fa rn ri n gp ra c ti cew herei nthe l egumi nous
plants which are benefited by the symbiotic
nitrogenfixing bacteriaare ploughedinto the soil Azolla
and a non-leguminouscrop is grown to take
benefitsfrom the already fixed nitrogen.Creen Azolia is an aquaticfern, which containsan
endophytic cynobacterium Anabaenaazollaein the
m a n u ri n gh a sb e e n i n p racti cei n Indi afor several
l eaf cavi ti esprovi di nga symbi o t icr elat ionship.
centuries.lt is a natural way of enriching the soil
But
is usefulas biofertilizer.
with nitrogen,and minimizingthe useof chemical Azollawith Anaebaena
due to certain limitation (listedbelow),the use of
fertilizers.Rhizobiumsp can fix about 50-150 kg
n itrogen/hectare./an num. Azolla has not become popular.
. Azollaplant requiresadequatesupplyof water.
ASYMBIOTIC NITROGEN FIXERS . lt can be easilydamagedby pestdiseases.
The asymbiotic nitrogen-fixingbacteria can . Azolla cultivationis labour intensive.
directlyconvertthe gaseousnitrogento nitrogen-
rich compounds.When theseasymbioticnitrogen PHOSPHATE SOLUBILIZING BACTERIA
fixers die, they enrich the soil rvith nitrogenous
Certain bacteria(e.g. Thiobacillus,Bacillus)are
compounds,and thus serve as biofertilizerses
capable of converting non-availableinorganic
Azobacter sp, Azospirillum sp. phosphorus presentin the soil to utilizable(organic
or inorganic)form of phosphate. Thesebacteriacan
Blue.green algae (cynobacteriaf
also produce siderophores, which chelateswith
Blue-greenalgae multiply in water logging iron, and makes it unavailableto pathogenic
conditions.They can fix nitrogenin the form of bacteria.Thus,besidesmakingphosphate available,
organic compounds(proteins,amino acids).The the plants are protectedfrom disease- causing
term algalization is used to the process of mrcroorSantsms.
cultivationof blue-greenalgae in the field as a
sourceof biofertilizer. Mycorrhizas
BIue-green algae, besides fixing nitrogen, Mycorrhizasare the fungus roots (e.g. Glomus
accumulatebiomass,which improvesthe physical sp) with distinctmorphologicalstructure.They are
propertiesof the soil. This is usefulfor reclamation developedas a resultof mutualsymbiosisbetween
of alkalinesoilsbesidesprovidingpartialtolerance certain root-inhabitingfungi and plant roots.
to pesticides.Cynobacteria are particularlyuseful Mycorrhizas areformedin plants,which are limited
for paddy fields. The most common blue-green with nutrientsupply.Theseplantsmay be herbs,
algae are Azobactersp and Azospirillum sp. shrubsand trees.
 lN PLANTS
BACTERIA
Chapter52 : GROWTH-PROMOTINC 647

For the developmentol mycorrhizas,the fungus . Freefrom environmental pollution.

may be located on the root surface . Besidesnutrientsupply,someother compounds,
(ectomycorrhizas) or inside the root 1e:ndo-
which promoteplant growth,are also produced
mycorrhizas). e.g. plant growth hormones,antibiotics.
In recent years/ an artificially produced o B i oferti l i zers i ncrease physi co-chemi cal
inoc ulum of my c o rrh i z a fu l n g i i s u s e d i n crop propertiesof the soil, soil texture,a4d water
fields. This practice improvesplant growth and hol di ngcapaci ty.
yield, besidesproviding resistanceagainst biotic
(pathogens)and abiotic (climatic changes)sfress" . R ecl ai mati on of sal i neor al kal i nesoi l i s possi bre
Mycorrhizasalso produceplant growth-promoting by usi ng bi oferti l i zers.
substances. . Biofertilizersimprove the tolerance of plants
againsttoxic heavymetals.
ORGANIC FERTILIZERS
r Plantscan better withstandb!otic and abiotic
There are severalorganic wastes,which are stresses and improvein productyield.
us ef ulas f er ti l i z e rs .-T h e si nec l u d e a n i ma l d ung,
ur ine,ur bang a rb a g es,e w a g ec/ ro p re s i d u easnd oi l LIMITA TION S OF B IOFE R TILIZE R S
cakes.A malorityof thesewastesremainunutilized
. Biofertilizers cannot meet the total needsof the
as organicfertilizers.Thereexistsa good potential
for the development of organic manures from these plants for nutrient supply.
wastes. . Theycannotproducespectacular results,as is the
casewith syntheticfertilizers.
B E NE F I T S O F B IO F E R T IL IZ E R S
Consideringthe advantages and disadvantages
o Low costand easyto produce.Smallfarmersare of biofertilizers, a realisticand pragmaticapproach
immenselybenefited. is to usecombinationof biofertilizers and synthetic
. Fertilityof the soil is increasedyear afteryear. ferti l i zers for opti mumcrop yi el d.
 f)lant
v
breadersare alwaysinterested
to improve Biochemical
I t he agr ic ult ur al pr oduc t i o n . T h i s i s p o s s i b l e t o
produce new/improved plant varieties with the
desired characters. An important task of plant
breedersis the correct identification,and continueo
maintenance of the plants with
improved
characters: There are different types of markers
used for this purpose. The basic requirement of a
marker is that it must be polymorphic i.e. it must
exist in different forms. The polymorphism can be
detected by three different types of markers
1. M or phologic al m ar k e r s
2. Bioc hem ic al m ar k er s
3. M olec ular m ar k er s .
Morphological markers
The morphological or phenotype markers
representthe quantitativetraits that can be visually
detected e.g. altered leaf morphology, albinism,
dwarfism etc. Morphological markers may be
dominant or recessive. These markers are not
widely used to detect polymorphism these days due
to the following limitations.
. M or phologic al m ar k er s i n f l u e n c e d b y t h e
environment, and thus may not represent the
des ir ed genet ic v ar iat ion.
. Majority of the visible markers may not be
required in the plant improvenrent programmes.
648
markers
The proteins produced by gene expression are
also used as markers in plant breeding programmes.
The most commonly used are isoenzymes, Ihe
different molecular forms of the same enzyme. The
isoenzymes can be separated by electrophoresis
and identified. However, the polymorphism of
isoenzymes is rather poor. Hence, they are not
widely used as markers.
A molecular marker is a DNA sequencein the
genome which can be located and identified. As a
result of genetic alterations (mutations, insertions,
deletions), the base composition at a particular
location of the genome may be different in different
plants. These differences, collectively called as
p o l y m o r p h i s m s c a n b e m a p p e d a n d i d e n ti fi e d .
Plant breeders always prefer to detect the gene as
the molecular marker, although this is not always
possible. The alternative is to have markers which
are closely associated with genes and inherited
togethe!'.
T h e m o l e c u l a r m a r k e r s a r e h i g h l y r e l i a b l e a n cl
advantageousin plant breeding programmes
. Molecular markers provide a true representations
of the genetic make up at the DNA level.
Chaot er53 : M OL E C U L AR
MA R K ER -AID ED
P LA N TB R E E D IN C 649

For instance,the diseaseresistantplant may have a

s h o r t e rD N A f r a g m e n tw h i l e t h e d i s e a s e- s e n s i t l v e
plant may have a longer DNA fragment (Fig. 53.1).
Molecular markers are of two types.
1. B a s e d o n n u c l e i c a c i d ( D N A ) h y b r i d i z a t i o n
( n o n - P C Rb a s e d a p p r o a c h e s ) .
2. Based on PCR amolification (PCR-based
approaches).

MARKERS BASED ON
DNA HYBRIDIZATION
The DNA piece can be cloned, and allowed to
hybridize with the genornic DNA which can be
detected Marker-based DNA hybridization is
widely used. The rnajor limitation of this approach
is that it requires large quantities of DNA and the
use of radioactivity (labeled probes).

Restriction fragment length

polymorphism (RFLPI

RFLPwas the very first technology employed for

the detection of polymorphism, based on the DNA
E
sequence differences.RFLPis mainly based on the
ShorterDNA
fragment altered restriction enzyme sifes, as a result of
m u t a t i o n s a n d r e c o m b i n a t i o n so f g e n o m i c D N A .
An outline of the RFLP analysis is given in
Fig. 53.2, and schematically depicted in Fig. 53.3.

Fig, 53,1 : Basic principle of molecular marker

detection (screening of genotypes for the lsolationof genomicDNA
identification of DNA markers).
ny
I oigestion
I restriction
endonucleases
. They are consistent and not affected by J
DNApieces
environmental
factors.
. Molecularmarkerscan be detectedmuch before
I
J
developmentof plantsoccur. Separatedby agar gel
electrophoresis
. A largenumberof markerscan be generatedas I
per the needs.
+
I
DNA piecestransferred
Basic principle of molecular to membranefilter
markeY detection I
I Incubatedwith
Let us assumethat there are two olantsof the I clonedand
labeledprobes
samespecies-onewith diseasesensitivityand the J
other with diseaseresistance.lf there is DNA RFLPbandsdetected
markerthat can identifythesetwo alleles,then the
genomecan be extracted,digestedby restriction Fig. 53.2 : An outline of restriction fragment length
polymorphism (RFLP) analysis as a molecular
enzymes,and separated by gel electrophoresis.
The
marker in plant breeding.
DNA fragments can be detectedby theirseparation
650 BIOTECHNOLOCY

Hindlll MARKERS BASED ON

I PCR AMPLIFICATION
200bp 800bp Polymerasechain reaction (PCR) is a novel
PlantA techniquefor the amplificationof selectedregions
of DNA (for detailson PCR,ReferChapterB). The
Hindlll EcoRl advantagewith PCRis that even a minutequantity
I I of D N A can be ampl i fi ed. Thus, PCR- based
molecularmarkersrequire only a smallquantity of
200bp 450 bp 350bp
DNA to startwith.
Plant B
markersmay be divided into two
PCR-based
types.
Digestionby
restrictionenzymes 1. Locus non-specificmarkers e.g. random
Gel electrophoresis
ampl i fi ed pol ymorphi cD N A (R A P D) ;am plif ied
fragmentl engthpol ymorphi sm (A FLP) .
,--/t-'-
2. Locusspecificmarkerse.g. simplesequence
\
TJ repeats(S S R );si ngl e nucl eoti depo lym or phism
With enzyme With enzyme (S N P ).
Hindlll EcoRl
Random amplified PolYmorPhic DNA
800bp (RAPDI markers
350bp R A P D i s a mol ecul armarker ba sedon PCR
amol i fi cati on.A n outl i neof R A P D i s depict edin
200bp 650bp Fig. 53.4. The DNA isolatedfrom the genome is
denatured,the template moleculesare annealed
Monomorphic Polymorphic
pattern pattern w i th pri mers,and ampl i fi edby P C R.Singleshor t

Fig. 53.3 : A schematic representation of restriction lsolationof

fragment length polymorphism (RFLP) analysis as genomicDNA
molecular marker.
II
+
Thg procedure_basically involvesthe isolationof DNA
Denatured
genomicDNA, its digestionby restriction enzymes, II
separation by electrophoresis, and finally +
h y b ri d i z a ti o nb y i n c u b a ti ngw i th cl oned and Annealingof DNA
labeled probes(Fig. 53.2). templalewith primers

Basedon the presenceof restrictionsites,DNA I

fragmentsof differentlengthscan be generatedby J
usingdifferentrestriction enzymes.ln the Fig. 53.3, strand
Complementary
two DNA moleculesfrom two plants(A and B) are synthesis
s h o w n . l n o l a n t A, a mutati onshas occurred II
leadingto the loss of restrictionsite that can be +
digestedby EcoRI.The resultis that when the DNA Amplifiedproducts
molecules are cligested by the enzyme by gel electrophoresis
Hindlll,thereis no differencein the DNA fragnrents and identified
separated. However, with the enzyme
Fig. 53.4 : An outline of random amplified poly-
EcoRl, plant A DNA moleculesis not digested
morphic DNA (RAPD) analysis as a
w h i l e p l a n t B D N A m o l ecul e i s di gested.Thi s molecular marker in plant breeding.
resultsin a polymorphicpatternof separation.
 Chapt er53 : M O L E C U L AR
MA R K ER -AID ED
P LA N TB R E E D IN C 651

oligonuc leo ti dperi m e rs(u s u a l l ya 1 O -b a speri mer) 5'ArCTTAGaac

catAtATT 3'
can be arbitrarily selected and used for the
3'TGAATCttg gtaTT{A 5'
am plif ic at ioD o f th e g e n o m e(w hi ch
n N A s e g m e n ts
m ay be in dis tri b u teth
d ro u g h o uth t e g e n o me).The
amplifiedproductsare separated on electrophoresrs
and ident if ie d .
Based on the nuclebtide alterationsin the
genom e/t he p o l y mo rp h i s ms
o f a m p l i fi e d D N A
sequences differ which can be identifiedas bends 5'CTTAGaac _catA 3'
on gel electrophoresis. Cenomic DNA from two 3',Cttg gtaTTA 5'
different olants often results in different I Lioation
with
amplificationpatternsi.e. RAPDs.This is basedon
the fact that a particularfragmentof DNA may be Jrinie'.
generated from one individual,and not from others. s',-CTTAGaac_63ttr_ t'
This represents polymorphlsmand can be usedas 3' Cttg - gtaTTA 5'
-
a molecuiarmarkerof a particularspecies. I
I Preselectiveamplification
Amplified fragment length (PCR)emOloVing single
|
polymorphism (AFLP| nucleotideextension
I
+
A F LP is a n o v e l te c h n i q u e i n v o l v ing a
combinationof RFLPand RAPD.AFLPis basedon
the principleof generation of DNA fragmentsusing o Cttg-gtaTTA- 5'
restrictionenzymesand oligonucleotideadaptors
( or link er s )a, n d th e i r a m p l i fi c a ti o b
n y PC R .Thus,
t his t ec hnio u e c o m b i n e s th e u s e fu l n essof Selectiveamplification(PCR)
restrictiondigestionand PCR. employingadditional
nucleotideextension
The DNA of the genome is extracted.lt is
subjectedto restrictiondigestionby two enzymes
(a rare cutter e.g. Msel; a frequent cutter e.g.
EcoRl).
The cut endson both sidesare then ligated 3' Cttg- gtaTTA- 5'
to known sequencesof oligonucleotides(Fig.53.5). cat-
PCRis now performedfor the preselectionof a
Fig. 53.5 : A diagrammatic representation of the
fragmentof DNA which has a single specific
amplified fragment length polymorphism (AFLP)
nucleotide. By this approach of preselective
(Note : The lower case lefters represent the
amplification, the pool of fragments can be reduced
from the original mixture.In the secondround of
amplificationby PCR,three nucleotidesequences
are amplified.Thisfurtherreducesthe pool of DNA
fragments to a manageable level (< 100). information. By AFLR a large number of
Autoradiographycan be performed for the polymorphicbandscan be producedand detected.
detectionof DNA fragments.Use of radiolabeled
primers, and fluorescently labeled fragments Sequence tagged sites (STSI
ouic k ensA F LP .
Sequencetaggedsitesrepresentunique simple
A F LP anal y s i s i s te d i o u s a n d re q u i re sthe copy segmentsof genomes,whose DNA sequences
inv olv em entof s k i l l e dte c h n i c apl e rs o n n e lH. ence are know n,and w hi ch can be ampl i fi edby usi ng
s om e peopleare n o t i n fa v o u ro f th i ste c h n i q u e.In PCR.STSmarkersare basedon the polymorphism
recentyears,commercialkits are made available of simple nucleotiderepeatse.g. (CA)n, (CT)n,
f or A F LPanaly s i s . (CAA)netc.on the genome.STShavebeenrecently
AFLPis very sensitiveand reproducible.lt does developedin plants.When the STS loci contain
not require prior knowledge of sequence simple sequence length polymorphisms (SSLPs),
652 B IOTE CHNO LO CY

S TS . The markerscreeningmethodsmustbe efficient,

th e y a re h i g h l yv a l u a b l ea s mol ecul armarkers.
loci havebeenanalysedand studiedin a numberof reproducibleand easyto carry out.
p l a n ts p e c i e s . . The anal ysi sshoul dbe economi cal .

Microsatellites MOLE C U LA R B R E E D IN G
Microsatellites are the tandemly repeated With rapid progressin molecularbiology and
multicopies of mono-, di-, tri- and tetra nucleotide geneticengineering, there is now a possibilityof
motifs.In someinstances, the flankingsequenceof
improvingthe crop plantswith respectto yield and
the repeatsequences may be unique.Primerscan quality.The term molecularbreedingis frequently
be designedfor such flankingsequences to detect used to represent the breeding methods that are
the sequencetagged microsatellites (STMS).This
coupled with genetic engineering techniques.
can be done by PCR.
lmprovedagricultureto meetthe food demands
Sequence characterized amplified of the world is a high priority area. For several
regions (SCARsl years,the conventionalplantbreedingprogrammes
(althoughtime consuming) havecertainlyhelpedto
SCARsare the modifiedforms of STSmarkers. improve grain yield and cereai production.The
They are developedby PCRprimerthat are made
develoomentof dwarf and semi-dwarfvarietiesof
for the endsof RAPDfragment.The STS-converted
riceand wheathavebeenresoonsible for the 'Green
RAPDmarkersare sometimes referredto as SCARs. Revolution',which has helped to feed millions of
SCARsare usefulfor the rapid developmentof STS poverty-strickenpeoplearoundthe world.
marKers.
Many developmenton the agriculturefront are
expected in the coming years as a result of
mol ecul arbreedi ng.

Li nkage anal ysi s

Selectionof the desiredtraitsand improvement Linkageanalysisbasicallydealswith studiesto
of crops has been a part of the conventional correlate the link between the molecular marker
breedingprogrammes. This is predominantly
based and a desiredtrait. This is an importantaspectof
on the identificationof phenotypes.lt is now an molecularbreedingprogrammes. Linkageanalysis
accepted fact that the phenotypes do not has to be carried out among the populationsof
necessarilyrepresentthe genotypes.Many a times several generationsto establishthe appropriate
the environmentmay markthe genotype.Thus,the l i nkage.
plant'sgeneticpotentialis not truely reflectedin
ln the earlieryears,linkageanalysiswas carried
the phenotypicexpression for variousreasons.
out by use of isoenzymesand the associated
The molecularmarkerassisted selectionis baseo polymorphisms. Molecularmarkersare now being
n f D N A markersthat l i nV used. The techniquesemployedfor this purpose
o n th e i d e n ti fi c a ti o o
representthe plant traits. These traits include have alreadybeen described.
resistance to pathogensand insects,toleranceto
abiotic stresses, and variousother qualitativeand QUANTITATIVE TRAIT LOCI
quantitative traits.The advantage with a molecuiar
These are many characteristics controlled by
marker is that a plant breedercan selecta suitable
severalgenes in a complex manner.Some good
marker for the desiredtrait which can be detected
examplesare growth habit, yield, adaptabilityto
well in advance. Accordingly, breeding
environment,and diseaseresistance.These are
programmes can be planned.
referred to as quantitative traits. fhe locations on
The following are the major requirements{or the chromosomesfor thesegenes are regarded as
r a rk e ds e l e c t i oni n pl ant breedi ng quantitativetrait loci (QTL).
th e mo l e c u l a m
. The marker should be closelv linked with the The major problem,the plant breederfaces is
desiredtrait. how to improvethe a complexcharactercontrolled
Chapt er53 : M O L E C U L AR
M AR KE R -A ID EPLA
D N TB R E E D IN C
by ma ny ge ne s . lt is not an eas y job t o m anipu l a t e
multip le g en es in genet ic engineer ing.Ther ef o r e ,i t
is a very d iffic ult and t im e c ons um ing pr oc es s F o r
instance, development of Colden Rice (with
e nriche d pro vi t am in A) inv olv ing t he ins er t io n o f
just three genes took about seven years.
The terms arid zone is used to refer to harsh
enviranmental conditions with extreme heat and
cold. The fileds have limited water and minerals. lt
is different task to grow plants and achieve good
cro p y;e ld in ar id z ones .
Semi-arid regions are characterized by
unpredictable weather, inconsistent rainfall, long
dry seasons,and poor nutrients in the soil. Most
parts of India and many other developing countries
(Africa, Latin America, SoutheastAsia) have semi-
a rid re gio ns.
Cro ps like s or ghum , m illet , gr oundnut a n d
cowpea are mostly grown in semi-arid tropics.
Besides unpredictable weather, biotic and abiotic
stressescontribute to crop loss in these areas.
The biotechnological approaches for the
b ree din g p rog r am m es in t he s em i- ar id r egi o n s
sho uld cover th e f ollowing ar eas .
. Development of crops that are tolerant to drought
a nd sa linity.
. lmprovements to withstand various biotic and
abiotic stresses.
r Micropropagation techniques to spread
econ omically im por t ant plant s whic h c a n
withstan d ha r s h env ir onm ent al c ondit ions .
653
Somesuccesshas been achievedin improving
sorghum,nri l l l etand l egumecropsthat are grow n
i n semi -ari dregi ons.Geneti c transformati onin
sorghumw as possi bl eby usi ng mi croproj ecti l e
method(Fordetails,ReferChapter49).
C reenhouse l i teral l ymeansa bui l di ngmadeup
of glassto grow plants.Creen housesare requireci
to grow regeneratedplantsfor further propagation,
and for growing plants to maturity. Creenhouses
are the i ntermedi ary stages i nvol vi ng the
transitionalstep betweenthe plant culturesand
plant fields. The purpose of greenhousesis to
acclimatizeand test the plants before they are
releasedinto the naturalenvironment.
The plantsare grown in greenhouse to develop
adequateroot systems and leavesso as to withstand
the fi el d envi ronment.The greenhousesare
normal l yequi ppedw i th cool i ngsystems
to control
temperature.
Creenhouses havechambersfittedwith artificial
lights.lt is possibleto subjectthe plantsto different
lighting profiles. In recent years many
improvements havebeenmadein the development
of more sui tabl egreenhouses. Thesei ncl udethe
parameters suchas soi l , and humi di ty.
The major limitationof greenhouse
technology
is an increasein CO, productionthat in turn
increases temperature. Some approaches are
availableto controltemoerature.
Greenhome technology is a recent
development In this case, temperature is
controlled by using minimum energy.
ENVIRONMENTAL
BIOTECHNOLOGY
 nviron men t m ay be c ons ider ed as o u r
f
l- su rrou nd ing swhic h inc ludes ev er y t hingar o u n d
us, i.e. Ilrc non-living (abiotic) and living (biotic)
environment. The abiotic environment consists of
a ir, wate r a nd s oil, while t he biot ic env ir onm e n t
includ es all the liv ing or ganis m s ( plant s , anim a l s ,
microo rga nism st)hat we r egular lyc om e in c ont a c t .

Fig. 54.1 : lnteraction of environmental components.

Th e en viro nm ent is c om pos ed of f our ba s r c
co mpo ne nts.
r Atmosphere reactions.The major components of the atmosphere
include the gases nitrogen, oxygen/ argon, carbon
. Hydrosphere dioxide, water vapour, and suspended particulates
. L ithosphere (dust, soot) The composition of the atmosphere
d e p e n d so n t i m e a n d s p a c e ,a n d i s h i g h l y v a r i a b l e .
. Bio sp he re.
A liter of air weights around 1.3 g.
The re is a c ont inuous int er ac t ion am ong th e The atmosphere is vertically divided into four
r arious components of the environmenl (Fig. 54.1). layes - troposphere, stratosphere, mesosphere,
And ultimately, it is the biosphere that gets and thermosphere.This division is mainly based on
influenced by the other components. the increase in the temperature.
The four components of the environment are
HYDROSPHERE
brie fly d escrib ed.
The hydrosphereprimarily consistsof the water
on the earth's surface. Thus, the hydrosphere
ATMOSPHERE
includes oceans, seas, rivers, streams, lakes,
The atmosphere consists of a blanket of gases, reservoirs and polar ice caps. Water is the most
su sp en de d liq uids and s olids t hat env elope t h e abundant substance on the earth's surface, which
earth. The atmosphereis basically derived from the m a y b e p r e s e n t a s i c e , l i q u i d a n d v a p o u r .
ea rth itse lf b y v ar ious c hem ic al and bioc hem i c a l Approximately, 71"/" of the earth's surface is

G57

3 ttechnology [42]
658 B IOTECHNO LO CY

coveredwith water,mainly in the form of oceans. possesses the requisitecharacteristics to sustainlife

e.g., ponds,seas,deserts,cities.
It is estimated that about 97% of the total
earth's water is in the oceansand inland seaswith
high salt content.And this water is not usefulfor
human consumption.Around 2"h of the water is
presentin the glaciersand ice caps. The actual
w a te ra v a i l a b l efo r h u ma nconsumpti on i s around
1% of the total earth'swater. This includestne
groundwater,water from lakesand riversand soil Man lives in two worlds-a natural world of tne
moisture. native environmentand a built-world created by
himself. The builrworld, an outcome of the
Humans uses water in the homes, industries, advancesmade in the science and technology,
agricultureand recreation.There is a continuous i s associ ated w i th envi ronment al pollut ion.
decrease in the consumable global water E nvi ronmental pol l uti oni s a gl obalphenom enon,
Therefore, thereis a needfor precioususeof water, and thereforea matterof concernfor everyone
and its conservation.
Pollution hroadly refers to the presence of
L IT H O S PH ER E undesirable substancesin the environment which
The outer boundary layer of the solid earth on are harmful to man and other organisms. fhe
which the continentsand the ocean basins rest Dresence of unwanted substances in tne
constitutesthe lithosphere.In a broad sense, environmentmay occur due to human activity
lithosphereincludesthe land massand the ocean dischargingbyproducts,a wide spectrumof waste
products
floor. However, in a general usage, the term productsand severalharmfulsecondary
(Note : lt must be rememberedthat the natural
lithosphererefers to the land surfacewhich is
approximately total surfaceof the earth. world of environmentis not totally free from
frth of the i mpuri ti esor undesi rabl esubstances. However ,
F ro mth e b i o l o g i c apl o i n tof vi ew ,the soi l i s the such i mpuri ti esare usual l ynot harmf ul) .
most importantpart of the lithospherebecauseit
containsthe organicmatterand supportsgrowthof SOURCES OF POLLUTION
plantsand microorganisms. Lithosphere is involved
in the productionof food for humansand animals, As already stated,environmentalpollution is
besidesthe decomposition of organicwastes. mostly due to direct or indirecthuman activities,
arisingout of the built-worldcreatedby him. There
BIOSPI{ERE are six major sourcesof environmental pollution.

The biospherecomprisesof all the zones on 1. l ndustri alsources

earth in which life is present.Biospl.rere is spread 2. A gri cul tural
sources
over the lower part of the atmosphere,the top of 3. B i ogeni csources
the lithosphere and the entirehydrosphere. In other
words, the broad spectrumof bioresourcesof the 4. Anthropogenic
sources
earth,supportinglife constitutes the biosphere.lt is 5. U nnaturalsources
estimatedthat the biospherecontainsmore than
6. Extra-terrestrial
sources.
3 .5 l a k h s p e c i e so f p l a n ts( i ncl udi ngal gae,fungi ,
m o s s ea s n d h i g h e rp l a n ts a
) nd morethan 110 l akh The relativeimportanceof each one of these
s p e c i e so f a n i ma l s(u n i c e l l ul ar,
mul ti cel l ul arand sourcesdependson the site-specific situation.For
h i g h e r a n i ma l s ).T h e b i o sphere provi des the instancein cities, anthropogenicsourcesare the
essentialrequisites(water, light, heat, air, food, majorcontributors while in ruralareas,agricultural
spaceetc.)for the existenceof life. y to pol l uti on .
sourcessi gni fi cantladd

The biosphereis very vast,and for the sakeof

Types of pollution
u n d e rs ta n d i n gi t, i s d i v i ded i nto smal l er uni ts
namely ecosystems. An ecosystem may be The environmentalpollutionmay be categorized
consideredas the smallestunit of biospherethar into six major groups.
 54 : E NV IR ON M EN T AL
C hA P I CT - CENERAL
P O L L U T IO N 659

pollution
1. Airlatmosphere moni tori ng of pol l uti on w hi ch i s a gl obal
2. W at eroollu ti o n phenomenon.W orl d H eal th Organi zati onand
U ni ted N ati on E nvi ronmentalP rosrammeare
3. Land/ s oilpo l l u ti o n
activelyinvolved.
4. Nois eoollu ti o n
5. T her m alpo l l u ti o n BIOTECHNOLOGICAL METHODS FOR
6. Radioac t ive
o o l l u ti o n . ME A S U R E ME N T OF P OLLU TION
T he det ailsof a i r p o l l u ti o na n d w a te rp o l l u ti o n In recent years, envi ronmental pol l uti on
are describedelsewhere(Chapters 55 and 56). detecti on and moni tori ng i s bei ng done by
approaches involvingbiosystems. Forthis,purpose,
NATURE OF POLLUTANTS several groups of pl ants, ani mal s and
The pollutantsthat occur in the environment microorganismsare utilized. The environmental
protection agencis (EPAs)consider biomonitoring
, i o l o g i c aal n d p h y s i c a il n th e i r
ma y be c hem ic al b
nature. of pollution as a useful device to monitor
envi ronmentalpol l uti on from the poi nt of
Chemicalpollutants: Caseouspollutants(sulfur diagnostic,preventiveand remedialmeasures.
dioxide,nitrogendioxide),toxic metals,pesticides,
herbicides, hydrocarbons, toxins,acidicsubstances, CBITER'A FOR B'OMONITOB'ilG
carctnoSens. OF POLLUTION
Biological pollutants : Pathogenicorganisms,
The parametersor the criteria chosen for
p roduc t sof biolo g i c aol ri g i n .
of pol l uti onare very cruci al .They
bi omoni tori ng
Physical pollutants : Heat (thermal),sound, should be reliable,reproducibleand cost-effective.
odours,radiationand radioactivesubstances. Three types of criteria are mostly adopted for
bi omoni tori ng of pol l uti on-vi sual rati ng,
genotoxicityratingand metabolicrating.

Visual rating
ln the visual rating, the growth rate and
Environmental pollutionposesa big threatto the productivity are considered.When m icroorganisms
h e alt hyex is t encoef h u m a n k i n dT. h e C o v e rn me nts are used in the test assay,the growth can be
worldover pay seriousattentionto continuously measuredby turbidometricanalysis.In case of
monit orand m ini m i z ep o l l u ti o n .T h e p u b l i c a n d higherplants,growth rate of differentparts,visual
non-governmental organizations(NCOs) are also damageto leaves,seed viability and germination
activelyinvolvedin this venture.Broadly,thereare frequencyare taken into account.
four levels of pollution monitoring agences or
A s regardsani mal s(fi shesare commonl yused),
environmental protection agencies(EPAs\.
the conceptof LDro is usedi.e. the doseat which
Primary level : This is at the districtor block 50% of the test organismis affected.
level.The non-governmental organizations and the
Sometimes,the presenceor absence of a
rural development agencies are involved in
particularspeciesof an organismservesas an
p ollut ionm onit or i n g . pol l uti on.
i ndi catorfor the envi ronmental
Secondary level : Existing at the state level, the
monitoringis done by the respective statepollution
Genotoxicity rating
control boards.
Genotoxicity tests measure the extent of
Tertiarylevel : This is at the national/country damage causedto an organism by environmental
level. Each country has its own environmental pollution at the cellular and sub-cellular levels.
protectionagencyto monitorpollution. The genotoxic lesions may be detectedon the
Quaternary level : International/inter-cel l ul arorganel l es(membranes commonl yused),
Covernmentalbodies are closely associated with genomes, immune systems, biomolecules, etc.
660 BIOTECHNOLOGY

Cytotoxic tests such as measurementof The mostcommonl yusedpl antsan d anim alsin
chromosomaldamage(includingbreakage),sister the bioassays
are brieflydescribed.
c h ro ma ti d e x c h a n g e (SC E ) and mi cronucl ei
counting are also useful for pollution detection. Plant test systems in bioassays
The cell viability can be measuredby detecting
Certain algae, bacteria, lichens, mosses and
in vitro lysosomalviability.In recentyeas, DNA
vascular macrophytesare commonly used in
probesare in use for the identificationof disease-
bioassavs.
causingorganismsin water.
Algal bioassays: Amongthe plantsystems, algal
Metabolic rating bioassays are the most commonlyused.Algaeare
fhe biochemical changeswith environmental consi deredto be rel i abl ei ndi cator sof pollut ion
pollution can be measured (qualitativelyand due to their high sensitivityand easy availability,
quantitatively) in selectedorganisms. The cr it er ia
In fact,certain besi dessi mpl e cul turi ngtechni ques.
metabolicparameterscan be usedas biomarkersto adopted for algal bioassaysare the growth rate,
assessfhe pollution sfress.The biomarkersused in biomass accumulation and ohotosvnthetic
metabolic rating include chlorophyll, proteins, efficiency.
n u c l e i c a c i d s (D N A a n d R N A ) and chansesi n The algae used in the test assays include
enzymeactivities. Chlorella, Microcystis, Spirulina, Navicula,
The biotechnoiogicalmethods adopted for Scenedesmus, Anabaena,Ulva, Codium,Fucusand
pollutionmeasurement are brieflydescribedin the Laminaria.In water, organic pollution can be
followingorder. detectedby usingthe blue greenalgae,Microcystis,
.l w hi l e metal pol l uti on can be m easur edby
. Generalbioassays
Navicula.
2 . C e l l b i o l o g i c aal s s a y s
Bacterialbioassays : Theseare commonlyused
3 . Mo l e c u l a rb i o l o g i c aassays
l for the detectionof fecalpollutionin potablewater,
4 . Bi o s e n s o rs . the most widely employedtest being coliform test.
Ames test that detects mutagenicpollutants is
E'OISSATS'N ENV'NONME}JTAL carried out by the bacteriumSalmonella.
MON'TON'NG Bacterialbioluminescenceis a recenttechnique
ln the earlyyears,the conventionalphysicaland usedfor the measurementof gaseous pollutantsand
chemical methodswere used for the detectionof othercompoundse.g.sulfurdioxide,formaldehyde,
environmentalpollution. Bioassaysare preferred ethylacetate.Photobacteriumphosphoreumis the
these days, since the biological responsesthat organismof choice for bacterialbioluminescence.
reflect the damagesto the living organismsare
Lichensin bioassays : Lichensare widely used
very crucial for the actual assessmentof
for the detection of atmosphericgas pollution,
p o l l u ti o n .
particularlyin cities.Lichensare very sensitivefor
The organismsemployed in the bioassaysfor the measurement of sulfurdioxide.
pollution detection are expectedto satisfy the
Mosses in bioassays: Environmentalmetal
followingcriteria.
pollution can be detectedby using certainforest
o lt shouldreadilytake up the pollutant(absorption and aquaticmossese.g. Stereophyllum,Sphagnum,
or adsorption). Brvnus.
o The organismshouldbe sensitive to the pollutant. Vascular macrophytes in bioassays : Water
o lt should possessmeasurablefeaturesto detect hyacinth (Eichormia crassipes)and duck weed
p o l l u ti o n . (Lemnaminor) are in use to detect aquatic metal
pollution.In fact, certainbiochemicalparameters
. The organismshouldhavewide occurrence,and
of macrophytesare usedto serveas biomarkersof
availableround the year.
pollutione.9., peroxidase activityincreases
due to
. The bioassay shouldbe simple,reproducible
and metal pollution; inhibition of nitrate reductase
cost-effective. activity by mercury. The other commonly used
 Chaot er54 : EN VIR O N M EN T AL -C E N E R A L
POL I-U T I ON 661

bioassayparameters are the estimationof soluble Biomonitoring of pollution with

pr ot einsnu
, c l e i ca c i d s ,c h l o ro p h y l la, n d a ssayof multiple species
enzyme(e.g.catalase,peroxidase) activities.
Most often, bioassays using a single organism
Pollution-inducedpeptides in bioassays: Very are not adequate to detect pollution. In such a
recently, someworkershaveidentifiedthe presence case, multiple species of organisms are used.
of s m allpep ti d e sw i th i n th e p l a n t c e l l sw h ich are
pollution-induced. These peptides,referredto as CELL B'OLOGY ENVIBONMENTAL
phytochelatins,are formed as a result of metal MONITOR'NC 'N
pollution. They are reasonablyreliable for the
Cell biology deals with the study of the structural
detection of metal pollution.
and functional aspects of cells and the cellular
organelles. lt is successfully exploited for
Animal test systems in bioassays
e n v i r o n m e n t a lp o l l u t i o n d e t e c t i o n ,p a r t i c u l a r l yw i t h
Amongthe animals,certainfishes,protozoaand referenceto mutagens and carcinogens.
helm int hsare e mp l o y e di n b i o a s s a y s .
The cell biological methods primarily aim to
Fishes in bioassays : Toxic effects of trace the harmful effects of pollutants on different
env ir onm en taplo l l u ta n tso n fi s h e sh a v e b e en i n c e l l u l a r c o m p o n e n t s- m e m b r a n e s , c h l o r o p l a s r s ,
usefor quite sometime as a measureof bioassays. m i t o c h o n d r i a , c h r o m o s o m e s . I n a d d i t i o n , t h e
In fact, the conceptof LDuo(i.e. the dose of the m a c r o m o l e c u l e sn a m e l y n u c l e i c a 'c i d s( p a r t i c u l a r l y
pollutantat which 50'h of the test organismsare DNA) and proteins are also used. Further, cell
affected)has originatedfrom the studieson fishes. b i o l o g i c a l m e t h o d s h e l p i n u n d e r s t a n d i n g t h e
The criteriaor parametersused for assessment of m e c h a n i s m so f t o x i c i t y o f p o l l u t a n t s .
f is h bioas s a yisn c l u d ec h a n g e si n th e mo rphol ogy
Some importantcell biological methodsused in
and organs,behaviouralpatternand modifications
environmentalpollution monitoring are described.
in metabolisms.The alterationsin Ihe enzvme
acetylcholine esteraseservesas a reliable marker
Membrane damage in bioassay
for pesticide pollution.
The plasma membrane, an envelope
The mostcommonlyusedfishesin bioassays
are
s u r r o u n d i n g t h e c e l l , p r o t e c t st h e c e l l f r o m h o s t i l e
Catla, Teleost,Labeo and Channa.
environment. lt is the first celiular component to be
Protozoain bioassays: The ciliated protozoa directly exposed to pollutants. Many toxic
serveas good bioassaysystemsfor the detectionof substancesthat cause damaee to cell structure and
environmentalpollution.The toxic effectsof the its functions are known
pollutantscan be measuredby the changesin the
For the purpose of bioassay, the physical
behavioural patternsof protozoa,recordedon an
danrages caused by pollutants or their deposition
ethogram
on the membranes can be detected by light, phase
Helminthsin bioassays : Rotifersare a groupof contrast and electron microscopy. This approach
nelminthsthat grow on aquaticvegetation.They may not be.always practicable. The alterations rn
are used for the detectionof organic matter in the semipermeable properties of the membranes
u'ater(givenby BOD).Rotifers, w'ithroundthe year Cue to pollutants can be detected by leakage of
availability,easvcultivat!on,slow growth rate and enzymes (e.g., lactate dehydrogenase),efflux of
easy recognitionare used for bionronitoringof electrolytes or uptake of trypan blue. Lysosomes are
\vater. also useful as biomarkers for measurement of cell
viability. This can be done by neutral red retention
Pollution-inducedpeptides in bioassays: As
fesf. The damaged lysosomes cannot retain this
alr eady des c ri b e d i n c a s e o f p l a n t b i o assays
dye.
above),pollution-induced smallpeptidesarefound
in anim alc e l l sa l s o .T h e v a re c o l l e c ti v e l vre ferreo I n r e c e n t y e a r s , a n i m a l a n d p l a n t t i s s u ec u l t u r e
to as metallothioneins (comparable to t e c h n i q u e s a r e a l s o u s e d f o r p o l l u t i o n m o n i t o r i n g .
phy t oc hela ti n si n p l a n ts ).M e ta l l o th i o n ei ns are T h i s i s m a d e p o s s i b l e b y n r e a s u r i n g c e l l u l a r
useful for the detection of metal pollution. damages observed in cell cycles. A good example
{'t

662 B IOTE CHNO LO CY

is the useof human lymphocytecultureto monitor Recently, the yeast cells (Saccharomyces
the personsexposedto toxic pollutants. cerevisae)are also used for the detection of
chemicalcarcinogens.
Gytogenetic bioassays
MOLECULAR B'OLOGY
The geneticdamagesof the cells,as reflectedby
'N
ENV'BONMENTAL MON'TOB|NG
changesin the chromosomes, can be effectively
used in biomonitoring of pollution. For this The use of molecular probes and immunoassays
purpose,animals(e.9.insectDrosophila) and plants i n moni tori ng pol l uti o nis gaining
of envi ronmental
(e.8. Arabidiopsrs)with shorter life cycles are importancein recentyears.Molecularbiological
preferred.
Other plantssuchas pea,maizeand soy bioassays are particularlyusefulfor the detectionof
bean are also used in cytogeneticbioassays. bacteria,virusesand other pathogenicorganisms
that causediseases.
Chromosomal damage : The pollutants may
cause several types of chromosomaldamages-
DNA probes
fragmentation, bridgeformation,disruptionin cell
division. The chromosomal alterations can be DNA probes and polymerase chain reaction
effectively usedfor pollution detection lt has been (PCR) can be effectively used for water quality
clearlyestablished thatthe severitvof chromosomal monitoring, particularly potable water. However,
damagedependson the chemical nature of the thesetechniquesare expensiveand not practicable
polIutant. at all olaces.For more detailson DNA probesand
PCR,ReferChapters9 and 8 respectively.
Micronucleus test : Severe damage to
chromosomesby pollutantsmay result in large lmmunoassays
scalefragmentationof chromosomes, followed by
micronucleiformation.The degreeof micronuclei lmmunologicaltechniquesare useful for the
developmentis directly relatedto the severityofdetectionof pollutants(pesticides,
herbicides)and
the damage.Micronucleustest (MNT) is used for identification of pathogens that exhibit
screeningof mutagenic compounds. immunological properties.
lmmunoassays are in use
for the measurementof several pesticidese.g.
Sister-chromatid exchange : The damages aldrin, triazines DDI glyphosate. Metabolic
caused by pollutantsresultsin misexchangeof productsof certainbacteriacan also be detectedby
chromosomalsegments(chromatids)during cell immunoassays. For instance/assaysystemshave
division.The sisterchromatedexchange(SCE)can been developed for the detection of toxins of
be detectedby usinga fluorescentdye technique. cholera and Salmonella.
ln recent years, use of monoclonal antibodies
Ames test in bioassays
(MAbs) in the detection and biomonitoringof
Ames test can be used for the detection of environmentalpollution is gaining importance.In
chemicalmutagensand their carcinogenecity.This fact, assaytechniquesare availablefor detectionof
is very widely used bioassayfor screeningof pesticideand herbicidecontaminationin water.
virious pollutants,drugs,cosmotics,food additives
and metals. Bioluminescent bioassays using
Ames test employsthe use of a specialmutant Lux reporte; genes
strainof bacteriumnamelySalmonellatyphimurium Certaingenes,referredto as Lux reportergenes,
(His-).This organismcannot synthesizehistidine, on the plasmids produce assayable signals.
hencethe sameshouldbe suppliedin the medium Wheneverthesegenesare expressedin luminescent
for its groMh. Addition of chemicalcarcinogens bacteria like Photobacterium and Vibrio. Some
causesmutations(reversemutation)restoringthe bacterialstrainshavebeendevelopedthroughgene
ability of this bacteriumto synthesizehistidine cloning (employingLux reportergenes)for the
(His+).By detectingthe strainof Salmonella(His+) detectionof pollutantsand their degradation.For
in the colonyof agarplates,the chemicalmutagens instance,geneticallyaltered Pseudomonas can be
can be identified.The Ames assaycan detect about used for detectingnaphthalene,xylene, toluene
90o/oof the chemical carcinogens. and salicvlate.
 54 : EN VIR O N M EN T AL
ChA O t CT -C E N E R A L
POL L U TION 663

B'OSENSORs'IV ENVIRONMENTAL
MONITON'NG Tlru 54.1 A selected llst of pollutants
measured by enploylng biosensors
A biosensor is an analytical device containing
an immobilized biological material (enzyme, Pollutant measured Biasensor-
o rga ne lle, c ell) whic h c an s pec if ic ally i n t e r a c t biological componenl
rvith an analyte (a compound whose concentration BOD Trichosparoncutaneun
is to be det er m ined) and pr oduc e p h y s i c a l ,
Soz Thiobacillus
sp
che mica l o r elec t r ic als ignalst hat c an be m e a s u r e d .
Biosen so rsar e highly s pec if ic and ac c ur at e i n t h e r r
cH+ Methanomonas flagellae
f u nctron . coz Pseudononas sp
Nitrate Azobactervinelandi
The details on biosensors-principles of
NH,andNO, Mixednitrifying
bacteria
working, types, various applications are described
Nervegas Acetylcholine
esterase
elsewhere (Refer Chapter 21) Some of the
imoo rtan t bios ens or s us ed in env ir on m e n t a l Ethanol NADHanddehydrogenase
po llutio n m onit or ing ar e br ief ly des c r ibed . Phenol Phenoloxidase
Parathion to parathion
Antibody
BOD biosensor Herbicide-induced
eleclrontransport
Biolo gic al ox y gen dem and ( BO D5) is a w i d e l y
chaininhibition Synechococcus
used test for the detection of organic pollution. This
test reo uir es f iv e dav s of inc ubat ion. A B O D
biosensor using the yeasl Trichosporon cutaneum Biosensorsfor the detection of polychlorinated
rvith oxyg en pr obe t ak es jus t 15 m inu t e s f o r b i p h e n y i s( P C B s )a n d c h l o r i n a t e dh y d r o c a r b o n sa n d
d ete ctin g o r ganic pollut ion. certain other organic compounds have been
developed.
Gas biosensors
Phenol oxidase enzyme (obtained from potatoes
Microbial biosensorsfor the detection of gases and mushrooms)containing biosensor is used for
su ch as su lf ur diox ide ( SO r ) , m et hane and c a r b o n the detection of ohenol.
dioxide have been developed. Thiobacillus-based A graphite electrode with Cynobacterium and
bio se nsor c an det ec t t he pollut ant SO r , w h i l e Synechococcushas been developed to measurethe
meth an e (CHr ) c an be det ec t ed by im m o b i l i z e o degree of electron transport inhibition during
Methalomonas. For carbon dioxide monitoring, a photosynthesis due to certain pollutants e.g.
particular strain of Pseudomonasis used. herbicides.
A selected list of environmental pollutants
lmmunoassay biosensors measured by employing biosensors is given in
lmmunoelectrodes as biosensorsare useful for Table 54.1.
the detection of low concentrations of pollutants.
Pesticide specific antibodies can detect the
presence of low concentrations of triazines,
ma lathion and c ar bam at es , by em p l o y i n g
rmmu no ass ay s .
The different approachesfor the managementof
Other biosensors environmental oollution are described in the
Biosensors employing acetylcholine esterase respectivechapters for air pollution, refer Chapter
55, and for water pollution Chapter 56.
lobtained from bovine RBC) can be used for the
d ete ctio n of or ganophos phor us c om pou n d s I n The general concepts of the biotechnological
rvater. In fact, portable pesticide monitors are approaches for the abatement of pollution n,ith
comme rcia lly av ailable in s om e de v e l o p e d special reference to the following aspects are
cou ntries. described in this chapter.
664 BIOTECHNOLOCY

1. AtmosphericCO2 reduction. Certainbiotechnological approacheshave also

2. Sewagetreatmentby bacteriaand algae. been made to improve the CO, utilization by
enhancing photosynthesis.The enzyme ribulose-
and phosphoruspollution.
3. Eutrophication
bisphosphatecarboxylase(RUBP-case)is closely
4. Managementof metal pollution. linkedwith CO, fixation.Thereare someattempts
5 . l mmo b i l i z e dc e l l s i n the manasementof to geneticallymanipulatethis enzymeso that the
p o l l u ti o n . photosyntheticefficiencyis increased.

ATMOSPHERIC CO2 REDUCTTON Microalgal photosynthesis

The lower atmospherecontains CO, at a
Certain microalgae are more efficient than
concentrationof 0.0314"/"bv volume.There is a
hi gher pl ants i n uti l i zi ng atmosphe r icCO , f or
continuousaddition of CO2, pirrticularlycoming
photosynthesise.g. Chlorellapyrenodiosa,Spirulina
from industrial processes.Any increase in the
maxima.Theseorganisms arecapableof generating
contentCO, hasto be viewedvery seriously, since
more O, than the amountof CO.,consumed.
it is the principalgasthat causesgreen houseeffect
and risesatmospheric temperature.lt is suggested It is advocatedthat growing microalgaein the
that the globaltemperature will increaseby around vicinity of industriesand power plantswhere the
2-5"C on doublingthe atmosphericconcentration productionCO, is very high will help to minimise
of CO2. The presentbelief is that during the past the polluting effects of COz. In addition,
.l
00-150 years,the CO, level increasedby about these microalgae, appropriately regarded as
25% with an increase in the atmospheric photobioreactors, accumulate as biomass which
temperatureby about 0.5%. Thus,CO, is closely can be utilized for the extractionof protein for use
linked with global warming. as food or feed.
For the reasonsstatedabove, the reduction in Cenetic manipulations are in progress to
atmospheric COz concentration assumes developnew strainsof microalgaethat can tolerate
significance.Thereare mainly two approaches for high concentrationsoI CO2, and yield larger
the biotechnologicalreduction of CO, in the biomass.Some successhas been reported in the
atmosphere. mutants of Anacystis nidulans and Oocystis sp.
1. Photosyntheiis
2. Biologicalcalcification. B'OLOG'CAL CALCIF'CAT'ON TO
nEDUCE ATMOSPHERTC CO2
PHOTOSYNTHESIS TO BEDUCE
Certain organisms present in the deep sea
ATMOSPHEBTC CO2
(corals,greenand,redalgae)are capableof storing
by plants CO, through a processof biological calcification.
Utilization of CO2 for photosynthesis
is the most significant process to reduce CO, The overall process of calcification may be
content in the atmosphere.Photosynthesis may be representedas follows.
representedby the following equation.
H rO + C Oz+ H 2C O3
Sunlight
6CO, + 6tl2o ----------+ CuH'rO. + 60, H2CO3,+Ca2* CaCO, + CO, + HrO
Chlorophyll
As the CaCO3getsprecipitated,more and more
Higher plant photosynthesis atmospheric CO, can be utilizedfor its formation.
The fast grorving trees are more efficient in
SEWAGE TREATMENT BY BACTERIA
utilizing CO, for photosynthesis,hence their
AND ALGAE
propagation is advocated. Further, micro-
propagation and syntheticseed production through The waste wafer resultingfrom various human
plant tissueculturetechniquesare also important. activities(domestic),agriculturaland industrialis
The major problemis the continuousdeforestation technically referredto as sewage.The sewage is
that is associatedwith a possible increase in mostly composed of organic and inorganic
atrnosphericCOr. The conservationof forestsand compounds, toxic substances,heavy metals and
plantationsis the need of the hour. pathogenicorganisms.
CT A O T54
CT : E N VIR O N M EN T AL
POL L U T IO N -C E N E R A L 665

. Certain metals (that are adsorbed from the

s'ewage)can be recovered.
. The load of pathogenic microorganisms is very
much reduced (they either dle or settle at the
bottom of the oond).

The algal species of different genera ideally

suiied for sewage treatment are Chlorella, Euglene,
Chlamydomonas, Scenedesmus, Uiothrix and
Thribonima.

EUTROPHICATION AND
PHOSPHORUS POLLUTION

Sewage, and waste waters from industries and

agriculture are rich in organic and inorganic
nutrients, containing phosphorus and to a lesser
extent nitrogen. These nutrients favour excessiye
growth of algae which results in oxygen depletion
(deoxygenation) and this phenomenon is referred
to as eutrophication. Eutrophication results in the
death of non-resistantorganisms (e.g. fishes), and
In the biological treatment of sewage, the
foul smell.
orqanic matter is subjected to biodegradation or
cacterial oxidation. In this way, the organic matter Water blooms : Certain species of algae
rs degraded to smaller molecules (CO2, NH3, PO4 (particularly
blue green algae e.g. Lvnhbia,
etc.), Biodegradationrequires a constant supply of Microcystis, Oscillatoria, Anabaena) that can
O, This can be done by c ont inuous bubbling o f tolerate eutrophication, grow well and form rvater
atmospheric O, through a specially designed blooms in waste/sewage water. From the extensive
equipment. This is an expensive process, besides formation of water blooms, one can predict that
the involvement of manpower. pond has accumulation of very high quantities of _
A continuous supply of O, can be achieved by phosphorus. The algae are capable of absorbing
growing microalgae in the ponds where sewage phosphorus, and store them as polyphosphates.
treatment is being carried out. The algae are very Phosphorus is a strong promoter of algal growth
erticient in photosynthesisand they release O, in and water bloom formation. Water blooms are
:o the ponds. This is the form of dissolved Ot associated with the oroduction of certain toxins
Fig. 54.A. Thus, algal-bacterial symbiosis is (e.g. lipopolysaccharides)that are harmful to fishes
responsible for photosynthetic oxygenation and and birds. These toxins may also cause certain
the biodegradation of sewage organic matter. fhis diseases in humans e.g. diarrhea, gastroenteritis,
is a natural and inexpensive process. nausea.
ln addition to the supply of COr, the algae used
in sewage treatment can adsorb certain toxic heavy Control of eutrophication : There are broadly
growth resulting
ne tals a lso . Th is is pos s ible due t o t he negatt v e two ways of controlling algal
in eutrophication. In the chemical approach,
cha rge son th e a lgal c ell s ur f ac ewhic h c an t ak e u p
algicides such are copper sulfate, sodium arsenate
:he positively charged metals. The algal treatment
a n d 2 , 3 - d i c h l o r o n a p h t h o q u i n o n e a r e u s ed. But
of sewage is useful in several other ways.
the chemical treatment is associated with
. Support fish growth, as the algal cells are good increase in sludge volume. In the biological
{ood for fishes, besides supplying dissolved Or. approach, cyanophages (i.e. the viruses that can
. Th e a lga l ce lls , r ic h in pr ot ein, s er v e as go o d k i l l a l g a l c e l l s ) c a n b e u s e d t o c o n t a i n
food and feed. eutrophication.
 666 BIOTECHNOLOCY

Highenergy \
comPounos Polyphosphates
---J-*;"",
carbon
PolYPhosPhates
|
r"-Enerov<--4
JJ
"| *r-l
"otpornOt

.Highenergy P; Metabolism P;
carDoncompounos \

P;
Anaerobic condition Aerobic condition

Fig, 54,3 : Biological removal of phosphorus by baeteria.

BIOLOC'CAL REMOVAL OF A s the l i vi ng organi sms(i ncl udi ngm an) ar e

PHOSPHORUS constantlyexposedto metals,they accumulateby a
processreferr,ed to as bioaccumulation.Continuous
As stated above, phosphorus signif icantly
exposureand accumulation of a given metalin the
contributesto algal growth in waste water. Removal
organismsresultsin its increasedconcentration, a
of phosphorus therefore can protect from
phenomenon referred to as biomagnification.
eutrophication. lt is possible to precipitate
phosphorus with salts of calcium, magnesium and
Biomagnificationusually occurs through food
chain and man is the ultimatevictim.
alum inium . This howev er , is n o t p r a c t i c a b l e o n
large scale. In addition, chemical methods add to Biomethylation is the process of transfer of
the sludge volume. Hence, biological methods are methylgroupsfrom organiccompoundsto metals.
preferred.
This is carriedout by microorganisms in the soil
and water.Althoughit is true that somemetalsget
Biological removal of phosphorus by bacteria is
detoxifiedby methylation(e.9. arsenic),some of
carried out through aerobic and anaerobic
them may even becomemore toxic (mercury).
pr oc es s es / as depic t ed in F i g . 5 4 . 3 . Under
anaerobic conditions, certain organic compounds
BIOSCAVENGEBS OF METALS
(e.8. acetate) obtain their energy from
polyphosphates to store as carbon reserves. ln Certain aquatic plants namely phytoplanktons
this process,free inorganic phosphorus is released. (plantsthat freelyfloat on water surface)or benthics
In the subsequent aerobic process, the organic (plantsattachedto some substratumat the bottom
carbon reservecompounds are oxidized to generate of aquaticreservoirs) and microorganisms can take
energy. A small portion of this energy is up the metalsfrom the bathingmedia(wastewater,
utiiized for the conversion of phosphates to ponds) and act as natuml bioscavengers.Besides
polyphosphates.The net effect is that phosphorus combating the pollution effects of metals, this
accumulates in the bacterial cell in the form of processis alsousefulfor the recoveryof industrially
polyphosphates. importantmetalsfrom the bioscavengers.
ln the recent years,environmentalprotection
MANAGEMENT OF METAL POLLUTIONagencies(EPAs) worldoverare advocatingthe use
of aquaticplantsand microorganisms as low cost
Environmentalpollution with heavy metals (e.g.,
bioabsorbantsfor the removalof toxic metalsfrom
lead, cadmium, mercury) causes several toxic
the envi ronmentalpol l uti on.
m anif es t at ions in liv ing or g a n i s m s , i n c l u d i n g
cancer. Bioaccumulation, biomagnification and Someof the plantsand microorganisms useful
biomethylation are some of the characteristic for the management of metal pollutionare briefly
featuresof metal oollution. described.
 Chapt er54 : EN VIR O N M EN T AL -C E N E R A L
POL L U TION 667

Plants in metal pollution management a n d / o r a d s o r p t i o n .T h e m e c h a n i s mo f a c c u m u l a t i o n

of metals by the bioscavengersis very complex and
Water hvacinth : This is the most common weeo
may involve one or more of the following
found in tropical waters and its control is a major
Drocesses.
problem. This plant can absorb and accumulate
cad mium, m er c ur y , lead and ev en s ilv e r t o a
Binding of metals to cell walls
significant extent.
B i o a c c u m u l a t i o no f m e t a l s o n t h e c e l l w a l l s i s
Water lettuce : This is a floating aquatic weed,
commonly observed in lower plants-algae,fungi
and is also a good metal bioscavenger.lt can take
and bacteria. Even some higher plants can
up la rge q ua nt it iesof lead, c adm ium , m er c u r y a n d
c o n c e n t r a t em e t a l s o n t h e c e l l w a l l s . T h e r e i s s o m e
arsentc.
evidence to show that metals are held to certain
Cat-tails : These plants possesslong erect leaves organic biopolymers on the cell walls, till the
and grow in marshy areas. Cat-tail leaves have binding sites get saturated.
been shown to be useful for the absorotion of
nicke l, cop per , bes ides c alc ium and m ag n e s i u m Accumulation of metals in cellular
salts. Water soinach that accumulates leao compartments
an d ch rom ium is eat en in s om e ar eas b y p o o r Plants are capable of transporting metals to
people. The toxic metals are certainly toxic to i n t r a c e l l u l a r a n d i n t e r c e l l u l a rf r e e s o a c e s .a n d t h e
n uma ns. c e l l u l a r o r g a n e l l e s ( l y s o s o m e s ,v a c u o l e s ) . C e r t a i n
Ferns : The fern namely Azolla filicoides metals occur as immobilized metal containing
accu mula tes c adm ium , c opper and ur a n i u m . crystals. For instance, heavy metal complexes of
Salvinia sp can absorb several metals including calcium oxalate crystals have been detected in
lea d, n ickel and c hr om ium . argae.

Microorganisms in metal pollution Synthesis of metal binding proteins

management Several algae and certain fungal species are
Algae : Several metals from the fresh water can known to synthesize metal binding proteins or
be absorbed by algal blooms. For instance, peptides. Phytochelatin is an ubiquitious protein
Chlorella vulgaris can take up copper, mercury and present in all the plants. lt is a metal chelating
ura niu m. The m ar ine m ic r oalgae ( e. 9. , U l v a , protein and is regared as a common buffering
Laminaria) are also useful for the abatement of molecu le for the homeostasis of metars.
metal oollutants. Phytochelatin is rich in cysteine and can form salt
metal complexes through sulfhydryl (SH) groups. lt
Fungi : Certain fungal species are good is comparable in its functions to metallothionein
absorbersof heavy metals (Pb, Hg). e.g. Rhizopus, found in animals.
Aspergillus, Penicillum, Neurospora.
Certain metals are known to induce the synthesis
Bacteria : A few bacterial species can of phytochelatin. Some workers, in recent years,
a ccumu latem et als on t heir c ell walls . For in s t a n c e , have suggested phytochelatin can be used as a
E. coli can take up mercury while Bacillus circulans biomarker for metal oollution detection.
ca n accu mulat e c opper .
IMMOBILIZED CELLS IN THE
Attempts are being made to improve the strarns
M A N A G E M E N T OF POLLUTION
of ba cte ria ( t hr ough genet ic m anipulat io n s ) f o r
more efficient uptake and accumulation of toxic The use of immobilized cells, paticularly
q-.etals.Further, such species will also be useful microbial whole cells, for the abatement of
'or the recovery of metals from the bacterial environmental pollution is a recent development.
f lo mass. The general aspects of cell immobilization,
techniques involved, and their applications are
,TECHANTSMS OF METAL SCAVENGING described elsewhere (Chapter 21).

The aq ua t ic plant s and m ic r oor ganis m s m a y The immobilized cells are useful for the waste
-3Ke up the metal pollutants through absorption water treatment, and for the recovery of metals
668 B IOTECHNO LO CY

from industrial effluents. A selected list of the

p o l l u t a n t s a n d t h e i m m o b i l i z e d m i c r o o r g a n i sm s
used for their abatement is given in Table 54.2. For
microorganismsused for their abatement instance, phenol and triethyl lead can be removed
respectively by immobilized Pseudomonasputida
Pollutant lmmobilized microorganism
and Arthrobacler so.
Phenol Pseudononas putida
Cryptococcuselinovii For more efficient management of a group
Triethyl
lead Arthrobacter
sp of e n v i r o n m e n t a l p o l l u t a n t s s i m ul ta n e o u sl y,
4-Chlorophenol E. nli immobilized systemswith more than one type of
microorganisms are used. In the i'ecent past,
Enterobacter
sp
genetically engineered microorganisms ( GEMs) are
Alcaligenes
sp
being developed for the treatment of polluted water
Ammonia Thiosphaera
sp and soils. Some details on this aspect are described
Copper Rhizopusarrhizus in Chaoter 59.
Jhe e arth' s alm os pher e c ont ains v ar iou s g a s e s .
I water vapor and suspended particles. Ihe dry Trnu 55.1 Compositionof the dry air in the
air of the atmosphere is composed of four major lower atmosphere
gases nitrogen, oxygen, argon and carbon dioxide,
that account for more than 99.5% (Table 55.1). fhe Cas Cornpasition
('/" by volume)
oth er g ases f ound in t r ac es in t he air i n c l u d e
he lium, m et hane, k r y pt on, hy dr ogen, c a r b o n Nitrogen 78.08
mo no xid e, nit r ous ox ide ( Nr O ) , nit r ogen c j i o x i d e Oxygen 20.95
(N0 2), a m m onia. , oz one, s ulf ur diox id e a n d
Argon 0.93
hydrogen sulfide. The lower part of the atmosphere
(u p to ab ou t 12 k m ) als o c ont ains wat er v a p o r a t a Carbondioxide 0.03
con ce ntra t ion, r anging f r om 0. 0. 1 t o 5. 0% . T h i s Tracegases > 0.02
water is mostly contributed by evaporationfrom the (He,CHo,Kr,Hr, CO,NrO,
hvdrosohere. N02,NH3,03, SO2,H2S)
The a ir is get t ing c ont am inat edby polluti o n d u e
the natural and unnatural activities of man. Air
welfare of a person or with the full use or
pollution is basically the presence of foreign
enjoyrnent of his property.'
substances in the air at a concentration that will
adversely affect the health and property of the The term pollutant refers to a substancewhich
in dividu al. increasesin quantity in the air and adverselyaffects
As per the World Health Organization (WHO) the environment e.g. carbon monoxide, sulfur
criteria, air pollution refers to 'the substancesput dioxide, lead. On the other hand, a contaminantis
in to th e air by t he ac t iv it y of m ank in d i n t o a substance which is not present in nature, but
concentration sufficient to cause harmful effects to released due to human activity e.g. methyl
his health, vegetables,property or to interferewith isocyanate. DDI malathion. However, this
th e e njo ym ent of his pr oper t y ' distinction is not very rigid, and most authors use
the term pollutant to represent both (pollutant as
Acco rdin g t o I ndian St andar ds I ns t it ut i o n 'a r r
well as contaminant).
po llutio n is t he pr es enc ein am bient at m os p h e r eo f
sub sta ncesgener ally r es ult ing f r om t he ac ti v i t y o f
SOURCES OF AIR POLLUTANTS
man , in suf f ic ient c onc ent r at ion. pr es e n t f o r
sufficien t tim e and under c ir c um s t anc es w h r c n T h e s o u r c e st h a t c o n t r i b u t e t o a i r p o l l u t i o n m a y
interfe,'esignificantly with the comfort, health or be broadly categorized into two types.

669
670 B IOTECHNO LO CY

CLASSIFICATION OF AIR POLLUTANTS

Air pollutantsareclassified
basedon theirorigin,
chemicalcomoositionand the stateof the matter.
Industry Major air pollutants
Classification based on origin
powerplants
Thermal NO2,N2O, SO2, Air pollutantsare divided into two categories,
particulates basedon their origin-primaryand secondary.
Steelindustries Smoke, particulates,
CO, Primary air pollutants : These pollutantsare
fluoride directly emitted into the atmosphere and present
there as such (i.e. in the form they are originally
Petroleum
refineries SOr,smoke, particulatesemitted).Primaryair pollutantscontributeto as
much as 90% global air pollution Particulates,
Melalsmelters SO2,NO2, NrO,smoke, carbon monoxide, (CO), oxides of sulfur (SO*),
particulates
oxides of nitrogen (NOr), hydrocarbons(HCs),
plants
Fertilizer s02,No2,N2o,NH3, radioactive compounds,pollenand bacteriaarethe
fluoride maj or pri maryai r pol l utants.
Secondaryair pollutants : These are produced
Acidplants s02,No2,N20 in the air as a result of interaction among the
plants
Cement SOr,smoke,particulatesprimary pollutants, or by a reaction that occurs
between a primary pollutant and a normal
plants
Soapanddetergent Particulates,
odour constituentof the atmosphere. Cood examplesof
secondary air pollutants are ozone (O3),
Papermills SO2,particulates,
odour
peroxyacetyl nitrate (PAN), formaldehyde and
smog.

1 . N a tu ra l s o u rc e s: T h ese i ncl ude vol canrc Glassification based on chemical

eruptions,sand storms,decompositionof organic composition
matter,forestfire, pollen grainsand cormic dust. A ccordi ng to chemi cal comp osit ion, air
The problemof pollutiondue to naturalsourcesin pollutantsare categorized as organicand inorganic.
g e n e ra l i, s c o n s i d e re to
d b e mi ni mal .
Organic air pollutants: These pollutantsare
2. Anthropogenic(man-made)sources : Air mainly composedof carbon and hydrogen. ln
'
p o l l u ti o nd u e to h u m a n -i nduced acti vi ti esi s very addition,oxygen,nitrogen,sulfur and phosphorus
h i g h . T h e s o u rc e so f p o l l u t i oni ncl udei ndustri es, may also be presente.g. hydrocarbons,organic
b u rn i n g o f fo s s i l fu e l s , e m i ssi onsfrom vehi cl es, sulfur compounds,aldehydes,ketones,carboxylic
agriculturalactivitiesand warfares. aci ds.
lnorganic air pollutants : These are purely
T h e s o u rc e s o f a i r pol l uti on may al so inorganicin nature e.g. carbon dioxide, carbon
considered as stationary (industries, open monoxide, oxides of sulfur, oxides of nitrogen,
combustion)or mohile (motor vehicles, trains, ozone.
aircraft)in nature.
Glassification based on the state
Industrial pollutants of matter
T h e m a j o r p ro b l e mo f ai r pol l uti oni s due to Air pollutantsmay be divided into two types,
industrialactivities.Among the severalindustries, based on the statein which thev exist-oarticulate
nine types of industries are considered to and gaseous.
be the major air pollutant generatingindustries Particulateair pollutants: The solidsand liquids
(Table55.2).Among these,thermalpower plants, dispersed in the atmosphere constitute the
steel industries,petroleum refineriesand metal particulateair pollutants.Solid particulatese.g.
smelters are the most dangerous polluting smoke, dust, fly ash. Liquid particulatese.g. mrsr,
i ndustries. fog, spray.
 Chapt er55 : A I R P O L L U T IO NA N D C O N T R OL 671

Gaseousair pollutants: Theseare the organic

and inorganicgasesthat are presentin the air as
pollutants.Organic gasese.g. methane,butane, air pollutants
aldehydes.Inorganic gasese.g. CO2, SO2, N02,
Pollutant Effects
\H 3, H2S .
Sulfur (SOr)
dioxide Noseandthroat initation,
EF F E CT S O F AIR POL L U T IO N respiratoryillness. Prolonged
Majority of the air pollutants a.re present exposure may lead to
normally in the atmosphere,although at low chronicbronchilis.
concentrations.Such pollutantsare unlikely to dioxide(NO2) Initation
Nitrogen of eyes,noseand
cause any significantharmful effects.When the andnitricoxide(NO) throat.NOcombines with
concentrations of the air pollutantsgo beyondthe O, andreduces supply of 02
acceptablelimits (variablefor each pollutant)they to tissues.
are dangerousand causeseveralharmfuleffects. Carbonmonoxide (CO) Binds to hemoglobin.and
The WHO has set guidelinesand fixed threshold drastically
reduces O, supply
limit values(TLV)for each air pollutant.The general Mayresult
to tissues. in
effectsof air pollutantson humans,plants,animals, cardiovascular andpulmonary
and glob a lc l i ma tea re b ri e fl yd e s c ri b e d.
ma t er ials diseases.
Heavymetals(Pb) Damage to liver, kidney and
Effects of air pollutants dn humans
brain;causes anemia,
On an average,man breathesaround 22,OOO neurobehavioural changes,
ti m es and inhale sa b o u t 1 6 k g o f a i r e a c h d a y. abnormalities in fertility
and
Prim ar ily ,t he air w e b re a th i s l i fe s u s ta i n i ng. pregnancy.
However,pollutantspresentin the air often cause Aliphatic hydrocarbons Cancer
harmful effects.The nature of the pollutant, its andpolycyclic
organic
concentration and durationof exposureare among c0mp0un0s
the factorsthat affect the health of humans.In
Suspended pulmonary
particulate Chronic diseases-
general, infants, elderly people and those with
matter(SPM) bronchitis,
asthma.
respiratory diseasesare more susceptible to air
pollution. Further, adverse health effects of air Radioactiveisotopes Anemia, andgenetic
cancer
p ollut antar
s em ax i m u md u ri n gw i n te rc o mp a re d
to 1;tst,p32,ge6o1 defects.
other seasons.
Someof the importantair pollutantsand their The entry of the gas air pollutants(SO2,NO2)
effectson human healthare given in Table 55.3. predominantly occurs through the stomata,
Sulfur dioxide is the most dangerous pollutant gas openingson the leaves.Stomataare locatedat the
to man. lt is produced in many industriesand bottom of the leavesthroughwhich CO, entersfor
causesrespiratorydisorders.Oxides of nitrogen photosynthesis. In the sameway as CO, entersthe
and carbon monoxide reduce O, supply to the leaves,the gaseouspollutantsalsoenterand cause
tissues.Lead pollution is associatedwith tissue various effects.
r'amage and abnormal behavioural pattern.
Suspended particulatematter(SPM)mainly causes The solid particlesare adsorbedon the surfaces
chronicpulmonarydiseases. Prolonged exposureto of leaves.Thismay resultin cloggingof the stomata
'adioactiveisotopesresultsin anemia,besidesa and a reduced intake of COr. Further, the
.eavy risk of cancerand geneticdefects. suspendedparticlesdepositedon the leaves,may
adversely affect the leaf functions (reduced
Effects of air pollutants on plants exposure to sun light, decrease in chlorophyll
content,interruptionof gaseousexchange).
At low concentration, the air pollutantsmay not
causeany visible damageto the plants.However, The effects of common air oollutants on leaves
evenat this level,the pollutantsmay be storedand are given in Table55.4. The sensitivityof plantsto
introducedinto the food chain. This in turn, may pollutants depends on many f actors such as
affectanimalsas well as humans. climatic conditions(light, temperature,humidity),
672 B IOTECHNO LO CY

Effects of air pollutants on materials

air pollutants on plants
Air pollutantscauseimmensedamageto various
Pollutant Effects materials- stone, metal, paint work, fibre
materials, glass, ceramic, textiles, rubber,
Sulfur
dioxide Bleaching
of leaves and
architectureetc. The adverseeffectsof air pollution
(killing
necrosis of leaves)
on materialsand propertiesare associatedwith
Nitrogen
dioxide Bleaching
andsuppressed severeeconomic lossesthroughout the world.
growth
Ozone Bleaching
andnecrosis Effects of air pollutants on global
Fluorides Marginal
necrosis climatic changes
Ammonia Leavesbecome dullgreen A i r pol l uti on i s associ atedw i th signif icant
Ethylene
and Leafcurlingandleaf changesin globalclimateand the relatedprocesses
pr0pyrene dropping e.g.,ozonedepletion,greenhouseeffects,acid rain
Peroxyacetylnitrate Suppressed growthand (ReferChapter60).
(PAN) silvering
of lowerleal
surfaces B IOMON ITOR IN G OF A IR P OLLUTI O N
Particulates Bleaching
andnecrosis Plants are used to monitor (biomonitoring)air
(withtoxicmetals) pollution and such plants are referred to as
indicator plants.This is basecjon the principleof
sensitivity and responseof the plants to ar
soil and water,and the individualplant'sresponse
pollutants. A list of the plants used tor
to a particularpollutant.The major air pollulants
bi omoni tori ng and the correspondi ng air pollut ant s
affecting plants are SO2, NO2, On fluorides,
is given in Table 55.5. Among these, lichensand
ammonia and ethylene, besides the particulates.
mossesare most commonly used to check the
They may damagethe plantsto varyingdegreesas
qualityof air.The patternof occurrenceof patches
outlinedin Table 55.4.
on lichensservesas an index'forbiomonitoringof
The sensitivityof certainselectedplantsto air ai r ool l uti on.
pollutantscan be used for biomonitoringof air
pollution(detailsgiven later). Bioluminescence in air pollution
monitoring
Effects of air pollutants on animals
Bioluminescence is the phenomenon of
The effectof air pollutionon animalsis mostly
emi ssi onof vi si bl el i ght by an organi smlt. occur s
indirect,as it occursafterthey eat pollutedplants
as an enzymati cal l ycatal ysed l i gh t em it t ing
or foliage. Fluorine,lead and arsenic are three
main air oollutantsthat cause harmful effectsto
livestock.
Tmrr 55.5 Alr pollutants and ttc platrts for
Fluorine : Fluorine causes loss of weight,
ttelr blomonttodng
muscularweakness, diarrhea,wearingof teeth,and
even death.Fluorosismainly affectsthe ruminants, Air pollutant Plant(s)
particularlydairy cows.
Sulfur
dioxide Lichens,
moss, pine
white
Lead: Leadpoisoningis commonlyobservedin
animals grazingnear lead mines. lt is associated Ozone garden
Tobacco, bean
with loss of appetite, difficulty in breathing,
Peroxyacetylnitrate Lettuce,
bean
d i a rrh e aa n d p a ra l y s i s .
(PAN)
Arsenic : Arsenic toxicity in animals is
Ethylene marigold,
Orchids, cucumber
associated with increased salivation, thirst,irregular
pulses,abnormalbody temperatureand paralysis. Fluoride peach
Apricot,
C h ro n i cp o i s o n i n gma y re s u l ti n anemi a,di arrhea,
paralysisand even death.
metals
Heavy lichens
Moss,
 Chaot er55 : A IR P O L L U T IO NA N D C O N T ROL 673

r eac t ionin li v i n g c e l l s . B i o l u m i n e s c e n cies an o Electrostatic

precipitators
indicatorfor the analysisof atmospheric gasessuch . Fabricfilters.
as SOr, formaldehydeand ethyl acetate. The
hicroorganism,Photobacteriumphosphoreumcan Gravity settling chambers
be usedas a specialphotodetector. The changesin
the emissionof light due to a pollutantcan be Settlingchambersarethe oldestand very simple
detectedby a sensor,amplifiedand recorded. type of equipment used for collection of solid
particles(Fig. 55.1A).As the air is passedthrough
the chambersat a low velocity,the dusf particles
(size40-100 pm diameter)settleby gravity and the
cl ean ai r comes out. A l though thi s techni que
is simple, inexpensive with low cost of
T he air p o l l u ti o n l o a d p a rti c u l a rl y from maintenance, it is not possibleto settlesmallersize
industries,can be reducedby severalmeasures- dust oarticles.
replacement of burningfuel by electricityor solar
energy, improvement in fuel burning process, Cyclone collectors
and dilutionof pollutants,
dispersion and reduction Cyclonecollectorsalso operateon the principle
at source by using control equipment. These of gravity settlement. The equipment mainly
measures,however,are useful only to a limited consist of a vertically placed cylinder with
extent. an invertedcone attachedto its base (Fig. 55.18).
ln the nature itself,there are certain devices, As the air entersthe cvlinder,it takes a helical
commonly referredro as atmosphericself-cleansing pathdownwards.Due to rapidspirallingmovement
processesfor the removal of air pollutants.These of the air, the particlesare thrown towardsthe
naturalprocesses are very slow and limited, and walls by centrifugaliorce. These dust particles
cannot cope up with the present increased settle down in the hopper at the bottom, while
demands of pollution control. However, the the dust free air passesthrougha pipe and comes
artificiallydevisedpollution control measuresare out.
basedon the same principlesof atmosphereself- The cyclonecollectorsare simple,low cost and
cleansing processes.These principles include easyfor maintenance. Theyare idealfor separation
dispersion, gravitational settling, flocculation, of dust particleswith sizes 15-50 pm. Cyclone
abmrption and rain out. collectorscannot settleoarticleslessthan 10 um
There are two categories of air control srze.
devices-devices to control particulate pollutants,
and to control gaseouspollutants. Dynamic precipitators
Dynamic precipitatorswork on the principle
CONTROL DEVICES FOR of centrifugal force, generated by rotating
PARTICULATE POLLUTANTS blades(Fig. 55.1A. The dust particlesof the air
In general,greateremphesisis given to control are concentratedon the rotating blades from
particulatepollutants,may be becausethey are where they are collected in a concentrated
visible.lt may however,be notedthat in termsof stream.By employingthis equipment,dustparticles
v olum e of a i r p o l l u ti o n , g a s e o u s p o l l utant of 5-20 pm size can be separated.However,
contributionis much higher. dynamic precipitatorsare not suitablefor sticky
or fibrous dust particles,as they stick to the
The important devices/equipmentused to blades.
control particulatepollutantsare listed.
. Cravitysettlingchambers Spray towers
r Cyclonecollectors ln spray towers, the dust particles(10 pm in
size) are made to settle down by spraying water
. Dynamicprecipitators
(Fig. 55.1D).This techniqueis particularlyuseful
o Spraytowers for separatinga heavy load of particles.

Biotechnology
[43]
 B IOTE CHNO LO CY
674

Water outlet
(D) SpraY tower

To high.voltage
caDle Shaking
arrangement
--) Clean
alr
-) Cleanair

Fabricfilters

U !U ! U

(E) ElectrostaticPreciPitator

Fig, 55.1: Controldeviceslor particulatepoilut!2

Chaot er55 : AIR P O L L U T IO NA N D C O N T R OL 675

Electrostatic precipitators
TmLr 55.6 A selected list of adsorbents and
Electrostaticprecipitators(ESPs)are very efficient their major applications
and versatile. They work on the basis of
electrostatic attraction. An ESP consists of a thick Adsorbent Major application(s)
cylinder fitted with an inlet at bottom and an outlet
or zeolates
Silicate of SOr,NO, and
Control
a t the top (Fig .55. 1D. An elec t r ode( m or e t han o n e
(asmolecular
sieves) mercury
electrodeare commonly used)fitted at the centre of
the cylinder is connectedto a high voltage cable. As
Activated
alumina air,gasesandliquids
Drying
the dust laden air passesthrough ESP,larger sized Activated
carbon Removing purification
odours,
particles settie down due to gravity. The smaller of gases
charged particles are attached to the oppositively Silicagel Dryingandpurifying
gases
chargedelectrodeswhich graduallyfall down to the Bauxite Drying of gasesandliquids,
bottom. The dust free air comes our. treatment of petroleum
Electrosiatic precipitators are said to be dry fractions
precipitators if the dust particles can be removed Fuller's
earth Refining
of oilsandfats
by scrapping. In case of wet electrostatic
precipitators, the particles are removed by using
water or any otlier fluid. Wet precipitatorsare more lf the adsorption is purely a physical
phenomenon (held by van der Waal's forces), it is
efficient, however, dry precipitatorsare preferably
referred to as physical adsorption. This process is
used for oractical reasons.
rapid and easily reversible. ln chemical adsorption
(chemisorption), the pollutant gas molecules form
Fabric filters
c h e m i c a l b o n d s w i t h t h e a d s o r b e n t .C h e m i s o r o t i o n
Fabric filters (Fig. 55.1D are the most efficient is very slow, usually requires the supply of energy
a nd can sep ara t epar t ic leswit h s iz es les s t han 0 . 5 and is irreversible. Pressure and temDerature
prm in diameter. As the air or gas is passed over significantlyinfluence chemical adsorption.
fabric like mats of wool, celluloss etc., the dust is
trapped while the gas passes out. Collection of In the Table 55.6, a selected list of tne
dust on the fabric filter resultsin the formation of a c o m m o r f l y u s e d a d s o r b e n t sa l o n g w i t h t h e i r m a j o r
dust layer (commonly referred to as filter cake). applications is given. The equipment used for
This in turn servesas a more efficient dust collector adsorption, referred to as adsorbers, may be fixed,
and helps to capture even fine dust particles. The moving or fluidized beds.
fabric filters have to be frequently cleaned Several
Multiple fixed bed adsorber : In this, adsorbent
models of fabric filters have been developed for
is arranged in the form of beds or layersas depicted
comme rcia l ap plic at ions .
in Fig. 55.2A. As the adsorption occurs, the
adsorbent gets saturated with the adsorbate. For
CONTROL DEVICES FOR GASEOUS reuse, the gas collected on the adsorbent must be
POLLUTANTS removed. lf this is not possible,the adsorbent must
The prin cip le gas pollut ant sar e SO r , N02, N2 O , be reolaced.
CO, hydrocarbons,and organic and inorganic acid
gases. The control devices used for gaseous Absorption
pollutants are based on the principles of adsorption,
absarption, condensation and combustion. fhe The pollutant gas (absorbate),when brought in
irrportant aspectsof selecteddevicesfor controlling contact with a solvent or liquid (absorbent),gets
gaseouspollutants are briefly described. adsorbed.The processof absorption may occur due
to physical or chemical phenomena.
Adsorption Absorption process is used to control the
When a stream of gas is passed through a pollutants SOr, NOz,HrS and hydrocarbons.
porous solid material (absorbent),it can attract and Ammonia is used as an absorbentfor controlling
hold the gas (adsorbate)molecules. soz'
676 B IOTE CHNO LO CY

Cleanair
-----J Steam
(duringcleaning)

Adsorbed
beds

--)Clean
all

Heated-----J
system
(forcleaning)
(A) Multiple fixed bed adsorber
(B) Perforatedtray tower

Cleanair Coolingliquid Clean Air with

inlet alr pollutants

:{- Liquid
inlet
bp,"y
Packing
material
Air with
pollutants

J IUE
Liouidoutlet Coolingliquid Condensate
outlet outlet
(C) Packed tower
(D) Surface condenser
Cleanair

{- Cold water
inlet

{- Air with
----* Clean
all

Waterand vapour
condensate Preheater
(E) Contact condenser (F) Thermal incinerator
 55 : A I R P O L L U T IO NA N D C ON T R OL
ChA O t Cr 677

Differenttypesof absorbersare usedfor control Combustion

of gaseouspollutants.Theseincludespraytowers,
plate or tray towers and packedtowers.The gas It is a fact that combustionor incinerationis a
absorptionunits are so designedthat thereoccurs maj orsourceof ai r pol l uti on.A nd surpri si nglthe
y,
an intimatecontactbetweenthe gasand the liquid. same combustionprocesseseffectivelyserve to
Thiswill ensureoptimalabsorptionof gas into the control ai r pol l utants.Thi s i s cari i ed out by
liouid. convertingcertain poliutants(e.g., hydrocarbons,
carbonmonoxide)to carbondioxideand water.For
Spray towers (Refer Fig. 55.1D) r They can effi ci entoxi dati onduri ng combusti on,suppl y of
etficientlyremovegaseouspollutants,besidesthe oxygen/ optimal tenrperature and time are
particulates. Thesedevicesare particularlyuseful necessary. Thereare differenttypesof cornbustion
for the removal of pollutant gases with high processes depending on the pollutant to
concentrations of oarticulates. be oxidized. The most imDortant ones are
Plate or tray towers : These absorbersare direct-flame combustion, thermal combustion and
designedwith horizontal trays or plates so as catalvtic combustion.
to provide large liquid-gas interfacial surface Direct-flamecombustion: In this device,the
Fig. 55.28). ln the perforated tray tower, the wastegasesare directlyburnt with or without the
abs or bent ent ersth e c o l u mn fro m th e to p , s p i l l s addi ti on of auxi l l ary fuel s. D i rect-fl ame
and f lows in a z i g z a g fa s h i o n .T h e p o l l u te da i r combustions are mostoften used in oetrochemical
entersthe column from an inlet at the side of the plantsand refineries.
bottom.As the air passesthroughthe openingsof
t he t r ay s ,it get si n c l o s ec o n ta c w
t i th Ii q u i dd u e to Thermal combusti on: Thi s processusual l y
repeatedexposure.This enablesthe removal of becomesnecessarywhen the gaseouspollutant
gaseous as well as particulatepollutants. The clean concentration is too low, and it is difficultto carry
qasemergesat the top. out di rect-fl ame combusti on. Thermalcombusti on
is carried out by a thermal incinerator or after
P ac k ed t owe rs : In th i s c a s e , th e l i q ui d
burner(Fig.55.2n.An heatexchangerpreheats the
rabsorbent) is sprayedover a packingmaterial(e.g.
air/gaswith gaseouspollutants.This preheatedgas
all rings,Berlsaddles) with largesurfaceto volume
is passedover to the incinerationequippedwith a
ratio (Fig. 55.2A. The air with gaseouspollutants
burner.The temperature of the incineratormay be
entersfrom the side of the bottom of the tower ano
i n the range 500-1000' C , and thi s actual l y
the clean air comesout from the top.
depends on the nature of tl.re pollutant to be
oxidized.Combustion with thermalincinerators nas
Condensation
to be carefullycarried out so that the oxidation
When the temperature of a gaseouscompound process i s effi ci ent and compl ete. Incompl ete
is reduced, its condensationoccurs. There are combustion may produce more pollutantse.g.
trvo devicescommonly used to control gaseous carbonmonoxi de.
pollut ant sbas , e do n th e p ri n c i p l eo f c o n d e n s a ti orr.
Catalytic combustion: This is requiredwhen
Surfacecondenser: In this device,the cooling the gaseous pollutants are too low in
mediumis passedthroughmetaltubesover which concentration. By ernploying a catalyst, the
the gaswith pollutantsis passed(Fig. 55.2D).fhe combustioncan be madefasterand complete,ano
\aoour condenses on the surfaceof the tubes.This the catalystcan be usedagairrand again.
g et sc ollec t eda s a fi l m o f l i q u i d w h i c h c a n be
drainedoff. Catalyticcombustionis usedfor the removalof
NOz (in the tail gas) from the nitric acid
Contact condenser: In contact condenser,the plants. Here platinum catalyst is used. Carbon
cooling m edium a n d th e g a s e o u sp o l l u ta n tsare nronoxidecan be removedby usingcopper(Cu2+)
made to have a direct contact with each other catalVst.
Fig. 55,2fi. As the vapors get cooled, they
condens eand are e a s i l yre m o v e da l o n g w i th the The maj or l i mi tati onof catal yti ccombuti oni s
rrater,and the cleangascomesout from the top of the high costof the catalystand maintenar.rce of tfre
the condenser. catalyticcombustion.
r-T -

678 B IOTECHNO LO CY

Water Thereare mainly two types of reactordesigns

+ for the biodegradationof VOCs- biofiltersand
bioscrubbers(Fig. 55.3).
H
U
M Biofilters
I
D A biofilter is composedof a porous medium
I (of compost, wood chips) that supports the
F mi croorgani sms formi ng a fi l m. The pollut ed
I air is first passed through a humidifier and
E Support
R material then through the biofilter. A great majority of
VOCs in the air (approximately9O%) get
biodegraded,and clean air comes out of the
Pollutedair biofilter(Fig. 55.3A).
Advantages of biofilters: They are simpleand
cost-effective.Certain poorly soluble pollutants
(hydrocarbons) can be easily rcmoved by this
Clean
alr approach.Biofilterscan be successfully used for
the biodegradationof several xenobioticse.g.
chloromethane.
Polluted Disadvantagesof biofilters : lt is ratherdifficult
atr to control processconditions(pH, temperature).
Bioscrubber
When compost is used as support material, it
(B) generates
unpleasant odours.Biofiltersrequirelarge
areas
Fiq.55.3 : Tteatmentprocesses for the remaval ot
volatile organic compounds{A) Biotilter (B) Biosuubber. Bioscrubbers
Bioscrubberis an improvementover biofilter
(Fig. 55.38). When the polluted air is passed
C O N T R OL D EV IC E S FOR V OLA TILE
througha' l i qui d streami n a spra ycham bert,he
ORGANIC COMPOUNDS (vOGsl pollutant(VOC)getstransferred to the liquid. The
There are severalvolatile organiccompounds, sprayed l i qui d contai ns a s uspension of
arisingfrom industrialand domesticactivities,that microorganisms which cycles betweenthe spray
c a u s ea i r o o l l u ti o n .C o od exampl esof V OC s are chamberand wastewater treatmentunit (activated
alcohols, ketones, organic acids, phenols and sludgeunit).The microbialbiodegradation of VOC
organicsolvents.The conventionalmethods(e.9. occursin the waste watertreatment unit. The clean
combustion,adsorption) of treatmentVOCsrequire air comes out, after recycle, from the spray
large quantities of energy, besides creating chamber.Besidesa continuousnutrientsupply,it is
secondarypollution. to maintainthe pH in the activatedsludge
essential
Biodegradation by microorganisms is a preferred uni t.
methodfor controliingair pollutionof VOCs.The
efficiency of biodegradationdepends on the Biotrickle filters
s n d basedon thi s,V OC sare
i n d i v i d u acl o m p o u n d a for more
Biotricklefilter is a recentdevelopment
broadlycategorized as follows : efficient biodegradationof VOCs. lI works on the
. Rapidly degraded VOCs-alcohols, ketones, principles of adsorption, followed by bio-
aldehydes,organicacids. degradation.The pollutantSasesare adsorbedon a
solid surface (e.g. activated carbon) and then
. Slowly degradedVOCs- hydrocarbons, phenols, sublectedto biodegradationby a streamof medium
organicsolvents(chloroethene). containingthe desiredmicroorganisms. As of now,
c Very slowly degraded VOCs-polyaromatic biotricklefiltersare at an experimentalstage,and
hydrocarbons, polyhalogenated hydrocarbons. soon they may be availablefor use.
\ fi /ater is absolutelyessentialfor the existence For the reasons stated above, it is well
V V of lif e (a n i m a l o r p l a n t). l t p ro v i desa recognized worldover that there is an urgent
won de rful ch em ic al m edium in whic h all biolo g i c a l need for water conservation and management.
and biochemical processesoccur'.Water dissolves T h e C o v e r n m e n t a l p o l i c i e s a l s o s u p p o r t t h i s
va riou s n utri ent s , dis t r ibut es t hem t o c ells a n d conceDt.
removes waste oroducts.

About 60% of the human body is composed of PI$SOLVED SXVGEN

water. One can survive without food for some
Oxygen is soluble in water, and this dissolved
weeks, but cannot survive without water for
oxygen (DO) is essential to support fish and other
more tha n a f ew day s . Bes ides dr ink ing, m a n
aquatic life in water. The solubility of atmospheric
req uire s wa ter f or bat hing, was hing, liv e s t o c k
oxygen in fresh water is around 7-15 mg/dl,
raising , in du s t r ial, agr ic ult ur al and v ar ious o t h e r
depending on the temperature and pressure.
purposes.
Increasein temperatureand salts in water decreases
O, solubility.
Water.a scarce natural resource
Oxygen gets dissolved in water by one or more
Water, covering more than 70% of the earth's
of the following ways.
su rface, exists in all t hr ee s t at es - liquid,s olid a n d
gas. Most of the water on earth is salt water, and is r Direct entry from the atmosphere.
un su itab lefor dr ink ing and ot her needs of m a n . l t o By aquatic plant (algae) photosynthesis.
is estimated that only around 0.007% of the earth's
water is available for direct human consumption. o lntroduction by artificial means (aerators).
lhus, consumable water is a scarce natural
re50urce. lnnportance of dissolned cxygen

Water is required for development and progress
.' people, and therefore a nation. In fact
^ ;iorically, the places of availability of water were
'-=
"rajor determinantsfor the settlementof people.
^ 5 is because besides regular use, water ts
::::^iial to raise the crops It is an accepted fact
-i. r co mmun it y wit h good wat er s upply has g o o d
:- '..f , progressand prosperity.
A minimum level of dissolved oxygen at a
concentration around a mg/cll is considered to be
necessary to support healthy aquatic life. If good
aerobic conditions are not maintained, anaerobic
microorganisms take over and cause harmful
effects And this is what happens in polluted 'waters,
particularly from the industrial and municipar
wastes.

679
>tr

68(} B IOTE CHNO LO CY

Determinationof dissolved oxygen is very The salient features of these pollutants and the
importantin water pollution.This is mostlyceirried sources of their contribution are briefly described.
out by measuring biochemical oxygen demand
for the oxidation ORGANIC POLLUTANTS
(BOD), utilizing r.'ricroorganisms
of organicmatter(Seep. 683). c o m p o u n d s a s s u c h a r e a b so l u te l y
Qrganic
esse'ntialfor the existence of life. However, their
entry into water causes pollution resulting in bad
o d o u r a n d u n p l e a s a n tt a s t e . I n a d d i t i o n , so m e o f
the organic compounds may be toxic or even
c a r c i n o g e n i ct o h u m a n s .
Sev er al k inds of nat ur a l , a n d m a n m a d e
ac t iv it ies ( dom es t ic , indus t r i a l , a g r i c u l t u r a l e t c . ) Sources of organic pollutants
c ont r ibut e t o wat er pollut ion . Wa t e r p o l l u t i o n i s
characterized by certain observable disturbances in There are several natural sources of organic
the normal properties and functions of fresh water. p o l l u t a n t s . T h e s e i n c l u d e t h e d e c a y o f l e a ve s,
These include offensiveodours, decreasein aquatic plants, and dead animals, besides the release of
life (e.g. fishes),bad taste and unchecked growth of o r g a n i c m a t e r i a l s f r o m a q u a t i c pl a n ts a n d
aouat ic weeds . mrcroorganisms.

Several synthetic organic pollutants (e.8.

Ground water pollution
pesticides,herbicides,ethylbenzene)and synthetic
The ground water is generally considered to be volatile organic chemicals (e.9. ca r b o n
s af e and is us ef ul f or dr ink i n g , a g r i c u l t u r a l a n d tetrachloride, tetrachloroethylene) pollute water.
industrial purposes. ln fact, ground water is less C e r t a i n o r g a n i c p o l l u t a n t s s u c h a s di o xi n a n d
prone to pollution. However, in recent years, it ts p o l y c h l o r i n a t e d b i p h e n y l s ( P C B s ) a r e ve r y to xi c,
r ec ogniz ed t hat c ont am ina t i o n w i t h f l u o r i d e , b e s i d e sb e i n g c a r c i n o g e n i ct o h u m a n s .
arsenic and nitrate in the ground water poses a
serious threat to human health. INORGANIC POLLUTANTS

In the natural fresh water (sourcemay be ground

Surface water Pollution
or surface),there occurs varying concentrationsof
The rivers, lakes and reservoirs are highly i n o r g a n i c c h e m i c a l s . M o s t o f t h e m , i n ge n e r a l , a r e
susceptiblefor pollution due to natural and man- within the acceptablelimits.
m ade ac t iv it es ( indus t r ial, d o m e s t i c , a g r i c u l t u r a l
etc.). The routes for the entry of pollutants include Sources of inorganic pollutants
sewageoutfalls, industrialoutfalls, and outfalls from
The industrial and agricultural outflows pollute
nuclear staiions.Surfacewater pollution is a major
water with inorganic compounds e.8. metal
threat for the survival of life itself. lt is therefore
complexes, inorganic salts, mineral acids, trace
necessarythat regular monitoring of various routes
elements. Among these, water pollution due to
of water contamination is done and effective
t o x i c m e t a l s a s s u m e s s i g n i f i c a n c e e .8 . l e a d ,
protective measures are taken to minimise the
m e r c u r y ,c a d m i u m , n i c k e l , a r s e n i c a n d a l u m i n i u m .
pollut ion.
MICROBIOLOGICAL POLLUTANTS
NATURE OF WATEB POLLUTANTS
A w i d e r a n g e o f m i c r o o r g a n i s ms ( vi r u se s,
There are a large number of water pollutants
bacteria, protozoa, algae, helminths) contribute to
whic h m ay be in dis s olv ed,s u s p e n d e do r c o l l o i d a l
water pollution. A majority of these organisms are
state.The pollutants may be broadly categorizedas
harmless to humans. However, there are certain
f ollows .
organismsthat cause diseases,referred to as water
Organic pollutarrts borne diseases.Such organismscausing some form
'
. I nor ganic pollut ant s of sicknessin humans are referredto as pathogens
or pathogenic organisms. A selected list of
. M ic r obiologic al pollut ant s pathogenic organisms and the corresponding
o Radioac t iv eoollut ant s . diseasesis given in Table 56.1 .
Chapter56 : WATERPOLLUTIONAND SEWACE 681

(domestic waste.watey) and the respective dlseases

Organism Disease Major diseasesymptom

Bacteria
Escherichia
coli Gastroenteritis Dianhea
(enteropathogenicl
Salmonellatyphi Typhoid Highfever,diarrhea
paratyphi
Salmonella Paratyphoid Fever
Salmonellasp Salmonellosis Foodpoisoning
Vibriocholerae Cholera Diarrhea,
dehydralion
Shrge//a
sp Shigellosis Bacillary
dysentery
pneunophila
Legionella Legionellosis Acuterespiratory
illness
Viruses
Adenovirus Respiratory
diseases Breathing
difficulty
Enteroviruses Gastroenteritis Diarrhea
Reovirus Gastroenteritis Dianhea
Rotavirus Gastroenteritis Diarrhea
HepatitisA Infectious
hepatitis Jaundice
Protozoa
Entanoebahistolytica Amoebic
dysentery Prolonged
diarrhea
Giardialanblia Giardiasis Milddianhea,
nausea
Balantidiuncoli Balantidiasis Dysentery
Helminths
Ascarislumbricoides Ascariasis infestation
Roundworm
Schistosoma
sp Schistosomiasis Hematuria,
dianhea
Fasciolahepatica Fascioliasis Jaundice
T::!!i:?s_i:?11
, Taeniasis Intestinal
infection

Algae
aeruginosa
Microcystis Gastroenteritis Dianhea
Aphanizomenon flosaquae Gastroenteritis Diarrhea
calciola
Shizothrix Gastroenteritis Diarrhea

Sources of pathogens wastes (particularlyfeces)from enteringpotable

water.
The pathogensare mainly presentin the waste
water and for most of them human feces are the RADIOAGTIVE POLLUTANTS
prime sourceof pollution. The human waste can Water may get polluted with radioactive
pollute the water from sewageoutfallsor from a
materialsarisingfrorn nuclearplants,radioisotopes
flow of waste from ground (mostly from failed used in medical,industrialand researchpurposes,
septicsystem). besides the processing of ores to produce
The best way to control water contamination radioisotpes. The radioactive pollutants are
with pathogenicorganismsis to preventthe human carcinogenice.g. uranium,radium,thorium.
682 B IOIE C HNO LO CY

Tnsre56.2 fypical compositionof domesticwaste water or sewage(untreated)

Constituent(s) Concentration (mg/l)*

Weak Medium Strong

Total solids (TS) 350 700 1,250

solids(DS)
Dissolved 250 500 900
solids(SS)
Suspended 100 200 350

Total nitrogen .U 45 on

Organic N 10 15 40
InorganicN (asfreeNH' 10 30 50

Total phosphorus + 8 16
Organic 1 6
Inorganic I

Chlorides 25 100
(asCaCO3)
Alkalinity 50 100 200
Grease 50 50 100150
Volatile (pg/ l)
compounds
organic <100 100-400 >400
Biochemicaloxygen demand(BOD) 100 400
Chemicaloxygendemand(COD) 250 1000
Total coliform(no/dl) 106-107 1o7-108 108-10e
* The unitsfor volatileorganb compounds
and totalcolitormare givenalongwith then.

living organisms- bacteria, viruses, algae,

fungi and protozoa. Some details on pathogenic
organismsand the correspondingdiseasesare given
Sewage is the liquid waste or waste arising in Table 56.1 .
niainly from domesfic (residential,institutional,
commercial)and industrial sources.
TYPES OF SEWAGE
C OMP O S IT IO N O F S EW A GE The waste water (sewage)is broadly categorized
The actualcompositionof sewagedependson as weak, medium and strong, based on the
the sourcefrom which it comes.In general,about composition (Table 56.2). lt must however, be
97-99% of sewageis composedof waterwhile the noted that the composition of sewage is
h i g h l y v a r i a b l e w h i c h m o s t l y d e p e n d s o n th e
re s t(1 -3 % )i s s o l i d s .T h e m o st i mportantorgani c,
i n o rg a n i c o mp o u n d sa, n d l i v ingorgani sms l o c a l c o n d i t i o n s . T h e s e w a g e m a i n l y co n si sts
found
in wastewater are listedbelow. of solids, organic and inorganic compounds of
nitrogen and phosphorus, chlorides, grease
Organic-carbohydrates, fats, proteins,amino
and volatile organic compounds. ln Table 56.2,
acids and urea, besidesthe products of their
the data on biochemical oxygen demand
degradation. (BOD), chemical oxygen demand (COD) and total
In o rg a n i c -s a n d ,m u d , mi neral ash, mi neral coliform for the three types of sewage are also
salts,lead,arsenic,mercuryand cyanides. qtven.
ChAPtCT
56 : WATERPOLLUTIONAND SEWACE
The measurementof water pollution in sewage
waste water with special reference to organic
ma tter an d p at hogenic or ganis m s is des c r ibe d .
MEASUREMENT O F O RG ANI C M ATTE R
OF SEWAGE
The organic matter present in the sewage is
regarded as biologically active, if it can be oxidized
by the bacteria. On the other hand, the organrc
matter resistant to bacterial degradation rs
considered as biologically inactive. For the purpose
of sewagetreatment,the biologically active organic
matter is imoortant.
The commonly used laboratory methods for the
measurementof organic matter in sewage are given
below.
o Bioche mica l ox y gen dem and ( BO D)
. Che mica l o x y gen dem and ( CO D)
o Total organic carbon (TOC)
. Theoretical oxygen demand (ThOD).
Biochemical oxygen demand (BODI
Bioche mic al ox y gen dem and is t he m os t wi d e l y
used parameter Io measure the organic pollution n
sewage as well as surface water. BOD basically
involves the measurement of dissolved oxygen
(DO) utilized by the microorganisms for the
biochemical oxidation of organic matter. fhe
demand for oxygen and the process of oxidation
depends on the type and quantity of organic matter,
temperature and type of the organism used.
In ge ne ral, bioc hem ic al ox y gen dem an d i s
measured for an incubation period of five days
(hence appropriately referred to as BODt) aI a
temperature of 20"C. lf the organic content of the
sewage is high, it needs to be diluted for the
measurementof BOD. Further,for waste water with
less p op ula tion of m ic r oor ganis m s ,s eeding w i t h
bacterial culture is necessary.
BOD indicates the amount of organic matter
present in the sewage. Thus, the more is organic
content, the higher is the BOD (Refer lable 56.2for
BOf) values of different types of sewages).lf the
a va ilab le oxy gen ( dis s olv ed 02) is les s t han t h e
BOD, the organic matter decomPoses
643
anaerobically,putrifies and produces foul smell.
fhus, BOD is a measure of nuisance potential of
sewag,e.
Limitations of BOD
1 . B O D m e a s u r e so n l y b i o d e g r a d a b l e o r g a n i c
matter.
2. A high concentration of bacterial load is
required.
3. For measuring BOD of toxic waste water,
pretreatment is necessary.
4. Requires long period of incubation i.e.5
0ays.
Despite these drawbacks, BOD is very widely
used worldover for oractical and economic reasons.
Chemical oxygen demand (COD)
Chenrical oxygen demand refers to the oxygen
equivalents of organic matter that can be oxidized
by using strong chemical oxidizing agents. [.lsually,
potassiumdichromate in the presenceof a catalyst,
i n a c i d i c m e d i u m i s e m p l o y e d f o r t h i s p u r p o s e .T h e
overall reaction is given below'.
Organic matter + CrrO2r-+ H+ ------+
C r 3 ++C O r +H r O
When compared with BOD, COD oxidizes more
organic compounds, hence COD valuesare higher
than BOD values. Chemical oxygen demand can
be determined in lust three hours, in contrast to
BOD requiring five days. Some workers determrne
COD and calculate BOD. This is possible since
there exists a reasonablygood correlation between
COD and BOD.
Total organic carbon (TOC)
Measurementof total organic carbon is required
when the concentration of organic matter is very
low. TOC can be determined by oxidizing organic
carbon, in the presenceof a catalystto COr, which
can be measured
Theoretical oxygen demand (ThODl
The organic matter of sewage is mainly
composed of carbohydrates, proteins, fats and
products of their decomposition. lf the chemical
formulae of the organic matter (i.e. individual
compounds) are known, the theoretical oxygen
demand can be calculated.
644 B IOTE C HNO LO CY

Innerfermentation
tube

Fig. 56.1 : Multiplelube fermentation technique for the detection of coliform bacteria.

DETECTION OF PATHOGENIC Among the indicatororganisms, the rod-shaped

ORGANISMS OF SEWAGE bacteria,commonly known as coliform organisms
(E. coli, Aerobactersp) are the most commonly
Hum an beings inf ec t ed wit h d i s e a s e - c a u s i n g
used. The intestinaltract of man is very rich in
microorganisms(i.e. pathogens)are the carriersand
coliform bacteria.lt is estimatedthat each person
they can discharge pathogenic organisms in waste
dischargesabout 100-400 billions of coliform
water. Severalspeciesof bacteria, viruses,protozoa
organismsper day. Therefore,the detection of
a nd helm int hs ar e r es pons ibl e f o r d i s e a s e s i n
coliform organismsis a clear indicationof fecal
humans. A selectedlist of the pathogenicorganisms
contamination and thusthe presence of pathogenic
and the major diseases is given in (Table 56.1).
organisms. On the otherhand,the absenceof these
Among the diseases (water-borne diseases)
organismsindicatesthat the water is free from
originating from sewage, typhoid, paratyphoid,
disease-causing organisms.Detectionof co[iform
cholera, gastroenteritis,diarrhea, and dysenteryare
organismsis basedon the fact that a great majority
important. All these diseasesare highly infectious
of water-borne diseaseshave fecal origin.
and are responsiblefor the death of thousands of
p eople in c ount r ies ( par t ic ular l yd e v e l o p i n g o n e s ) LABORATOBY METHOOS FOB
wit h ooor s anit at ion. DETECT'ON OF COLIFONM ORGAN'SMS
There are three methodscommonly employed
lndicator or index organisms of
for the detectionof coliform organismsin water
sewage
sampl es-mul ti pl etube-fermentati ontechnique,
I t is t edious and t im e c ons um i n g t o i s o l a t e a n d membranefi l tertechni queand col i l erttec hnique.
identify pathogenicorganismsin water waste. Some
other non-pathogenic bacteria of sewage polluted Multiple-tube fermentation (MTFI
water are employed for this purpose e.g. E. coli, technique
Streptococus faecalis, Clostridium perfingens, Thi s i s basi cal l y a test that i nvol v es acid
Klebisella sp. These organisms are collectively fermentation.Lactosebroth fermentationis testeo
referred to as indicator or index organisms. at around 35'C for 1-2 days in a seriesof tubes,
Chapter56 : WATERPOLLUTIONAND SEWACE 645

Advantagesand disadvantages : The membrane

filtrationtechniqueis rapid and can be carriedout
outside the laboratory (fields) also. This is in
contrastto multiple tube fermentationtechnique.
To vacuum The major limitationMFT is that it cannotbe used
for turbid and/or heavily polluted water. This is
becausethe membraneporesget clogged.
Membrane Bacteriaon
filtrationunit membranefilter Golilert technique
Although commonly used, both the tests
Fig. 56.2 : Membrane filtration technique for the described above (MFT and MF) sometimesgive
detection of coliform bacteria. false-positiveand false-negativeresults.Colilert
techni quei s a recentand noveltechni quedesi gned
to specificallydetect E. coli and other coliform
inv olv ing s eq u e n ti a l te s ts -p re s u mp ti v etest, bacteria.This is basedon the principlethat when
confirmedtest and completedtest. certain substratesare digestedby microorganisms,
chromogens(coloured substances)are produced.
Presumptivetest : The waste water sample is Thus, when O-nitrophenylB-D-galactopyranoside
s er iallydilut ed .On e m l o f th e s a mp l eo f e ach (ON P C ) i s used, i t i s convertedto a yel l ow
dilution is then transferredto each of the five compoundby coliformbacteria.With the substrate
fermentationtubes. These tubes contain lactose 4-methyl umbel l i ferylp-D -gl ucuroni de,E . col i
culture medium and invertedgas collectiontubes producesa fluorescentcompound.
(Fig. 56.1). At the end of 24 hour incubation
period, if thereoccursg'ascollectionin the inverted TE C H N IQU E S TO D IS TIN GU IS H FE C A L
tube, the test is positiveindicating the presenceof FROM NON.FECAL BACTERIA
coliform organisms.
A positivelaboratorytest for coliform bacteria
Confirmedtest : lf no gas formationoccurs in need not necessarilybe due to pathogenic
the presumptivetest, the incubationis continued organisms, originatingfrom intestines.For instance,
for another 24 hours. lf gas formation is detected Enterobacter (Aerobacter\ aerogenes, found in
now, the test confirmsthe presenceof coliform decayingplant materialscan fermentlactoseand
bacteria.And if no gas is producedeven in the give a positivecoliformtest.Thus,the presenceof
confirmedtest,it can be assumedthat the samples E. aerogenes in water does not indicate
are free from coliform. contaminationwith coliformbacteria.
Completedtest : In this, a samplefrom positive A seriesof laboratorytests are availableto
coliform test is grown on eosin methyleneblue differentiate fecal from non-fecalorganisms. These
(EMD)agar.The incuhationis carriedout at 35"C
techniquesare collectivelyreferredlo as IMViC
for 24 hours. The presence of E. coli can be test,with the followingconnotations.
detected by the occurrence of greenish metallic
sheenon the plates. I stands for indole test
M representsmethy red test
Membrane filtration technique (MFTI
V is for Voges-Proskauertest
The presence of coliformorganisms in watercan
C stands for citrate test
be detectedby usingcelluloseacetateesterwith a
pore size of 0.3 to 0.5 pm. For this purpose,the (Note : i is usedonly for phoneticpurpose).
d r 2 O mi n u te sa t B 0 ' C .
m em br aneis f irs ts te ri l i z e fo Thesefour testsare usedto distinguishE. coli
The filtration is carried out in asepticconditions from Enterobacteraerogenes.E. coli gives positive
unoerv ac uum . indoleand methylred tests,and indicatesthat the
As the filtrationoccurs,rhe bacteriaare held on sourceof pollutionis of fecalorigin.On the other
the membrane surface (Fig. 56.2). The trapped hand, Enterobacter gives positiveVoges-
aerogene.s
bacteriaare dried and stainedwith a dye such as Proskauerand citratetestswhich shows that the
erythrosine, and detected. water pollution is due to non-fecalorigin.
 wat er pollut ant s , c om pos i t i o n o f s e w a g e /
Jh e
I waste water, and the measurement of
po llut ion in s ewage ar e des c r i b e d i n t h e
previous chapter. Discharge of sewage or
waste water to the environment is a mafter
of concern, as it poses serious threat to public
health.
ln t he r ur al c om m unit ies , r ecy c l i n g o f w a s t e s
(h um an, anim al, v eget able)has be e n p r a c t i s e df o r
centuries. Such recycling processes provide
fertilizers and fuel. However, these waste
and the crude recycling processes are very
dangerous, as they are the major sources of
d is eas e- c aus ing pat hogens . A n d in many
d ev eloping c ount r ies , c r ude, a n d u n s c i e n t i f i c
recycling of wastes is still in use exposing the
people to health hazards. In some countries,
th e was t es ( of r ur al or ur ban or ig i n ) a r e c a r e f u l l y
treated and d isposed off . Waste waLer/
sewage treatment was started in 19th century
(150 y ear s ago) , and c er t a i n l y i t i s t h e
most important factor for the well being of
pr es ent day m an, bes ides inc r e a s i n g t h e l i f e
expectancy
The process designed for the treatment of
se wage s hould hav e t he f ollowing o b j e c t i v e s .
. Removal of floatable and suspended particles
o Treatmentof biodegradable organic materials.
o Elim inat ion of dis eas e- c aus i n g ( p a t h o g e n i c ) rags, papers, animals etc ) settleable inorganrc
or qanr s m s .
686
CI.ASSIFICATION OF TREATMENT
PROCESSES
The sewagetreatment methods may be classified
in two different ways based on the principle of
operaticn or the stagesof treatment process.
Based on the principle of operation
Physical unit operations : These methods
i n v o l v e t h e a p p l i c a t i o n s o f p h y s i c a l f o r ce s fo r
sewage treatment. The physical processesinvolved
include screening, mixing, s e d i m e nta ti o n ,
flocculation and filtration.
Chemical unit operations : These processes
operate on the principles of chemical reactionsthat
m a y i n v o l v e p r e c i p i t a t i o n ,d i s i n f e c t i o n , a d s o r p ti o n
etc.
Biological unit operations : The removal of
p o l l u t a n t s , p a r t i c u l a r l y t h e b i o d e g r a d a b l eo r g a n i c
m a t e r i a l s , c a n b e c a r r i e d o u t t h r o u g h b i ol o g i ca l
procesSes.
tsased on the treatment process
The sewage treatment processes are more
conveniently classified based on the stages of
treatment as preliminary, primary, secondary and
tertiary treatments.
Preliminary treatment : This process basically
involves the removal of floating materials (leaves,
s o l i d s ( s a n d , g r i t e t c . ) , a n d g r e a s e ,f a t s a n d o i l s.
 Chapter57 : SEWAGEAI/ASTE
WATERTREATMENT 687

Primarytreatment: Thisprocess, usuallycarried

out by sedimentation,involves the removal of
suspended organicsolidsfrom the sewage. + Etfluenl
.HJ
Secondary or biological treatment : The
removalof fine suspendedand dissolvedorganic Bars for Perforatedmetal
material present in the sewage constitutes screen olatform
(A)
secondarytreatment.This processcan be carried
out by aerobic or anaerobic biological units.
Sewageinlet
Tertiaryor advancedtreatment: This is the final
treatmentprocessof sewage,and is carried out
mostly in the developedcountries.In general,
sewage is disposed off after the secondary
treatment.When needed,the tertiary treatmentis
c ar r iedout by d i s i n fe c ti o (i
n .e .c h l o ri n a ti o n ).
Somedetailson the varioustreatmentorocesses
are brieflydescribed,in the following pages

Fine screen
As alreadystated,preliminarytreatmentinvolves
the removalof floating materials(leaves,papers,
rags)and settleableinorganicsolids (sand,grit),
besidesoily substances(fats,oils, greases).The
three major types of equipment-screeners, grit
Sewage
c ham ber s ,and s k i mmi n g ta n k s , e m p l o y e d i n outlet
(B)
pr elim inar sy c re e n i n g
a re b ri e fl yd e s c ri b e d .
Fig. 57.1 : Diagrammatic representation of screeners
Screeners (A) Fixed bar screen (coarse or medium)
A s c r eene irs a d e v i c ew i th o p e n i n g s(u s u al l y (B) Shredder.
uniform in size)to removethe floating materials
and suspendedparticles.The processof screening
can be carried out by passingsewagethrough Grit chambers
different types of screeners(with different pore The heavy inorganic materiai.ls (specificgravity
sizes). The screeners are classified as coarse, 2.4-2.7)like sand,ash and otherscan be removed
medium or fine, depending on the size of the by usinggrit chambers.Thistechniqueis basedon
openings.The coarsescreenhas largeropenings the processof sedimentation due to gravitational
( 75- 150 m m ) . T h e o p e n i n g sfo r m e d i u ma n d fi ne forces.Crit chambersmay be kepteitherbeforeor
screensrespectively are 20-50 mm and lessthan afterthe screens(described above).A diagrammatic
20 m m . representation of a typicalgrit chamberis depicted
in Fig. 57.2A.
Different types of screens-fixedbar screen
(coarseor medium) disc type fine screen,drum
Skimming tanks
type fine screen are in use. A diagrammatic
Several grcasy and oily materials (fats, oils,
reoresentationo( a fixed bar screen is shown rn
waxes,soapsetc.)from the domesticor industrial
Fig. 57.1A.
outletsfind their entry into the sewage.They can
A shredderor comminutor is a special screen be removedby using a skimmingtank which is
that can cut and retain the floatingand suspended fitted with baffle walls that divide the tank
materials(Fig.57.1B). (Fig. 57.28). The skimmingtank is divided into
688
Flushing
pump
Stilling
compartment can settle down at the bottom.
Slotted
baffles
Air pushed
up-waros
(B)
Fig. 57.2 : (A) Grit chamber (B) Skimming tank
(cross section).
three comoartmentsthat are interconnected.As the
compressedair is pushed from the floor of the tank,
t he r ais ing air bubbles c oagu l a t e a n d s o l i d i f y t h e
oily and greasy materials present in the sewage.
This material is pushed to the side compartment
referred to as stilling compartment from where it
c an be r em ov ed m anuallv or m e c h a n i c a l l v .
Primary treatment is aimed at the removal of
f ine s us pended or ganic s ol i d s t h a t c a n n o t b e
r em ov ed in t he pr elim inar y t r e a t m e n t . P r i m a r y
treatment basically involves the process of
sedimentation or settling. In the normal process of
sewage treatment, sedimerrtationis usually carried
out twice-once before the secondary treatment,
referred to as primary sedimentation, and then
after the secondarytreatment is complete, a process
known as secondary sedimentation lt is sometimes
necessaryto use chemical coagulants to facilitate
or aid sedimentation,and this process is referredto
B IOTE CHNO LO CY
as chemical precipitation or coagulation-aided
sedimentation.
Principle of sedimentation
The solid particle of the sewage tend to settle
down due to gravity. How.ever,most of the solid
particles of organic compounds remain in a
suspendedstate in a flowing sewage. lf the flow of
the sewage is stopped and if it is stored in a tank
referred ro as sedimentation tank, the solid particies
The process of sedimentation is influenced by
several factors. These include the size, shape and
specific gravity of particles, besides viscosity and
flow velocity of sewage.
Types of settling
There ar four major types of settlinS-discrete
s e t t l i n g , f l o c c u l a n t s e t t l i n g , h i n d e r e d o r zo n e
s e t t l i n g a n d c o m p r e s s i o n . T h i s c a t e g or i za ti o n i s
mainly based on the tendency of the particles to
interact and form solids.
Discrete settling : The particles which do not
change their size, shape and weight are referredto
as discrete particles or granular particles. The use
of grit in sewage may be consideredas an example
of discrete settling.
Flocculant settling : The flocculant particlescan
change their size, shape and weight, and thus lose
their identity. These particles actually coalesce
d u r i n g s e t t l i n g . S e t t l i n g o f b i o f l o c s , a n d ch e m i ca l
flocs in secondary sedimentation tanks are good
examplesof flocculant settling.
Hindered or zone settling : The particles as
such, tend to remain in a fixed position with
respectto each other. When flocculated, the whole
mass of oarticles settle as a unit or a zone. In the
hindered settling the concentration of particles
increasesfrom top to the bottom and this resultstn
t h e t h i c k e n i n g o f t h e s l u d g e . Z o n e se ttl i n g r s
e m p l o y e d i n c o n j u c t i o r r w i t h b i o l o g i c a l tr e a tm e n t
f a ci l i t i e s .
C o m p r e s s i o n : S e t t l e m e n t o f p a r t i c l e s i n th e
l o w e r l a y e r s c a n o c c u r b y c o m p r e s si o n o f th e
w e i g h t o f t h e p a r t i c l e s o n t h e u p p e r l a ye r s. Th i s
p r o c e s s f a c i l i t a t e s s l u d g e t h i c k e n i n g a t th e
bottom.
 Chapter57 : SEWACEA//ASTE
WATERTREATMENT 689

For the sake of illustration,some sedinrentatron

tanks are depicted in Fig. 57.3.
--lOutlet

Chemical.aided' sedimentation

flOW It is not always possibleto remove the colloidal

- .' ,9 ' .r .' Od l
Rectangular sedimentation tank wastes in sewage by plain sedimentation.However,
a d d i t i o n o f c e r t a i n c h e m i c a l s a i d s s e d i m e n t a t i o n ,a
Inlet process referred to as chemical precipitation or
\ c h e m i c a l - a i d e d s e d i m e n t a t i o n .B y t h i s t e c h n i q u e ,
about 60-80% of the suspended particles can
be removed. Chemical precipitation involves
three stages-coagulation, flocculation and
sedimentation.
Coagulation is mainly a chemical process
YI
,t.! lOutlet wherein the charged particles are destabilized (by
the addition of chemical agents). On the other
hand, flocculation involves the physical
phenomena of aggregatingthe destabilizedpartictes
Center-fed circular sedimentation tank t o f i n a l l y f o r m s e t t l e a b l es o l i d s ( i . e . s e d i m e n t a t i o n ) .
Inlet The chemicals used in chemical-aided
\ sedimentation are of two types-coagulants and
coagulant aids.
Coagulants : These are the chemicalls (normally
positively charged) which form insoluble and
Radialflow
gelatinous precipitates with colloidal particles
(negatively charged ones present in sewage). The
most commonly used coagulants in sewage
treatment are alum (aluminium sulfate) iron salts
(ferric sulfate, ferrous sulfate, ferric chloride), lime
and soda ash (sodium carbonate), sodium silicate
and sodium aluminate.
Peripheral-fed sedimentation tank
Coagulant-aids : These chemicals aid or
facilitate the processof coagulation.This is brought
57.3 : out by enchancing the action of coagulants and
reducing the amount of sludge formed. The
common coagulant aids are activated silica,
Types of sedimentation tanks (clarifiersf weighting agents (e.g. powdered lime stone or
There are different ways of classifying silica) and polyelectrolytes.
sedimentation
tanks.
circularand
Basedon the shape- rectangular,
square.
Based on the flow of sewage- longitudinal,
vertical, radial and spiral.
Biological treatment of sewage is required for
Basedon the purpose and position - primary, the removal of dissolved and fine colloidal organic
secondary,coagulation-cum-sedimentation tanks, matter. This orocess involves the use of
grit chambers,septicand lmhoff tanks. microorganisms (bacteria, algae, fungi, ptotozoa,
Based on the operation-batch type and rotifers, nematodes) that decompose the unstable
continuousflow type. organic matter to stable inorganic forms.

Biotechnology
[44]
690 B IOTECHNO LO CY

Activated sludge process

Tnsu 57.1 A list of major biological (secondary)
treatment processesused for sewage treatment Aerated lagoons

Sequencing batch reactor

Category Common process(s)
Aerobic processes . Aerobic digestion.
Suspended-growth Activated sludgeprocess
Among these, activated sludge process is the
syslems Plug{low process
most widely used for the secondary treatment of
Sequencing batchreactor
sewage.
Contact stabillzation
Oxidation ditch
ACTIVATED SLUDGE PROCESS
Deepshaft
Suspended-growth ication
nitrif The activated sludge process,first developed in
Aerobic lagoons E n g l a n d i n 1 9 1 4 , c o n t i n u e s t o be th e m o st
Aerobic digestion commonly used modern process for the biological
Attached-growth Tricklingfilters treatment of sewage.
systems Roughing filters
Rotating biological contactors I n t h i s m e t h o d , t h e s e w a g e c o n t ai n i n g o r g a n i c
(RBC) matter with the microorganisms is aerated (by a
Packed-bed reactors mechanical aerator)in an aerationtank. The reactor
Suspended-attached Activated biofilters contents are referred to as mixed liquor. Under
growthcombined (e.9.trickling filtersolids- aerobic conditions, the microorganismsmetabolise
sysrems nnnfant nrnnocq\ the soluble and suspended organic matter. The
generalized metabolic reaction is as fclllows
Anaerobic processes
Suspended-growth Anaerobic
digestion(single- Bacteria
stage/two-stage) coHNS + O, + Nutrients )
systems
Anaerobic
contacl process (organic mattefl
filterprocess
Anaerobic C O r +N H r + H r O
Attached-growth
(new cells + other Products)
:Y?19*'- Flry9:9e99':":'_
Pond processes As given above, a part of the organic matter is
ponds
Aerobic utilized for the synthesis of new bacterial cells
ponds
Anaerobic w h i l e t h e r e m a i n i n g g e t s o x i d i z e d to C O, a n d
ponds
Facultative HzO. The newly formed microorganisms are
agglomerated to form flocs, technically referred to
which is not in
The biologicaltreatmentprocesses of sewage as sludge. The separated sludge
contact with organic matter becomes activated' lt is
are broadly classifiedas aerobic, anaerobic and
Dependingon the natureof the sepa.rated from the settlingtank, and returnedto the
pond processes.
aeration tank, and recycled. fhe activated sludge
useof the microorganisms, the biologicalprocesses
recycled in aeration tank serves as a seed or
are categorized as suspendedgrowth systemsand
inoculum. The excess and waste sludge can be
attached growth systems.A list of the major
secondary (biological)treatmentprocesses is given removeo.
in Table57.1, and the most importantones are For efficient operation of activated sludge
brieflvdescribed. process, it is necessary to maintain a constant
s u p p l y o f O , w h i c h c a n b e d o n e b y m e ch a n i ca l
aeration or through the use of rotating paddles.
Crowth of protozoa in a sludge is an indication of
its healthy condition.

The most important suspended-growth T h e d i s p o s a l o f a w a s t e s l u d g e i s a p r o b l e m . l t

biologicaltreatmentsystemsusedfor the removal may be used as a fertilizer in crop lands or as
of organicmatterare listed. landfills, after drying.
 ChAPtCT
52 : SEWACEA//ASTE
WATERTREATMENT
Fig. 57.4 : Flow diagram of activated-sludge process.
Factors affecting performance : There are
several factors that influence the efficiency of
activated sludge process,the most important being
the type of the reactor, aeration, food
microorganism (F/M) ratio, nutrients, sludge
recirculation rate, besides pH and temperature.
Advantages : The activated sludge process is a
very compact, low-cost and an efficient biological
treatment system for sewage treatment. lt is worked
o ut th at un de r ideal c ondit ions , up t o 95% o f
BOD', 98% of bacteria (particularly coliform) and
95% of suspendedsolids can removed by activated
sludge process.The excessand waste sludge has a
higher fertilizer value compared to other treatment
processes.
Disadvantages : There is production of large
r olumes of sludge which sometimes becomes
ctifficultto ha nd le. Power c ons um pt ion is r elat iv e l y
h igh for o pe ratio n.Super v is ionby s k illed per s on n e l
s necessary.
Conventional activated sludge process
ln the normal treatment of sewage,the activated
sludge is preceededby primary sedimentationtank.
The conventional activated sludge system consists
of a separationtank, settling or sedimentationtank
and sludge removal line (Fig. 57.4\. fhe sewage
after the primary treatment is introduced at the
h ea d of the tan k . lt is des ir able t o s upply O,
u nifo rmly thro ug hout t he t ank .
Modified activated sludge processes
For increasingthe performance of the activated
'several
sludge system, modifications have been
done in the recent years. Most of them are directed
to bring out efficient aeration Aeration can be
691
-l
-----------* ----____-l>
Erf
ruenl
lr."jltr
Excessand
waste sludge
done by step aeration, tapered aeration, high rate
aeralion by complete mixing and extended
aeration.
AERATED LAGOONS
Aerated lagoons, also called as aerated ponds,
are the faculative stabilization pcnds wherein
surface aeratorsare installed to overcome the bad
adours (due to overload of organic materials).The
microbiological treatment of aerated ponds is
comparable to the activated sludge process. The
major difference is the large surfacearea in aerated
p o n d s a n d t h i s i s m o r e s u s c e p t i b l ef o r t e m p e r a t u r e
effects.
It is possibleto carry out continuous nitrification
in aerated lagoons. This however, depends on the
design and operating conditions of the pond
(particularly the temperature).
SEQUENCING BATCH REACTOR
S e q u e n c i n gb a t c h r e a c t o r( S B R )i s a m o d i f i c a t i o n
of activated sludge treatment system.The processes
namely aeration and sedimentationare carried out
in both the systems.The major difference is that
while in the conventional activated sludge system,
a e r a t i o na n d s e d i m e n t a t i o no c c u r s i m u l t a n e o u s l yi n
separatetanks, these two processesare carried out
sequentially in the same tank in SBR. Thus, the
sequencing batch reactor may be regarded as fill-
and-draw activated sludge process.
The operating sequence of a typical SBR is
depicted in Fig. 57.5. The process is carried out in
a sequence of five sreps-- filling, aeration
(reacting) sedimentation (settling), decanting and
idle. Severalmodifications and impi'ovementshave
been made in the SBR for more efficient or;eration.
692 B IOTECHNO LO CY

Aeration digesters.More details on aerobic digestion are

status eiven elsewhere(Referp . 7 1 1 ) .

Air onlolt

Aerobic attached-growthtreatment processesare

commonly used to remove the organic matter
Air on found in the sewage. These processes are also
useful for the nitrification (conversion of ammonia
to nitrate). The commonly used attached-growth
processesare listed.
. Trickling filters
. R o u g h i n gf i l t e r s
Reacting(aeration)
. R o t a t i n gb i o l o g i c a l c o n t i a c t o r s
Air off
. Packed bed reactors.

A m o n g t h e s e ,t r i c l <l i n gf i l t e r i s m o s t w i d e l y u se d .

TRICKLING FILTERS

Trickling filters, also known as Percolating ol

sprinkling filters, are commonly used for the
biological treatment of domestic sewage and
industrial waste water. In a strict sense, trickling
filters are not filters, but they are oxidation units.

A diagrammatic representationof trickling filter

Effluent
removal
Air on/off
Fig. 57.5 : Operating sequence of a typical
sequencing batch rcactor (SBF).
AERO BI C DI G ESTI O N
The organic sludges produced from various
treatment processes (activated sludge treatment,
trickling filter-sludge) are subjected to aerobic
digestion in special reactors referred to as aerobic
is depicted in Fig. 57.6. It has a bed of coarse, hard
and porous material over which sewage is sprayed.
In about two weeks time, the biomass attached to
the media surfacegrows and forms a layer, referred
to as biological film or microbial s/ime. This film
h a s a t h i c k n e s s o f 0 . 1 t o 2 . 0 m m a nd i s r i ch i n
m i c r o o r g a n i s m s . A s t h e l i q u i d ( s e wa g e ) tr i ckl e s
through the biof ilm, the organic matter gets
oxidized to CO, and NO, by the microbial
metabolism. This oxidation is carried out by
the aerobic organisms (particuJarlybacteria) that
are present on the upper portion of the biological
film.
T h e b i o l o g i c a l f i l m i s r i c h i n th e b a cte r i a -
Pseudomonas, Flavobacterium, Alcaligenes, and
algae-Chlore!la, tJtothrix, and Stigeoclonium,
besides some fungi and yeasts. Biofilms with a
thickness in the range of 7O-100 pm are efficient
for the treatment process.
As the biofilm ages, its thickness increasesand
it automatically settlesto the bottom of the tank.
 WATERTREATMENT
Chaoter57 : SEWACE/WASTE 693

Influentin--__)
(sewage)
i:ri ifu
li :.:;:.:.<-
Biologicalfilm
, r,
', .l:l ,r,,ii;;
,,r:ri ( m i c r o b i asll i m e )
+ ||

OO O
a--\ O
^
o Ao n
^
"UVV QO O
o 00 0 oo ooo

Porusbed

Fig. 57.6 : A diagrammatic representation of trickling filter.

The waste waters obtained in milk processing,
paper mills and pharmaceutical industries are
treated by trickling filters.
Types of trickling filters
The trickling filters are classifiedas low rafe (the
converrtional one), high rate and super rate. This
ca teg orizati on is m ainly bas ed on t he hy d r a u l i c
and organic loading rates of the sewage. The low
rate filters are suitable for the treatment of domestic
sewage,while high rate filters and super rate filters
are useful for industrial sewage.
Factors affecting performance of
trickling filt er s
The type of the media and its depth, organic and
hydraulic loading, filter staging, recirculation rate
and flow distribution are the important factors that
influence the perforn.ranceof the trickling filters.
Advantages : Trickling filters are simple, occupy
less space and the operating costs are low. They
operate efficiently in hot climate and thus are
suitable for most developing countries (like India)
Disadvantages : Removal of BOD is moderate
around TOoh), and disposal of excess sludge is
ne ce ssary.Pr im ar y s edim ent at ionis r equir ed, s i n c e
trickling filter s c ar r not handle r aw s ewage.
ROUGHING FI LTERS
Rou gh ing f ilt er s ar e a s pec ial t y pe of t r ic k l i n g
filtersthat are designedto operate at high hydraulic
Ioading rates. These filters are mostly used to
reduce the organic matter in downstream
p r o c e s s i n g ,b e s i d e s n i t r i f i c a t i o n a p p l i c a t i o n s . D u e
to high hydraulic loading, there is a continuous
s l o u g h i n go f t h e b i o l o g i c a l f i l r n . I n s u c h a c a s e ,t h e
unsettled filter effluent can be recycled, and this
increasesthe efficiency of the treatment process.
The retention time on the biofilm being less,
organic materials that are not readily degradable
remain unaffected.
BOTATING BIOLOGICAL
GoNTACTORS (RBCI
Rotating biological contactor (RBC) is a recent
device for the biological treatment of sewage. lt
operates on the principle of aerobic attached-
growth system operated on the moving media.
RBC are suitable for the treatment of domestic ano
indusirial sewage in small and medium towns.
A rotating biological contactor is composed of a
series of closely spaced and light weight circul'ar
discs (Fig. 57.V. They are made up of inert
materials such s polystyrene or polyviriyl chloride
(PVC) or polyethylene.These discs are mounted on
a horizontal shaft in a tank through which waste
water flows. The shaft is rotated slowly (less than
10 revolutions per minute) by a low speed motor
The discs of the shaft, i'eferred to as biodiscs, are
partially (40-60%) submerged in sewage. As the
biodiscs are rotated, the biornassattached tc them
is alternately submerged in sewage. This enables
the discs to pick up a thin layer of sewage, and
then to oxidize the absorbed substrates The
unoxidized substratesfall back into the sewage.
694 B IOTECHNO LO CY

Common Rotation
shaft (B)

Biodisc

Shaft

Sewage

Fig. 57.7 : A diagrammatic reprcsentation of (A) rotating biological contactor (HBC) and (B) A single biodisc.

And this processin repeatedagain and again by the The sewage along with air (or pure oxygen)
rotating biodiscs. is introduced from the bottom of the reactor
(Fig. 57.8). The main advantage with packed bed
Rotating biological contractor is basically a film
reactors is that they have high surface area of
flow bioreacfor. The microbial biofilm is built
biofilms for unit of reactor.
upon the partly submerged support medium
c ont aining biodis c s
The RBC is very efficient for the removal of
organic matter in the sewage (about 90% of BOD).
As and when there is an excessgrowth of biomass,
it has to be removed. This process can be carried
in a s im ilar f as hion, as it is d o n e f o r t h e t r i c k l i n g
A n a e r o b i c p r o c e s s e s b a s i c a l l y i nvo l ve th e
filter (described already).
d e c o m p o s i t i o n o f o r g a n i c a n d i n o r g n i c m a tte r i n
RBC are commonly used for the treatment of the absence of oxygen. Anaerobic processing
municipal waste water. In addition, they are widely systemsare important for the treatment of sludges,
used for the biological processing of industrial
wastes, coming from several industries such as
vegetables, pulp, meat and textiles.
Factors affecting performance of RBC : The
treatment process in RBC is influenced by rotation
speed of shaft, waste water retention time, Overflow
temperature, disc submergence, organic loading,
media density, and number of stages.
Adv ant ages : RBC is c o m p a c t a n d r e q u i r e s
moderate energy input. lt has high BOD removal
efficiency.
Disadvantages: Disposal of sludge formed is a
major problem with RBC. The treatment process is
frequently associated with odour formation. RBC
oper at ion r equir es s k illed pe r s o n n e l .

PACKED.BED REACTORS
Sewage+ Air
Packed-bed reactors or fluidized bed reactors
are used for the removal of BOD and nitrification.
Fig. 57.8 : A diagrammatic tepresentation of a
A reactor is oacked with a medium to which tne packed-bed reactor
microorganisms get attached and form biofilms.
ChAOICT
57 : SEWACEAVASTE
WATERTREATMENT 695

Fixed complete mix, single stage digester is depicted in

CHa + CO2 Fig. 57.9.

The processof anaerobic digestion is carried out

in an air tight reactor. Sludge is introduced
continuously or intermittently. In the high-rate
digestion system, the contents of the digester are
Sludge{
t |r_\ heated and mixed completely. And it takes about
inlets \+
ll
LLy'
fstuose 15 days for the process to be complete.
outlets

Mixer Biodegradation of organic matter of

Fig. 57.9 : High-rate complete mix, single-stage
anaerobic digester.
and high strength organic sewage. Several reactor
systemsfor anaerobic suspended-growthtreatment
process have been developed. Among these, the
complete-mix anaerobic digestion process is most
widely used for the treatment of sewage.
sludge (or sewage)
The biological degradation of organic matter of
sludge occurs in three stages (FiC. 57.10) -
h y d r o l y s i s ,a c i d o g e n e s i sa n d m e t h a n o g e n e s i s .
Hydrolysis : In the enzyme-catalysedreactions,
high molecular weight compounds (proteins,
polysaccharides, lipids and nucleic acids) are
degraded to low molecular weight compounds
(amino acids, monosaccharides,fatty acids, purines
and pyrimidines). The latter serve as substratesfor
energy supply and microbial growth.

Acidogenesis : The low molecular weight

ANAEROBIC DI G ESTI O N
Anaerobic digestion is mostly useful for the
stabilization of concentrated sludges that are
produced on the treatment of industrial sewage. A
diagrammatic representationof a typical high-rate,
compounds (referredabove) are converted to acidic
products (propionate, butyrate, lactate).
Methanogenesis : This is the third and final
stage and involves the production of methane and
carbon dioxide, from the intermediatesformed in

Carbohydrates Lipids Nucleicacids \ (r'

(polysaccharides) lo
It
;
ts
Monosaccharides Fatty acids Purinesand
pyrimidines
Jr
Acidogenesis

Fermentation products
(propionate,butyrate,
lactate,succinate)

Methanogenicsubstrates
(methanol,acetate,H2, CO2)

Methanogenesis

METHANOL+ CO2

Fig. 57.10 : A diagrcmmatic view of the degradation of organic materials in anaerobic digestion.
696 B IOTECHNO LO CY

. et hane gas i s h i g h l y i n s o l u b l e a n d
ac idogenes is M
its departure from the digester represents the
stabilization of sewage or sludge.
Microorganisms to degrade organic
matter of sludge (or sewagef
A consortir-rmof anearobic microorganismswork
together for degradation of sludge (or sewage)
organic matier. They may be categorized into two
rypes.
1 . Acid-forming bacteria : These are also
known as acidogens or non-methanogenic
bacteria. They bring out the hydrolysis of
m ac r om olec ules ( e. B c ar b o h y d r a t e ) t o s i m p l e
substrates(e.g. monosaccharides),and the latter to
acids e.g. Clostridium sp, Corynebacterium sp,
Lactobacillus sp, Actinomyces sp, Staphylococcus
sp, Peptococcus anaerobus, Escherichia coli.
2. Methanogenic bacteria : These bacteria,also
referred to as methanogens ot methane formers are
r es pons ible f or t he c onv e r s i o n o f a c e t i c a c i d
and hydrogen to methane and carbon dioxide.
The most important methanogens belong to
the genera Methanobacterium, Methanobacillus,
Methanococcus and Methanosarcin a.
( s e w a g e ) f l o w s u p w a r d s t h r o u g h t h e co l u m n
c o n t a i n i n g a n a e r o b i cb a c t e r i a .D u e t o th e p r e se n ce
of solid media, the bacteria are retained in the
column. This makes the treatment process more
efficient.
EXPANDED-BED PROCESS
The sew'age can be treated by pumping lt
through a bed of inert materials (sand or coal
expanded aggregates)on which the bacteria have
grown and formed a film. The effluent that comes
out can be recycled to maintain the flow rate.
Pond treatment processesfor the treatment of
sewage (containing biodegradable wastes) are
c a r r i e d o u t b y s p e c i a l l y d e s i g n e d a n d co n str u cte d
ponds. These ponds, referred to as stabilization
ponds, are large, shallow earthen basins. The
treatment process is a natural one involving the
combineci use of bacteria and algae. The
stabilization ponds are classified as aerohic,
anaerobic and facultative ponds.

ANAEBOBIC CONTACT PROCESS AEROBIC PONDS

Anaerobic contact process is carried out in a T h e a e r o b i c p o n d s , a s t h e n a m e i n d i ca te s,

specially designed reactors.The treatment process m a i n t a i n c o m p l e t e a e r o b i c c o n d i t i o n s .Th e se p o n d s
consistsof mixing of sewage with recycled sludge u s u a l l y h a v e a d e p t h o f a b o u t 0 . 5 t o 1 .5 fe e t ( 1 5 0
s olids and t hen diges t i c n u n d e r a n a e r o b i c to 450 mm) and allow the penetration of light
conditions. After the digestion is complete, the throughout the liquid depth. A second type of
supernatant effluent is discharged and the settled aerobic ponds with a depth of 5 feet (1.5 m) are
sludge is recycled. Anaerobic contact process is a l s o i n u s e . I n a l l t h e s ep o n d s , o x y g e n i s m a i n ta i n e d
successfully used for efficient industrial wastes t h r o u g h c o n t i n u o u s a t m o s p h e r i c d i ffu si o n ( b y
with high BOD e.g meat packing wastes. surface aeratorsor pumps), besidesthe production
by algae grown in the pond.

T h e a e r o b i c s t a b i l i z a t i o np o n d s c o n ta i n b a cte r i a
a n d a l g a e i n s u s p e n s i o n T h e y a r e p a r ti cu l a r l y
useful for the treaiment of soluble wastes.

T h e a l g a e a n d b a c t e r i a e x h i b i t a s ym b i o ti c a n d
There are mainly two treatment processesunder
c y c l i c r e l a t i o n s h i pi n t h e a e r o b i c p o n d s. Th e a l g a e
the anaerobic attached-growth treatment system-
can carry out photosynthesisand releaseoxygen to
anaerobic filter processand expanded bed process.
m a i n t a i n a e r o b i c c o n d i t i o n s i n t h e p o n d . Th e
bacteria degrade the organic matter to produce
AhI AERO BI C FI LTEB PR O C E S S
CO, and other nutrierrts to be utilized by algae
Anaer obic f ilt er c ons is t so f a c o l u m n f i l l e d w i t h (Fig. 57.11). Some higher organisms like protozoa
solid media for the treatment of organic matter in and rotifers present in the pond are responsiblefor
sewage. In this process system, waste water t h e p o l i s h i n g o f t h e e t T l u e n t .
 WATERTREATMENT
Chapter57 : SEWAGEMASTE
Algae
(photosynthesis)
(aerobicdegradation)
Fig. 57.11 : The symbiotic relationship between algae
and bacteria in an aerobic pond.
Factors affecting aerobic ponds
The speciesof the algae and bacteria present in
the pond significantly influence the efficiency of
the aerobic oonds. The other factors such as the
quality and quantity of organic material, degree of
p on d mixing , nut r ient s , s unlight , pH and
temperature are also important for the successful
operation of these ponds.
ANAEROBIC PONDS
Anaerobic ponds are useful for the treatment of
high-strength organic matter and solid containing
sewage/waste water. These ponds are completely
devoid of dissolved Or. They are very deep (up to
30 feet i.e. about 9 m). so that heat conservation is
possible,besides requiring minimum land area (for
pond construction).
When the sewage is added to the pond,
precipitation and anaerobic conversion of organic
waste to COr, methane and other gases, organic
acids etc. occurs. Under suitable conditions, 75%
of the BOD can be removed in anaerobic ponds.
The clarified effluent is usually dlscharged for
further treatment.
FACULTATIVE PONDS
In facultative ponds, the treatment of sewage is
carried out by a combination of both aerobic and
anaerobic processes. Three types of microorganisms s k i l l e d personnel.
aerobic, anaerobic and facultative (both aerobic
and anaerobic) are employed in facultative ponds.
The\erm oxidation pond or stabilization pond
is frequently used for facultative ponds.
697
A diagrammatic view of a facultative pond is
depicted in Fig. 57.12. lt consists of three zones.
1 The surface aerobic zone : It has the aerobic
bacteria and algae, existing in a symbiotic relatton
(as explained on p. 696).
2. The bottom anaerobic zone : lt contarns
anaerobic bacteria and solids that u ndergo
decomposition.
3. Intermediate facultative zone : This zone ts
partly aerobic and partly anaerobic and contains
both types (aerobic and anaerobic) of bacteria.
Processes that occur in facultative
ponds
The sewage organic matter is stabilized by both
aerobic and anaerobic processes.The algae present
in the aerobic zone carry out photosynthesisand
release Or. This oxygen is utilized by the aerobic
and facultative bacteria to oxidize soluble and
colloidal organic matter. The organic solids present
in the anaerobic zone (bottom sludge)are degraded
to dissolved organic compounds (organic acids)
and gasessuch as CO2, CH4 and HrS. The organic
acids can be oxidizeci by the aerobic bacteriawhile
the gasesproduced (CO2, CH4, H2S, NH3) may be
vented to the atmosphere. In fact, most of CO, is
utilized by the algae for photosynthesis,while l-1rS
combines with 02 to form sulfuric acid.
HrS + 20, ----------)H25O4
In the absence of adequate O, in the upper
layers of the pond, gaseswith unpleasantand foul
odours (HrS) are vented to the atmosphere.These
foul smells often cause nuisance to the
surroundings.
It is generally possible to maintain good O,
supply by the algae and facultative bacteria.
Someltimes,surface aerators are used to enhance
the efficiency of facultative ponds (particularly
when the sewage contains high organic content).
Advantages: For the facultativeponds, the initial
and operating costs are low. There is no need for
Disadvantages : Unpleasant odours and
mosquite breeding are frequently seen in facultative
ponds. The requirement of land area for
construction of these oonds is more.
 B IOTECHNO LO CY
698

Sunlight

H 2S , C H 4
Sewage
o'nf"
----*"""'
a't^
oeaJcerts
/ \
coz O2
Settleable
solids
\
\ ------...--- /-orsanic
matter
Bacteriak---t'
'->
t-t
Newcells Deadcells

BOTTOMSLUDGE

Bacteria Low molecular Bacteria

^..^^,r;^
vluar rrv
;:lbrii;s;;T; co2 + cH4 + H2s + NH3 Anaerobic
maner
compounds zone
(acids,alcohols)

Fig. 57.12 : A diagrammatic representation of a lacultative pond'

. For the removal of nitrogen and phosphorus

Tertiary treatment or advanced treatment is
sometinres needed for the removal of suspended
ancl dissolved substances, after the conventional
pr im ar y and s ec ondar yt r e a t m e n t s .I n S e n e r a l ,t h e
effluent of the sewage obtained after secondary
t r eat m ent c an be c onv en i e n t l y d i s p o s e d w i t h o u t
causing any nuisance. However, tertiary treatment
is needed under t he f ollow i n g c i r c u m s t a n c e s .
o When the qualitv of the effluent to be discharged
does not meet the standard requirements
( par t ic ular lyin t he dev e l o p e d c o u n t r i e s ) .
. When there is a necessaryto reuse the sewage/
waste water (reclamation of water is quite
ex pens iv e,but is r equir e d i n c e r t a i n s i t u a t i o n so f
water shortage).
compounds.
Tertiary treatment process broadly involves the
removal of suspendedand dissolvedsolids,nitrogen,
phosphorus and pathogenic organisms' In the
conventionalheirarchyof sewagetreatment,the unit
operationsare carried out in the order of preliminary,
primary, secondary and finally tertiary treatment'
However, sornetimesadvanced (tertiary)treatment
process may be directly carried out bypassingthe
other unit operations.This mainly depencison the
composition of waste water and the requirements'
There are four major processesunder the tertiary
treatment.
1. S o l i d s r e m o v a l
2. Biological nitrogen removal
3 . B i o l o g i c a l P h o s P h o r u sr e m o v al
4 Disinfection.
 WATERTREATMENT
Chapter57 : SEWACEAVASTE 699

Some authors use the term biological nutrient To load

removal for the removal of nitrogen and activatedcarbon
phosphorus.

SOL IDS REM O VAL )

The techniques for the removal of suspended (
and dissolved solids in waste water treatment are Backwash{_:
outlet (influent)
comparable with those employed for the prclcessing
of potable (drinking) water. Cylindrical
tank

Removal of suspended solids

Granular
The effluentsobtained from secondarytreatment activated
carDon
mav con t ain s us oended s olids in t he s ize 0 . . 1 t o
1 00 um. The c onc ent r at ion of t hes e s o l i d s i s Carbon
-| discharge
varia ble ,a nd is us ually 20- aO m gl. The r e m o v a l o f
suspended solids is carried outby granular medium --* Etfluenl
(sand) filtration and microscreening. Sometimes,
diatomaceous earth filters and coagulation-cum
sed imen tat iont ec hnioues ar e als o us ed.

Removal of dissolved solids

Backwashinlet
The d is s olv eds olids c an be r em ov ed m a i n l y b y
two techniqu es-adsorption and ion-exchange. Fig. 57.13 : A diagrammative representation of
granular activated carbon contactor (GAC).
Adsorption by activated carbon : Activated
carbon is highly porous and provides large surface
area for the adsorotion of dissolved solids in the Powdered activated carbon (PAC), instead of
advanced treatment. The comoounds that can De granulated carbon, is sometimes used for
removed by adsorption include organic materials absorption process. PAC is added to an aeration
(h erb icid es ,pes t ic ides ,t annins , lignins , c o l o u r a n d tank (of secondary treatment) which can adsorb
odour producing substances), inorganic materials several organic compounds.
(toxic trace metals) and several other pollutants.
lon-exchange for dissolved solids
Granular activated carbon contactors removal

Cranular activated carbon contactors (GAC) are
the most widely used for the advanced treatment of
sewage. The CAC consists of a tank (usually
cylind rica l)whic h is loaded wit h gr anular a c t i v a t e d
carbon (Fig.57.13). The waste water/sewage
(influent) is passed from top through the activated
carbon bed and the effluent comes out from the
bottom. The solids attached to the carbon bed
hamper further adsorption process.These particles
can be removed by backwashing.
The activated carbon requires periodical
regeneration. This can be done by heating in a
furnance at about 800'C in the absence of Or. At
this temperature,the adsorbed organic compounds
are converted to gases and released. The carbon
granulates are reactivated for reuse.
As the name indicates, ion-exchange involves
the displacement of one ion by another. The
exchange occurs between the ions of insoluble
exchange material (ion-exchangematerials)and the
ions of different speciesin solution (i.e. waste water
for advanced treatment).
The ion-exchange process is carried out by
employing two types of ion-exchange materials-
cation exchangers and anion exchangers
(Fi9.57J4. The synthetic resins with strong
acidic (H+) and basic (OH-) functional groups serve
as ion exchangers.The cation exchangers(with H+
or Na*) can replace the positively charged
ions (Ca2+, Mg2*; in water by hydrogen ions.
This is what is done for removinq the hardnessof
water.
700 B IOTE C H N OLO CY

Influent--_-.1 H2SOa NaOH

;
(for reqeneration)
.l__l_ (for regeneration)
vv'

Ca2+ so+F
M92+ ct-
c"?-
Fig. 57.14 : Ion-exchange process for the removal of dissolved solids-

The anion exchangers (with OH ) can remove Nitrification and denitrification

negatively charged ions (SOl-, NO3, CO23-).
Nitrification: A m m o n i a n i t r o g e n f i r s t g e ts
The waste water is first passedthrough a cation oxidized to nitrite (NO;) by the bacteria
exch anger and t hen t hr ough an an i o n e x c h a n g e r Nitrosomonas sp. This is followed by further
packed in two separate columns. When the ion oxidation nitrite (NOt) to nitrate (NOa) by
exchange capacity of the resin is exhausted,it has Nitrobacter sp.
to be regenerated for further use. For cation-
exchange resins, regeneration can be done with NHj (ammoniaN)
stron g ac ids ( H2SO 4, HCI ) , whi l e f o r a n i o n I Nitrosomonas
e xchange r es ins ,alk ali ( NaO H) is u s e d . I
J
For an effective removal of dissolved solids by NO, (nitrite)
ion exchange, the waste water should not contain
hig h c onc ent r at ion of s us pended s o l i d s a s t h e y I Nitrobacter
I
block the ion exchange beds +
No; (nitrate)
BIOLO G I CAL NI TRO G EN REM O V A L
The bacteria involved in nitrification are
Decomposition productsof protelnsand the urea process is
auxotrophs. The nitrification
present in sewage are the major constituents of
accomplished by aerobic suspended growth ahd
bio logic al nit r ogen Alt hough, nit r oge n i s a n u t r i e n t ,
aerobic attached-growth systems. In the general
its excessconcentrationcauseseutrophication,and practice, nitrification is carried out along with the
thus its removal is required. Biological nitrogen
BOD removal in the secondarv treatment with
removal (BNR) is carried out by the methods based
suitable modifications. Trickling filters, rotating
on the f ollowing pr inc iples ( Fig. 57.1 5 ) .
biological contactors and packed towers can be
1. As s im ilat ion of nit r ogen used for nitrification process.
2 . Nit r if ic at ion and denit r if ic at io n . Denitrification : The removal af nitrogen in the
form of nitrate by converting to nitrogen gas is
Assimilation of nitrogen referred to as denitrification. This process occurs
Sinc e nit r ogen is a nut r ient ,t he m i c r o o r g a n i s m s under anaerobic conditions and is brought out by
in the sewage can assimilate ammonia nitrogen, certain genera of bacteria-Aerobacter, Bacillus,
and grow. As some of these cells die, a portion of Brevibacterium, Lactobacillus, Micrococcus,
this am m onia nit r ogen will be r e t u r n e d t o t h e Pseudomonas and Spirillum. These bacteria are
sewage. heterotrophs and require no oxygen, but the
 WATERTREATMENT
Chaoter57 : SEWACEAA/ASTE 7{J7

Ammonianitrogen
N
I
T
R
I
Lysisand F
autooxidation I
Organicnitrogen C
(bacterialcells)
T
S
S o
I N
[,1
I
A D
T E
I N
o
N T
Organlcnitrogen R
(net growth) I
F
I
C
T
I
N

Fig. 57,15 : Biological nitrogen removal.

presence of organic carbon is essential. The The flcu, diagramfor the removal of biological
presence of even minute quantities of O2 nitrogen is depicted in Fig. 57.16. The process
suppresses denitrification. The heterotrophic primarily consistsof the removal of organic carbon
bacteria can reduce nitrate in the following stages, (aerobic), followed by nitrification (aerobic) and
to fina lly nitr ogen gas . denitrification (anaerabic).

No; (nitrate) BIOLOGICAL PHOSPHORUS REMOVAL

+
NO; (nitrite) Fhosphorusin the sewageis most{ypresentin
t" the form of orthophosphate (PO34 ), polyphosphate
+
NO (nitricoxide) (PrO,) and organic bound phosphorus.ln fact,
J phosphorus i s an essenti al nutri ent for
NrO (nitrousoxide) Thus,duri ngthe normalsecondary
mi croorgani sms.
J- treatment process, 10-30% of the sewage
N2 (nitrogengas) phosphorusis Lrtilizedby the microorganisms for
growth and energypurposes.
The processdenitrification can be carried out by
suspended growth and attacl.red growth systems. Phosohorusremoval from waste water is
The plug flow type of activated sludge system is and to maintain
requiredJo controleutrophication
commo nlv us ed. water quality.
 7|J2 B IOTE C HNO LO CY

Effluent{-

Fig. 57.16 : An overview of biological nitrcgen removat (BNR) process.

Frinciple of phosphorus removal The process of phosphorus removal

The biologic al r em ov al of ph o s p h o r u s i s b a s e c l Based on the principle d e scr i b e d

on the principle of exposing the microorganisms
ta alternating anaerobic and aerobic conditions
W hen t he m ic r oor ganis r ns a r e s u b j e c t e d t o
a naer obic c ondit ions , t hey r elea s et h e p h o s p h o r u s
c ont ent and f or m a s ludge w h i c h i s d e f i c i e n t
in phos phor us . W hen t his s lu d g e ( d e f i c i e n t r n
phosphorus) is returned to aerobic tank of
sewage treatment, the microorganismsaccumulate
lar ge quant it ies of phos phor us i n t h e i r c e l l s ( a s
poly phos phat es ) . Thes e m ic r o o r g a n i s m s w h e n
s ublec t ed t o anaer obic c onditi o n s r e l e a s e t h e
phos phor usand t his c y c le ( of alt e r n a t i n ga n a e r o b i c
and aerobic processes)can be continued for the
biologic al r em ov al of phos phor u s .
Acinetobacter sp are mainly responsiblefor the
biological removal of phosphorqs.
a b o v e , p r o c e s s i n g p l a n t s h a v e b e e n d e ve l o p e d
f o r t h e b i o l o g i c a l r e m o v a l o f p h o sp h o r u s.
Phostrip (phosphate stripper) process system,
used for this purpose is depicted in Fig. 57.17.
The anaerobic phosphate stripper removes
the phosphate, and the resultant sludge rs
returned to aeration tank which takes Iarge
q u a n t i t i e s o f p h o s p h o r u s . A f t e r s e d i m e n ta ti o n ,
t h e p h o s p h o r u s e n r i c h e d s l u d g e a g a i n p a sse s
through Phostrip, and the cycle is repeated again
and again.
T h e p h o s p h o r u s e n r i c h e d s u p e r n a t an t th a t
comes out of the phosphorus stripper is treated
w i t h l i m e t o p r e c i p i t a t e t h e p h o s p h o ru s. Th e
r e s u l t a n tl i q u i d s u p e r n a t a n tc a n b e r e t u r n e d to th e
aeration tank for further treatment

Influent -----+ l4o"tio6 -- I ------+ Secondarysedimentation ------) Effluenl

(fromprimarytreatment) | ' .- ----" t*1-] tank

.-'. .-.'->
Wastesludge

Phosphorus
Phosphorusstripped enrichedsludge
returnsludge
(";;),
phosphorLrs
i /
\Jrflpper/

Supernatant
return

J
Wastesludge

I
I
Fig. 57.17 : Biological phosphorus removal by Phostrip (phosphate sttipper) process.

I
I
 Chapter57 : SEWAGEA//ASTE
WATERTREATMENT 7o3

DISINFECTI O N Disinfection with chlorine

Disinfection broadly refers to the selective
destruction or inactivation of disease-causing
(pathogenic) organisms. ln the process of
disinfection, all the organisms are not destroyed.
This is in contrast to sterilization which invorves
the destruction of all the organisms.
C h l o r i n e i s a v e r y w i d e l y u s e d d i s i n f e c t a n t ,a s i t
satisfies the criteria (listed above) of an ideal
disinfectant. The most commonly used chlorrne
compounds are- chlorine gas (Cl2), calcium
h y p o c h l o r i d e [ C a ( C O C l 2 ) 1 ,s o d i u m h y p o c h l o r i t e
(NaOCl) and chlorine dioxide (Cl02).

There are severalwater borne diseases(typhoid, The disinfectionefficiencv of chlorine deoenos

cholera, dysentery) caused by bacteria, viruses on the number of microorganisms in the water
and other pathogens (Refer Table 56.1). The being treated, pH and temperature.
very purpose of disinfection is to control these
d iseases SEWAGEAIIIASTE WATER
TREATMENT_A SUMMARY
Agents for disinfection
The various treatment processesof sewage i.e.
Disinfection is accomplished by using chemical preliminary, primary, secondary and tertiary (with
and physical agents, besides mechanical and details of operations processing units in each
radiation means. category),have been described in this chapter.The
treatmentof sludge is dealt with in the next chapter.
Chemical agents : Chlorine and its compounds
They were, however, discussed as independent
a re mo st co m m only us ed. The ot her c hem i c a l s -
ooerations.
b romin e, io dine, oz one, alc ohols , phenols , h e a v y
metals, hydrogen peroxide, alkalies and acids A conventional sewage treatment plant has the
are sometimes employed. After chlorine (regarded requisite operating units arranged one after another
as most universal disinfectanl, bromine and for treatment and final disposal of sewage. The flow
iodine are in use. In recent yearst ozone as a chart of a conventional sewage treatment plant is
disinfectant is gaining importance, since it is very depicted in Fig. 57.18.
enective.

Physical agents : Heat and light can be

effectively used as disinfectants. Sunlight
(particularly ultra-violet rays) is in fact a good
disinfectant.
The characteristicsof waste water are highly
Mechanical means : The pathogenic organisms
on the i ndustri al
vari abl edependi ng source,w hi ch
can also be removed by mechanical means, during
in turn dependson the industrialactivity.Thus,
the course of waste water treatment.The processes
treatmentof wastewater differs,and this is largely
involving screens (coarse and fine), grit chambers
dependenton the industry.The treatmentschemes
and sedimentation can partly remove the disease-
of waste watersof dairy, distillery,tannery,sugar
ca usrngorg an r s m s .
and antibioticindustriesare brieflydescribed.
Radiatioh means : The gamma rays emitted
frorh radioisotopes can serve as effective WASTE WATER TREATMENT
d isinfectants. FOR D A IR IE S
Dairy wastewater is pollutedat variousstages
Gharacteristics of an adeal disinfectant by the activitiesof dairy operation.This hasto be
An ideal disinfectant should possess the appropriately treatedbeforebeingdischarged. This
following characteristics. is essentialto avoid water pollution.A schematic
. Toxic to pathogensat low concentration represntationof dairy waste water treatment is
depicted\n Fig.57.19, and briefly describedbelow.
. Solubleand stable in water
,r The processingof dairy waste water is carried
o Non-toxicto man and higherorganisms out by a two-stagepond system.The treatment is
. Cheapand e a s i l ya v a i l a b l e . first done in an anaerobicoond. and then in an
 7o4 B IOTE C H NO LO CY

l*-- Preliminary
-) { Primary.-,J {- Secondary
' lreatment treatment treatment

Seconciary

Fig. 57,18 : A diagrammatic representation of a flow chart of conventional sewage treatment plant

aerobic pond. The treated water coming out of the The treatnrentprocess is carried out in two phases
a naer obic pond is r ic h in nut r ien t s ,h e n c e i s u s e f u l ffig. s7.21).
for irrigation purposes.At the end of the two-stage
Phase | : This phase involves biological
treatment, abour 95"h of the BOD material rs
acidification and production of sulfides from
removed from the waste water.
sulfates present in the waste water. The sulfide
recovered in the form of sodium hydrogen sulfide ls
WASTE WATER TREATMENT
reused in tanneries.
FO R DI STI LLERY
P h a s e l l : T h i s i s c a r r i e d o u t i n a n a na e r o b tc
The waste water from distilleriesmostly consists
sludge blanket reactor. ln this phase, the organic
of dissolved O, and organic wastes (predominantly
fermented starches and organic nitrogen). The
treatment is carried out by a two-stage bacterial
oxidation in anaerobic reactors. The complex
organic wastes are first degraded to organrc
m olec ules ( ac ids , aldehy des , a l c o h o l s , k e t o n e s
et c . ) . Thes e m olec ules ar e f ur t h e r o x i d i z e d t o
s m aller or ganic ( CHo) and inor g a n i c ( C O 2 , N H 3 ,
H25, H2O) compounds (Fig. 57.20)-

The products formed in the first stage of

bacterial oxidation will promote the gro'wth Anaerobicpond
treatment
a nd m ult iolic at ion of new bac t e r i a l c e l l s . T h i s
I
is advantageous for more " efficient bacterial
f=-* lrrigation
ox idat ion. + Purpose
Aerobicpond
WASTE W ATER TREATM EN T treatment
FO R TANNERY

Tanner ies ar e indus t r ies i n v o l v e d i n t n e

p r oc es s ingof anim al s k ins . For th e t r a n s f o r m a t i o n
o f s k ins t o leat her s ,a lar ge num b e r o f c h e m i c a l s ,
dyes, tanning agents etc., are used. Tannery waste
Fig. 57.19 : A ciiagrammaticrepresentation of
water poses a serious environmenta! threat since
dairy waste watet trcatment.
many of the ingradientshave low biodegradability.

I
 57 : SEWACEA//ASTE
ChAPICT WATERTREATMENT
Acids,alcohols,
New bacterial
aldehydes, cells
ketones etc.
Fig. 57.20 : A diagrammatic representation of
distiilery organic waste water treatmenL
acids are converted into gases.The left over sulfide
from phase I is converted into solution, and
processed to producesulfurwhich will be usefulrn
c hem ic alindu s tri e s .
With the two phase systemsfor waste water
treatmentof tanneries,it is possibleto remove207o
of chemicaloxygendemand(COD)and 90% of the
sulfur compounds.Sometimes,additionalaerobic
bioreactorsare usedto removemore COD.
WASTE WATER TREATMENT
F O R S UG A R IN D U S T R Y
The wastewater from sugarindustriescontains utilized for irrigationpurposes.In the developing
high COD materialswhich are biodegradable. The countriesat some places,the raw domesticsewage
treatmentprocessis comparableto that described (completelyuntreated)
for waste water treatmentof distillery (Fig. 57.2O). of crop land. Such practicesexposethe peopleto
Biological
acidification
Fig. 57.21 : A diagrammatic representation of
tanneN waste water treatment.
Biotechnology [45]
7o5
TREATMENT OF ANTIBIOTICS
IN WASTE WATER
There is a widespread use of antibiotics in
human h e a l t h c a r e , 'a n i m a l h u s b a n d r y a n d
agriculture. In addition, the drug and antibiotic
industries also directly contribute to pollution.
Antibiotic pollution has become a major
environmental threat. This is mostly due to the
development of antibiotic resistance by the
pathogenic bacteria. The conventional waste water
treatment processesare ineffective in removing the
antibiotics.
Special tertiary treatment processesare required
to remove the antibiotic pollutants. The treatment
involves the degradation of target molecules by
oxidation, exposure to ultraviolet light etc. Some
pharmaceutical industries have introduced a new
technique namely membrane filtration to remove
even trace amounts of antibiotics from the waste
water.
There is shortageof water throughoutthe world.
Therefore,there is a need for recyclingof waste
water (i.e. the use of reclaimed waste water)
wherever,possible.
Approximatelytwo thirds of world's water is
is directlyusedfor irrigation
risk of infectionsby pathogenicbacteria,viruses
and prions.lt is thereforedesirableto developlow-
cost technologies to produce irrigation water from
waste waters.
Large quantitiesof water is also utilized in
industries.For instance,to make one ton of steel,
about 300 tonesof water isneeded.Consumptron
of water is alsohigh for manyother industries such
as food, paperand textiles.At someplaces,and for
some industries,processes for recyclingof water
have been developed.The technologyfor the
recycl i ngof w ater i s vari abl e,and thi s mai nl y
dependson the industry.
Recycling of waste water is useful for
agriculture,horticultureand wateringof lawnsand
golf courses,besidessupplyingto lakes(fishingand
706 B IOTECHNO LO CY

boating purposes). However, the use of recycled

wat er f or dr ink ing pur pos ess h o u l d b e a v o i d e d

The large scale sewage/wastewater of treatment

processes have been described. They are not
s uit able f or t he t r eat m ent o f s m a l l s e w a g e o r
was t e wat er , par t ic ular ly c o m i n g f r o m s m a l l
c om m unit ies .
The on-site systems of sewage treatment for
indiv idual r es idenc es( or c om m u n i t i e s )w i t h s p e c i a l
referenceto oxidation ditch, septic tanks and lmhoff
Excess
t ank s ar e br ief ly des c r ibed. sludge

O XI DATI O N DI TCH
O x idat ion dit c h m et hod , f i r s t d e v e l o p e d I n
Netherlands,is a suitable method for the treatment
of s ewage in s m all c om m un i t i e s . T h i s i s b a s i c a l l y
an aeration type of activated sludge process (See
p. 590) wit h a m ec hanic a l s y s t e m o f a e r a t i o n .
Howev el t her e is no pr im a r y s e d i m e n t a t i o n o f
s ewage,c ons equent lyt he pr o b l e m o f h a n d l i n g a r r d
t r eat m ent of pr im ar y s ludge i s e l i m i n a t e d .
O x idat ion dit c h c ons is t s o f a e r a t i o n u n i t s ,
nam ely dit c h c hannels ( 2 or m o r e ) c o n s t r u c t e ds i d e
by side (Fig. 57.22). The sizes of the ditch channels
ar e v ar iable- lengt h150- 1 000 m , w i d t h I - 5 m a n d
dept h 1- 5 m . They ar e c ons t r u c t e d w i t h b r i c k o r
stone masonary. A special type of rotors (cage
rotors) are fitted into each ditch channel
f or c ont inuous ly agit at ing a n d c i r c u l a t i n g t h e
s ludge,bes idess upply ingO r . T h e s l u d g e i s a l l o w e d
to settle down in a separate sedimentation tank'
The activated sludge is returned to ditch channels.
lnstead of using a separatesedimentation tank,
the sewage may be allowed to settle down in
tl-re ditch channels by stopping the rotors
( us ually dur ing night ) The s u p e r n a t a n t f r o m t h e
dit c h c hannelsc an be t ak en o u t . T h e e x c e s ss l u d g e
c ollec t ed ( in t he s edim en t a t i o n t a n k o r a t t h e
bot t om of dit c h c hannels )ca n b e s t a b i l i z e d .
SEPTI C TANKS
Sept ic t ank s ar e r ec om m e n d e d f o r i n d i v i d u a l 1 . 5 m a n d a b r e a d t h o f 0 . 7 5 m i s r e co m m e n d e d .
hous es and f or s m all c om m u n i t i e s a n d i n s t i t u t i o n s F o r s u c h a t a n k , t h e c l e a n i n g i n t e r va l i s 2 - 3
wit h a c ont r ibut ing populati o n a r o u n d 3 0 0 .
Fig. 57.22 : A diagrammatic representation of
oxidation ditch-
Septic tanks work on the principle of anaerobic
digestion.This occurs as the solids of the sewage
settle at the bottom of the tank. Under anaerobic
c o n d i t i o n s , t h e b i o d e g r a d a b l e o r g a n i c m a tte r i s
converted to gases(CH4, CO2, HrS etc.) and liquid
c o m p o u n d s . T h i s r e s u l t s i n a d r a s t i c re d u cti o n tn
t h e v o l u m e o f s l u d g e .A t h i c k c r u s t o f s cu m fo r m e d
on the surface of septic tank maintains anaerobic
c o n d i t i o n s . T h e e f f l u e n t c o m i n g o u t of th e se p ti c
t a n k c o n t a i n s s o m e o r g a n i c s o l i d s a n d p a th o g e n s
Therefore, disposal of the effluent from septic tank
has to be-dealt with very carefully.The septic tanks
a r e d e s l u d g e d a n d c l e a n e d a t r e g u l a r i n te r va l s,
u s u a l l y o n c e i n 2 - 5 y e a r s ( d e p e n d i n g o n th e ta n k
size and its use).
Gonstruction and operation of
septic tanks
S e p t i c t a n k s a r e u s u a l l y c o n s t ru cte d w i th
b r i c k s , o r s t o n e m a s o n a r y . T h i c k - w a l l p o l yth e n e
a n d f i b r e g l a s s t a n k s a r e a l s o i n u se i n r e ce n t
y e a r s . Wh a t e v e r m a y b e t h e c o n s t r u c ti o n m a te r i a l
u s e d , t h e s e p t i c t a n k m u s t b e w a t e r - ti g h t a n d
m u s t f u n c t i o n e f f i c i e n t l y . T h e s i z e o f th e ta n k i s
v a r i a b l e , d e p e n d i n g o n t h e n u m b e r o f u se r s. Fo r
a f a m i l y o f f i v e m e m b e r s , a t a n k w i t h a l e n g th o f
years.
ChAPtCT
57 : SEWACEA//ASTE
WATERTREATMENT 7(,7

A conveniional septic tank has t'wo

compartments, the first compartment being twice
the size of the second one (Fig. 57.23). For effective
Sewage-} .J Effiuent
sedimentationof solids,the tank should be designed influent
to prevent the short-circuiting at the top and the
bottom of the tank. Further,the location of inlet ano
outlet should be such that the contentsof the septic
tank are not disturbed while the sewage enters or
effluent leaves.Septic tank should be provided with
a ve ntila tion pip e, t he t op of whic h s hould oe Fig. 57.23 : Schematic representation of a
covered with a mosquito proof wire mesh. conventionaltwo-compartmentseptic tank.
As the sewage enters the septic tank, the solids
settle to the bottom while grease and other light Gas vent
Water level
materials float on the surface, and form a scum. (withscum)
The bottom-settled organic material undergoes Settling
facultative and anaerobic decomposition to form compartmenl Sludgeout
more stable compounds and gases (Cll4, CO2, Valve
Slot Slope
HrS). In th is wa y, ther e is a c ont inuous r educ t ion of
solids of sewage entering the septic tank. However, - Sludge
sludge accumulates at the bottom of the tank. withdrawlpipe
Desludging of septic tank has to be carried out Digestion
periodically (once in 2-5 years). compartment

Sludge
IMHOFF TANKS

lmhoff tank is an improved septic tank. lt

basically consistsof a two storey tank in which the
sedimentation (settling) occurs in the upper tank
while the digestion of the settled solids takes place
in the lower compartment (Fig. 57.24.
As the sewageentersthe sedimdntationtank, the
solids settle down to the bottom and the sewage
flows into the digestion tank through slopes and
slot of the sedimentationtank. Cas oroduced in tne
Fig. 57.24 : A diagrammatic view of imhotf tank.
digestion process escapes through gas vent. The
sedimentationtank is designed in such a way that
the gases and the gas-buoyed sludge particles
raising from the sludge layer do not enter into it.
The sludge collection at the bottom can be
withdrawn periodicallv.
 slud,Se s a semi-liquid mass that is
$ewape
ufproduced during the course of sewage/waste
water treatment processes(Chapter57). Sludge may
be regarded as a semisolid liquid that contains
s olids in t he r angeof 0 5 t o 1 2 p e r c e n t ( b y w e i g h t ) .
The c om pos it ion of t he s l u d g e i s h i g h l y v a r i a b l e
and depends on the source of raw sewage and its
treatment processes.Some authors use the term
organic slurries for sewage sludge.
SO URCES AND CHARA C T E R I S T I C S
O F SLUDG E
f a t s , c e l l u l o s e , h y d r o c a r b o n s , p h o sp h o r u s, i r o n
silica, organic acids, heavy metals, pathogens and
pesticides
The various methods of sludge treatment and
disposal are given in Table 58.2. The important
aspectsof selected processesare briefly described
PRELIMINARY OPERATIONS
T h e p r e l i m i n a r y o p e r a t i o n s a r e ca r r i e d o u t to
provide a constant and homogeneous sludge for
further processing.

The following unit operations are the major Sludge grinding

sourcesof sludge. C r i n d i n g o f s l u d g e i s c a r r i e d o u t to cu t a l a r g e
. Sc r eening m a s s o f s l u d g e i n t o s m a l l p i e c e s . F o r th i s p u r p o se ,
hardended steel cutters and overload sensors are
o Cr it r em ov al
used.
o Pr im ar y s edim ent at ion
. Sec ondar ys edim ent alion Sludge degritting

r Sludge- pr oc es s ing
unit s .
The details on the above processeshave already
been described. The important sludges and their
characteristics are given in Table 58.1. The
characters of sludge are variable and depend on
t he or igin of t he s ludge, a n d t h e a g i n g a f t e r i t i s
produced
Chem ic al c om pos it ion of sludge
The c hem ic al c om pos it i o n o f s l u d g e i s v a r i a b l e .
The major constituentsof sludge are proteins and
ot her nit r ogen c ont aining c o m p o u n d s , g r e a s e a n d
Sometimes, removal of grit (degritting) is
necessary for the effective treatment of sludge.
D e g r i t t i n g c a n b e c a r r i e d o u t b y a p p l yi n g
centrifugal force to the flowing sludge. Cyclone
degrittersare usually employed for this purpose to
separategrit particles from the organic sludge.
Sludge blending
Sludge produced in primary (settleablesolids),
secondary (settleablesolids and biological solids)
and tertiary (biological solids and chemical solids)
treatmentsis mixed (blended)to produce a uniform
mixture.

708
 58 : S L U D C EAN D SOL IDWA ST E S-TR E A TME NATN D D IS P OS A L
C hA P I CT 709

SLUDGETHICKENING
(coNcENTRATTONI
characteristics
The solid content of the sludge is variable and
SIudge Characteristics may range from 0.5 to 12oh. It is necessary to
c o n c e n t r a t et h e s l u d g e ( p a r t i c u l a r l yw i t h l o w s o l i d
Screenings Containsorganic
and
content) by removing some amount of liquid.
inorganicmaterials
thatcan
Physical methods are employed for sludge
be easilyremoved.
thickening.
Grit Richin heavierinorganic
solidsanda goodquantityof Gravity thickening
organicmatter(fats,grease).
Cravity thickening is the most commonly used
Primarysludge Obtained fromtheprimary m e t h o d f o r t h e t h i c k e n i n g o f p r i m a r y s l u d g e .l t c a n
settling
tanks,greyin colour be carried out in the conventional sedimentation
andoffensive in odour. t a n k ( c i r c u l a r t a n k i s p r e f e r r e d ) .A s t h e s l u d g e g e t s
Activatedsludge concentrated by gravity, the supernatant can be
Brownishto darkin colour
(i.e. primary settling
appearance, r e t u r n e dt o t h e t r e a t m e n tp l a n t
withflocculant
hasearthy t a n k )
usually odourand
canbe digestedeasily.
Trickling-filter
sludge Brownish
in colourwith
Tnslr 58.2 Sludgetreatment and disposal
odour(fresh
inoffensive
methods
easily
sludge), digestible.
Scum materials
Floatable skimmed Type of process Treatment methods
fromthesurfaces of primary Preliminary
operationsGrinding,
degritting,
blending,
andsecondary settlingtanks. storage
Scumcontains oils,fats, Thickening Gravity
thickening
foodwastes,plastic
Flotation
thlckening
materials,
cottonetc.
Centrif
ugalthickening
Aerobically
digested Brownishto blackin colour Rotarydrumthickening
sludge withflocculant
appearance. Stabilization Limri
ilairitiiiiiil
Nooflensive
odour. Heattreatment
Anaerobically
digested Darkbrownto blackin Anaerobicdigestion
sludge colour,
containslarge Aerobicdigestion
quantities
of gas.0n drying Composting
thegases
thesludge, Conditioning Chemicalmethod
escape. Heattreatmentmethod
Septage Sludgeof septictanks,black Disinfection Pasteurization
in colour.
Usuallywell Dewatering Vacuum filter
digesteddueto longstorage. Centrif
uge
Offensiveodourdueto Sludge dryingbeds
hydrogensulfide. drying Flashdryer
Slowdryer
Rotarydryer
Sludge storage
Thermal reduction Multiple-hearth inciner
ator
Storageof sludge becomes necessarywhen the Fluidized bedincinerator
p ro ce ssingun its ar e not in oper at ions ( night s hif t s ,
Ultimatedisposal Landapplication
rveekends). Short{erm storage of sludge rs
Distribution andmarketing
a ccomp lish ed in s et t ling t ank s while f or long -
Landf
ill
term storage, specially designed tanks have to be
Lag00nrng
u s eo .
71(, B IOTECHNO LO CY

Flotation thickening sludge,the pH raisesto 12 or higher.At high pH,

the microorganisms cannotgrow.Consequently, the
F l o ta ti o nth i c k e n i n gi s the techni queof choi ce
sludge will not putrify,create unpleasantodours
for the treatmentof sludgesfrom suspended-growth
not oose healthhazards.
biological treatmentprocesses.There are three and does
types of flotation thickening processes - dissolved In the lime pretreatment,addition of lime is
air flotation, dispersed-airflotation, and vacuum done prior to dewatering,while in lime post-
flotation. Among these,dissolved-airflotation ls treatment, it is done after dewatering.
w i d e l yu s e d .In th i ste c h n i q ue,ai r i s passed
through
the sludgethat is held at high pressure.The air Heat treatment
raisesto the top of the sludge in the form of
Heat treatmentinvolvesheatingof sludgefor a
bubblesalong with attachedparticlesto form a
period (250'C for about 30 minutes)under
sludgeblanket.This can be skimmedoff at regutar short
intervals.The efficiency of flotation thickening
pressure. By this process,sludgeis dewateredand
sterilized.Heattreatmenthelpsin stabilization and
dependson the air to solidsratio.
condi ti oni ng (di scussedl ater) of t he sludge.
Gentrifugal thickening However,due to high cost, it is less frequently
U SC O.
Centrifugalthickeningis basedon the principle
of settlementof sludgeparticlesunderthe influence Anaerobic sludge digestion
of centrifugalforces. Two types of centrifuges-
solid bowl centrifuge and basket centrifuge are Anaerobicdigestionof sewageis one of the
used for this purpose.Althoughconcentrationof oldestformsof biologicaltreatmentprocesses. The
sludge by centrifugalthickening is efficient, it same operati nguni t i s equal l y usef ul f or t he
involves high cost of maintenanceand power stabilizationof sludge.Thereare differentways of
consumption. carryi ngout anaerobi sl
c udgedi gestion- st andar d
rate digestion,single-stage
high rate digestionand
Rotary drum thickening two-stagedigestion.

Thickeningof the sludgecan be carriedout by Standardrate digestion: This is a single-stage

use of rotary drums. As the sludge is passedover
the rotatingscreendrums,the solidsseparatefrom
the water arrdthe thickenedsludgerolls out at the
d ru m e n d s .
SLUDGE STABILIZATION
digestionprocess(ReferFig. 57.9. fhe sludgeis
addedand heatedby externalheatexchangers. As
the sludgegets digested,the gas releasedcomes
out to the surfaces.Along with the gas, certarn
sludgeparticles(fats,oils, grease)also raiseto the
top to form a scum which can be removed.

Sludge requires stabilizationto achieve the S i ngl e-stagehi gh rate di gesti on: This is
followingobjectives. characterized by high rate of sludgeloading,and
i ts di gesti onunder anaerobi ccon dit ions.The
. To reducethe load of disease-causing organisms
sludgeis continuouslymixed by gas, heatedand
(pathogens).
recirculated for optimal digestion.
. To inhibit the potentialfor putrefaction.
Two-stagedigestion : There are two digestion
o To eliminateoffensiveodours.
tanks used in this processof high-ratedigestion.
Sludge stabilizationcan be carried out by The first tank is used for digestionproper (with
appropriatebiologicaland chemical means.The mi xi ng and heati ngfaci l i ti es)w hi l e t he second
techniquesusedfor sludgestabilization are- lime digesteris employedfor storageand concentration.
stabilization, heat treatmenl anaerohic digestion,
Thermophilicanaerobicdigestion: Sometimes,
aerohic digestion and composting.They are briefly
sludgedigestioncan be carried out at relatively
described. higher temperature (45-60"C) by thermophilic
bacteria.In fact, the digestionprocessis fasterwith
Lime stabilization
increased bacterial destruction and improveo
When lime, in the form of hydrated lime dewateringat higher temperature.But the major
l C a (O H )rlo r q u i c k l i m e (C aO) i s added to the limitation is the requirementof high energy for
 58 : SL U D C EA N D S O L IDWA STE S -TR E A TME NATN D D IS P OS A L

continuous heating. For this reason, thermophilic

anaerobic digestion process is not widely used.

Aerobic sludge digestion Water

surface
Supernatant€ after
Ae rob ic diges t ion of s ludges f r om v a r i o u s
sources(primary, secondaryand tertiary treatments)
I decanting

is becoming popular in recent years for the

I
following reasons.
o The digestion of volatile solids is as effective as
in anaerobic digestion.
a Fertilizervalue of the sludgeis much higher. Fig. 58.1 : Diagrammatic representation of an
a Lowercapitaland operationalcosts. aerobic digester.

However, the major limitation in aerobic

digestionis the continuoussupplyof O, which rsa
certain advantages of thermophilic aerobic
costly affair. d i g e s t i o n .T h e s e i n c l u d e k i l l i n g o f m o r e p a t h o g e n i c
The process,aerobic digestion of sludge is organisms and digestion of more solids, w,ith
comparable to activated sludge process (Refer m i n i m a l u n p l e a s e n to d o u r s i n s t a b i l i z e d s l u d g e .
p. 690) .
Principle of aerobic digestion : When the
nutrient(substrates)
supplyto the microorganisnisis
depleted,they start oxidizing their own cellular
(protoplasmic)materialsfor energy supply and Compostingbasicallyinvolves the processof
biological degradation of solid organic waste
maintenance. About 70-80%of the cellularorganic
material to stable end products. Although
matter can be oxidized aerobicallyto produce
compostingcan be carriedout under aerobicand
carbon dioxide, nitrate (NO;) and water.
anaerobicconditions,aerobiccompostingis more
Approximately2O-3Oohof the cellular organic
frequently used. Thus, composting may be
matter cannot be oxidized, as it is not bro-
considered as an aerobic microbiological process
degradable.
for convertingsolid organic wastes into stable
A diagrammatic representationof an aerobic sanitary,nuisancefree, humus like materialthat
digesteris depicted in Fig. 5B.l . The processcan can be safelydisposedinto the environment.
be carriedout by batchor continuousflow system
Compostingis a cost-effectiveand environment
of operation.With regardto the aerationprocess,
friendly process for stabilization and ultimate
aerobic digestionis of two types-conventional
disposalof sludge.The product of compostingis
aerobicdigestionand high purity oxygenaerobic
usefulfor soil improvementand the productionof
digestion.
mushrooms. Thus,compostingis ultimatelyhelpful
Conventionalaerobic digestion : Atmospheric for reuseand recyclingof organicwastematerials
air is used in the conventionaldigesterfor the from domestic,agricultureand industry.
processof aerobicdigestion.
Organisms involved in composting
High-purityoxygenaerobic digestion: In this
case,a pure gradeof oxygeninsteadof air is used. A w i de vari etyof organi sms(both uni cel l ul ar
The digestionprocessis usually carried out In and mul ti cel l ul ar)are i nvol ved i n the process
c los edt ank s . compostingThe bacteriamake up 80-90% of the
microorganismsfound in the compost. These
Thermophilic aerobic digestion : This rs
bacteria possessa broad range of enzymesto
refinementof the abovetwo techniques,and can
degradea wide rangeof organiccompounds.
effectivelydigestup to 2O"hof the biodegradable
organics. The procersis carriedout by thermophilic The other organisms actively involved in
bacteriaat a temDerature around 45'C. Thereare compostingare actinomycetes(a filamentoustype
712 B IOTE CHNO LO G Y

of bacteria), fungi (molds, yeasts), and protozoa,

besides earthworms, insects, mites and ants.

M ECHANI SM O F CO M PO S T I N G

Com pos t ing is a v er y c om pl e x p r o c e s si n v o l v i n g

t he par t ic ipat ion of s ev er a l m i c r o o r g a n i s m s -
bac t er ia, ac t inom y c et es and f u n g i . T h e b a c t e r i a
br ing out t he dec om pos it ion c - r fm a c r o m o l e c u l e s Compost Sludqeano
nar nely pr ot eins and lipids . b e s i d e s g e n e r a t i n g cover agent
bulkin-g
energy (heat). Fungi and actinomycetes degrade
c eilulos e and ot her c om plex o r g a n i c c o m p o u n d s . Fig. 58.2 : A diagrammatic representation of aerated
static pole of comPosting.
Com pos t ing m ay be div ide d i n t o t h r e e s t a g e s
with re{erence to changes in temperature--
m es ophilic , t her m ophilic and c o o l i n g , Aerated static pile - 55%
M es ophilic s t age : The f ung i a n d a c i d - p r o d u c i n g Windrow - 30%
bac t er ia ar e ac t iv e in t hi s s t a q e , a n d t h e
In - v e s s e l _ 15%
temperature increasesfrom ambient to about 40'C.

Thermophilic stage : As the composting Aerated static Pile system

proceeds, the temperature raises from 40'C to
70' C. Ther m ophilic bac t er ia , t h e r m o p h i l i c f u n g i The dewatered sludge is mixed with a bulking
and ac t inom y c et es ar e ac t i v e i n t h i s s t a g e . agent (wood chips) and placed over a grid of
Ther m ophilic s t age is as s oc ia t e dw i t h h i S h r a t e a n d aeration or exhaustpiping Air is supplied through
m ax im um degr adat ionof or ga n i c m a t e r i a l s . blowers for efficient aeration.A layer of compost ts
kept over the top of the aerated static pile for
Cooling s t age : The m i c r o b i a l d e g r a d a t i v e insulation and good aeration (Fig. 58.2)- lt takes
ac t iv it y s lows down and t he t he r m o p h i l i c o r g a n i s m s
about 3-4 weeks for composting, and another 4-5
ar e r eplac ed by m es ophilic b a c t e r i a a n d f u n g i . weeks for curing. The cured compost is screened[o
Cooling stage is associatedwith formation of water, reduce the quantity, besides the recovery of the
pH s t abiliz at ion' and c om ple t i o n o f h u m e i c a c i d
bulking agent
f or m at ion.
Windrow system
M ETHO DS O F CO M PO STI N G
Wi n d r o w s a r e a t y p e o f s t a t i c p i l e s w i th
The oper at ion of c om po s t i n g i n v o l v e s t h e p e r i o d i c a l t u r n i n g a n d m i x i n g o f s l u d g e d u r i n g th e
f ollowing s t eps
c o m p o s t i n g p e r i o d ( 3 - 4 w e e k s ) T h e m i xi n g i s
o M ix ing of dewat er eds ludge w i t h a b u l k i n g a g e n t u s u a l l y c a r r i e d o u t a t w e e k l y i n t e r va l s a n d r s
( s aw dus t , r ic e hulls , s t r aw o r r e c y c l e c o m p o s t ) . associatedwith releaseof unpleasantand offensive
The bulk ing agent im pr ov e s t h e p o r o s i t y o f t h e odours. The windrorvs may be open or covered and
mixture for good aeration t h e a e r a t i o n i s c a r r i e d o u t b y m e c h a n i c a l m e a n s.
. Cr eat ing aer obic c ondit i o n s ( a e r a t i o n ) b y
In-vessel system
m ec hanic al or ot her m ean s . T h i s i s n e e d e d f o r
the supply of oxygen, to control temperature,and In the in-vesselcomposting system, the process
for the remo'zalof water (moisture) is carried out in a closed vessel or container. This
. Rem ov al of t he bulk ing ag e n t , i f p o s s i b l e . is an advanced method and is designed to control
the environmentai conditions (ter-nperature, air flow
o Storage and disposal of the compost. m i n i m i z i n g t f r e r e l e a seo f
and O, supply), b e s i d e s
There are three maicr methods of composting- offensive odours. The advantages of in-vessel
aerated static pile, windrow, and in-vessel systems. s y s t e m i n c l u d e h i g h e r e f f i c i e r r c y o f c o m p o sti n g ,
As per a r ec ent s ur v ey ,t he app r o x i m a t ed i s t r i b u t i o n w i t h l o w e r l a b o u r c o s t s , a n d s m a l l e r ar e a fo r th e
of different composting methods is given. plant.
 713

ChaPter
in'vesse':
..J[::,J:Jil!,'T#'J".'IT:H'J' tt/ t''

|owi"*".":l':f::"i:9,::l$T":li::
Plug-f
aretwo tvpesot l:T-';;"J u"a
tiii. sa.se
ComPosting
ml x
cvlindrical'o*"t "no,r,'if,''"' .i'.j'"thip between
r"f
is.sn).In.both:f :itfi.;i"s-mass is maintained
theparticles n, thesewase rs
the same
'"'l]:.:T5;;.Err.
throughoutrnc of first
oc i;:;"n" principle
---* Outfeed
fed, the comPosting
in and first out'
Dvnamic, '1;-]:;
inlv1*'.:"1H,T[t'T:f lnteed

are also known

"ri.Po"ng In these units
'eactors.' mixed d
materialis mechantcallY circul: -4
Outfeed
DYnamic
"i'o-t"ttt* rectangular reactors \
Turlu,ri'
"t!i.i'if^,"in
'oi.* i;:
t'*. :Hi[ or thereaction
augurs rotate arouno ':^"r;r
material' As
tne comp-oslii2,.-^'"t)^^n,",
veiser ancl mix
the '".t"?3,:';, iffi-
res,ards tnecompost, (B)
,in,"yo'is responstote .i"l compost the outlet
l^-^I.t to tne lnteed
l"i'ui':*" disciarge of the
conveyor'
aerobic composting
Factors affecting
rhereareseverat ^r1;to;;"'ffi#l;,":::, ff
ng
sludge-comPostl Proc€
brieflYdiscusseo'
of,srudee'3o[r:
1 rvpe "11""1""1J;t"1'f::::
compostcu'
sludges can be and emitsunpreas
^'^l'--".unpleasant
oxvsen'
more ns
:iffi;;i'es Odieed
odours. (c)
: ror lnteed
nitrogen ratio
2. carbon
';
.;;li;;';i".;F:H"?T'i'':'5il):ff
^.eiii;ielt I
the ranget)t LJ .' this ratio.
check an'J maintain .lable
PeriodicallY
agents: Cheapand ':idllt:l^"'
3. Bulking
b"Iki"; asentsaa1 i ::ilT:;
.l;*;,i,:ii .lTi"'],
Extraction
an conveyor
Their Particlesize
coniPosting'
: A moisture^::-:::"t "t
4' Moisture content ior composttng'
Air
(D)
ilan oo7" is ideal
,l;r"i;; of
5 Aeration J':I:il
: I,';lffi:?:::,':1:'1" ,,t;;"''"t'^'matic,reoresentation
supplyis.
reaches comPos ,.nut"'iur.
-"'::ll,i;;
the
of
;Wtr;";;;*t:"Yj:;;;w7
compostinsreacors-','"i?''!,Y^7liloJ,,ii1,,
and pH t e1t ^111tts ls
6' Temperature the temperature
compostingu'" '""i"-*h"n
714 B IOTE CHNO LO CY

between 45-55'C. lf the temperature goes beyond c o m m o n l y u s e d c h e m i c a l s a r e a l u m , l im e , fe r r tc

60'C, the process almost gets halted. The optimal chloride and organic polymers.
pH is between 6-9.
Heat treatment method
7. Mixing and turning : For appropriate
c om pos t ing, m ix ing and t ur nin g a r e r e q u i r e d . T h i s Heat treatment can stabilize and condition the
prevents drying and caking of the compost. sludge.This processis carried out for a short period
under pressure. Heat treatr.nent results in the
VERM I CO M PO STI NG c o a g u l a t i o n o f s o l i d s , b e s i d e s r e d u c i n g th e w a te r
affinity of sludge solids.
Vermicomposting refers to the process of
compost formation by earthworms. In fact,
earthworms are known to play a significant role rn
t he nat ur al c y c ling of s oil o r g a n i c m a t t e r a n d
m aint enanc eof por os it y of t h e s o i l .
I n r e c e n t y e a r s , d i s i n f e c t i o n o f sl u d g e i s
Earthworms are very efficient in nutrient b e c o m i n g s i g n i f i c a n t d u e t o i t s r e u se o r i ts
recycling. They can consume organic matter, a p p l i c a t i o n o n t h e l a n d . T h i s i s b e ca u se th e
approximately 'lO-20% of their own biomass per pathogenic organisms of the sludge should be
day. Earthworms can utilize organic matter with destroyed to protect the health of the inhabitants
variable carbon nitrogen ratio (C : N ratio) and who are exposed to it. There are a large number of
convert to lower .[ : N ratio. In other words, disinfection methods to cnoose.
vermicomposting involves the conversion of
. Pasteurization
carbon-rich organic compounds to nitrogen-rich
organic compounds. This is highly advantageous . Heat drying
f or s oil enr ic hm ent . .lrradiation
In recent years, vermicomposting of cow and . High pH treatment
buffalo dung has become a profitable, low (bio)
. Addition of chlorine
technology industry. The earthworm namely
Drawidia nepalensis is most commonly used for o Long-term storage of digested sludge
t his pur pos e. Ver m ic om post s a r e c o m m e r c i a l l y . Complete composting
available now, Introduction of earthworms into the
soils to bring out a natural process of
v er m ic om pos t ing ( f or s oil e n r i c h m e n t ) i s a l s o
advocated.

Dewatering basically involves the process of

reducing the moisture content of sludge.
Dewatering can be carried out by vacuum filters,
c e n t r i f u g e sa n d s l u d g e d r y i n g b e d s .
Condit ioning of s ludge is n e c e s s a r yt o i m p r o v e
its dewatering characteristics.Chemical and heat
Vacuum filtration
treatrnent methods are most commonlv used for
t his pur pos e. The ot her c o n d i t i o n i n g m e t h o d s D e w a t e r i n g b y v a c u u m f i l t r a t i o n i s r a th e r o l d ,
inc lude ir r adiat ion,f r eez ing an d s o l v e n t e x t r a c t i o n , but has been discontinued in recent years due to
whic h ar e les s f r equent ly us e d . h i g h o p e r a t i n g a n d m a i n t e n a n c ec o s t s , b e si d e sth e
complexicity of the process. In fact, improved and
Chernical method more efficient alternative methods have been
developed during the past ten years.
By use of chemicals, certain solids in lhe sludge
can be coagulatecl with a release of absorbed
Centrifugation
wat er . Chem ic al c ondit ionin g c a n r e d u c e t h e
moisture content of the sludge by about 15-30% Centrifugation is commonly used for the
(i.e. from about 959/oto about 65%). The most dewatering of sludges obtained from industries.By
Chapt er58 : SL U D C EA N D SOL IDWA STE S -TR E A TME NATN D D IS P OS A L 715

the process of centrifugation, it is possible to Land applications of sludge

iemove liquids from solids. Different types of {as a fertilizer}
centrifuges (solid bowel centrifuge, basket
fhe spreading of sludge on or just below the
cen trifug e) ar e c om m er c ially av ailable f or s l u d g e
soil surface is considered as land applications of
dewatering.
sludge. Sludge may be used in the agricultural
lands, forest lands, and dedicated land disposal
Sludge drying beds
s i t e s .T h e p a t h o g e n sa n d t o x i c o r g a n i c c o m p o u n d s
Drying b eds ar e widely us ed in dev e l o p e d present in the sludge can be respectivelydestroyed
countries (USA, Britain) to dewater the digested by sunlight and soil microorganisms. Sludge
slud ge .This m et hod pr oduc eshigh s olid c on t a i n i n g a p p l i e d t o l a n d i s t h u s u s e f r : la s a s o i l c o n d i t i o n e r
dried product which can be e.asily disposed off to imorove the characteristicsof land-nutrienr
(in a lan dfil l or as s oil c ondit ioner ) Sludge d r y i n g transport facilitation and increasedwater retention.
beds are cost-effective. Thus, sludge can replace the expensive fertilizers.

The quanrity of organic materials and the

pathogens must be reduced before the sludge ls
applied on the land. A high content of organrc
matter will result in offensive odours while the
Whe n th e s ludge is s ubjec t ed t o m ec h a n i c a l pathogens spreaci diseases. There are in fact
h ea t drying , t he wat er c ont ent c an be s ubs ta n t i a l l y regulatory requirements to control pathogens of
reduced. The ultimate purpose of heat drying is to s l u d g e b y v a r i o u s m e a n s .
prepare a sludge, free from moisture that can be
Distribution and marketing
incine rate de f f ic ient ly .M ec hanic al heat dr y i n g c a n
be carried out by flash dryers, spray dryers, rotary Distribution and marketing of sludge for
dryers an d m ult iple hear t h dr y er s . b e n e f i c i a l p u r p o s e si s g a i n i n g i m p o r t a n c e i n r e c e n t
years. ft is estimatedthat about 1O-20% of the total
THERMAL REDUCTI O N O F SLUDG E sludge produced is utilized in this fashion. The
marketed sludge is used as substitution for topsoil
Thermal reduction basically involves the total or
pa rtial co nver s ionof or ganic s olids t o ox idize o 'e n d and peat on parks, lawns, golf courses and in
ornamental and vegetable gardens.
oroducts such as carbon dioxide and water. This
may b e car r ied out by inc iner at ion or by w e t - a i r There are regulatory requirementsto reduce the
oxidation. Thermal reduction of sludge is associated p a t h o g e n i c o r g a n i s m s f o r distribution and
with destruction of pathogenic organisms, m a r k e t i n g o f s l u d g e .
detoxificationof toxic compounds and reducing the
vo lume o f dis pos abJ es ludge. LANDFILLING
The major processes employed for thermal Landfilling is a method for the final disposal of
red uction of s ludge ar e r nult iple - h e a r t h sludge that is not useful any more. fhe sanitary
incine ratio n,f luidiz ed- bed inc iner at ionand w e t - a r r landfill method is most suitable for the disoosal of
o xid atio n. solid domesiic wastes. This involves a low-cosr
anaerobic technology. ln this method, the sludge or
solid wastes are deposited in low-lying and low
v a l u e s i t e s .T h e d e p o s i t i o n i s d o n e a l m o s t d a i l y a n d
the deposits are covered with a layer of soil
(Fig. 58.4. With the coverage of the new waste
While considering the iinal disposal of sludge,
d e p o s i t s .n u i s a n c e c o n d i t i o n s s u c h a s b a d o d o u r s
its beneficial uses are first taken into account.
and flies are minimized.
Sludge is useful for the supply of nutrients to the
soil, be sid es pos s es s ing t he pr oper t ies o f s o i l It is desirablethat the sludge is dewateredso that
conditioning. Thus, attempts are made to dispose its transportation becomes easy. Further, the
the slud ge in a benef ic ial m anner . lf t his i s n o t generation of leachate (liquid that percolates out
oossible. alternatesare considered. due to leachingj)is minimal from a dewatered sludge.
716 B IOTE CHNO LO CY

Soillayer
Gas outlet
Monitor Sealinglayer

J I Leachateto
treatment

First layer

Secondarylayer
Leachatecollection

Fig. 58.4 : A diagrammatic representation ot a landfill

At leas et wo im per m eablelay e r sa r e b u i l t b e l o w t h e be stored indefinitely in lagoons or may be

landfill to prevent the leakage of leachate to the removed periodically.
s ur r ounding lands The ac c um u l a t e d l e a c h a t e c a n
be taken out and treated by appropriate methods SEPTAGE AND SEPTAGE D I S P O SAL
The c om plet e f illing of land f i l l s m a y t a k e s e v e r a l Septage is a combination of sludge, scum and
months or even years, depending on the size of the liquid coming out from a septic tank (Refer Chapter
site and the quantity of waste being deposited. 57\. lt must be disposed under controlled
Landfills can be used for the generation of methane c o n d i t i o n s t o a v o i d e n v i r o n m e n t a l p o l l u ti o n .
gas for commercial use. However, methane
pr oduc t ion us ually c or nm enc e ss e v e r a lm o n t h s a f t e r The most commonly used methods of septage
t he landf ill is c or nplet ely f illed . disposal are listed below.

ln some countries, there are strict regulationsto
us e landf ills f or s ewagedis pos a l .T h e s e i n c l u d e t h e
air- and water tight sitesto protect the environment
. Land application (surface or subsurface).
c Co-treatment with waste water (biological or
chemical treatment processes)'

LAG O O NI NG . Co-disposal with solid wastes (composting and

A lagoon is a shallow lake (or earth basin) landfilling).
usually located near a river or a sea. Lagooning . lndependent processing facilities (composting,
( dis pos al of s ludge int o lagoo n s ) r s a c o n v e n i e n t b i o l o g i c a l t r e a t m e n t , c h e m i c a l o x i d a ti o n , l i m e
m et hod of s ludge dis pos a l i f t h e t r e a t m e n t stabilization).
plant is loc at ed at a r em ot e p l a c e . I n l a g o o n i n g ,
t he s ludge is s t abiliz ed b y a n a e r o b i c a n d For more details on these treatment processes,
aer obic dec om pos it ion whic h i s a c c o m p a n i e d the reader must refer the preceeding pages.
by r eleas e of objec t ionable o d o u r s . F o r t h i s
reason, lagoons should be located away from TREATMENT AND DISPOSAL OF
dwelling ar eas and high ways t o a v o i d n u i s a n c e S O L I D WA S T E S
c ondit ions .
Solid wastes are mostly being treated by
The s t abiliz ed s olids of t he s l u d g e s e t t l e t o t h e i n c i n e r a t i o n a n d l a n d f i l l i n g , a l t h o u g h th e se
bottom of lagoon and accumulate. The sludgescan methods have certain limitations.
Chapt er5 8 : S L U D C EAN D SOL IDWA STE S -TR E A TME NATN D D IS P OS A L 717

lncineration : This requirescostly equipment After the separationof the importantreusable

and high p o w e r c o n s u m p ti o nIn . c i n e ra t ors
do not materi al s, the l eft out i n the sol i dw astesi s mai nl y
allow recoveryof any usefulmaterials.Further,it is the biodegradable organic matter.
f r equent lay s s o c i a tewdi th e n v i ro n me n tal p ol l uti on.
Gomposting
tandfilling : The variousaspectsof landfilling
A goodaccounton the pri nci pl es and processes
hav e bee n d e s c ri b e d(R e fe rp . 7 1 5 ). T h e maj or
of compostinghave alreadybeen described(See
lim it at iono f l a n d fi l l si s th e p ro b l e mo f l eachates
p. 711).The solid wastesare mostlycompostedby
and gas emissionswhich pollutethe environment.
anaerobicprocessand to a lesserextentby aerobic
Further,they are not efficientproducersof biogas.
means.
It is not possibleto recyclethe reusableproducts
(paper, plastic, construction materials etc.) In Dry anaerobic
composting
landf illing .
{DRANCOI process
SEPARATION AND ln recent years, some companies have
COMPOSTING PLANTS developed specific anaerobic digesters for
composti ng sol i dw astes.The desi gni ng
of di gester
T he ind u s tri aal n d m u n i c i o a ls o l i d w astescan is mairrlybasedon the solid contentof the waste,
be effectivelytreatedby separation,followed by the temperature(from 35'C to 55"C) and the
composting.In fact, hugeplantsare constructed to numberof stages(1 or 2).
servethe dual purposes.
Dry anaerobic composting process is a
Separation commercially designed anaerobic digester. lt
is employedfor compostingof high solid (200-400
It is possibleto recoverseveralusefulmaterials g/l) wastes at thermophilictemperature(around
f r om t he s o l i d w a s te s b y e mp l o y i n g physi cal 55" C ) by usi ng a si ngl estagereactor.The marn
orocesses. advantageof DRANCO processis the high rate of
. P las t icm a te ri a l fo
s r re u s e . composting under controlled conditions. This is
. Sandand gravelfor constructionpurposes. evident from the fact that the compostingof solids
, can be comoletedin abouttwo weekstime. This is
. Paperand cardboardfor use in paperindustry. in contrastto a landfillwhich
takesseveralyears,
. lr on and a l u mi n i u mfo r me ta l l u rg y . sometimeseven decades(10-30 years)!
lJ iodegr adat ionor biologic a l d e g r a d a t i o n i s t h e
L) phenomenon of biologicat transformation of
organic compounds by living organisms,
particularly the microorganisms. Biodegradation
bas ic ally inv olv es t he c onv e r s i o n o f c o m p l e x
or ganic m olec ules t o s im p l e r ( a n d m o s t l y
non-toxic) ones The term biotransformation is
used for incomplete biodegradation of organic
c om pounds inv olv ing one or a f e w r e a c t i o n s .
Biotransformationis employed for the synthesisof
c om m er c ially im por t ant products by
m ic r oor ganis m s .For det ails on b i o t r a n s f o r m a t i o n ,
refer Chaoter 22.
Bioremediation refers to the process of using
microorganisms to remove the environmental
pollutants i.e. the toxic wastesfound in soil, water,
air etc. fhe microbes serve as scavengers n
bioremediation. The removal of organic wastes by
microbes (or environmental clean-up is the essence
of bior em ediat ion.The ot her na m e s u s e d ( b y s o m e
authors) for bioremediation are biotreatment,
bioreclamation and biorestoration.
I t is r at her dif f ic ult t o s h o w a n y d i s t i n c t i o n
bet ween biodegr adat ionand bio r e m e d i a t i o n ,h e n c e
they are consideredtogether in this chapter.Further,
in biotechnology, most of the reactions of
biodegr adat ion/ bior em ediat ioinn v o l v e x e n o b i o t i c s .
Thus , t his c hapt er m ay be apor o p r i a t e l yc o n s i d e r e c l
as m ic r obial degr adat ion of x e n o b i o t i c s . F o r t h e
bioremediation of metal polluiants, the reader must
Ref er Chapt er 32) .
718
Xenobiotics
Xenobiotics (xenos-foregin)broadly refer to the
u n n a t u r a l ,f o r e i g n a n d s y n t h e t i cc h e m i c al s su ch a s
pesticides, herbicides, refrigerants, solvents and
o t h e r o r g a n i c c o m p o u n d s M i c r o b i a l d e g r a d a ti o no f
x e n o b i o t i c s a s s u m e ss i g n i f i c a n c e ,s i n c e i t p r o vi d e s
an effective and economic means of disposing of
t o x i c c h e m i c a l s , p a r t i c u l a r l y t h e e n v i r o n m e n ta l
pollutants.
P S E U D O M O N 'A S _ T H E P R E D O M I NAN T
M I C R O O R G A T {I S M F O R
BIOREMED!ATION
Members of the genus Pseudomonas (a soil
microorganism) are the most predominant
microorganismsthat degrade xenobiotics. Different
strains of Pseudomonas, rhal are capable of
d e t o x i f y i n g m o r e t h a n 1 0 0 o r g a n i c c o m p o u n d s,
have been identified. The examples of organic
c o m p o u n d s a r e s e v e r a l h y d r o c a r b o n s, p h e n o l s,
o r g a n o p h o s p h a t e s i p o l y c h l o r i n a t e d b i p h e n yl s
( P C B s )a n d p o l y c y l i c a r o m a t i c s a n d n a p hth a l e n e .
A b o u t 4 0 - 5 0 m i c r o b i a l s t r a i n s o f m i cr o -
o r g a n i s m s ,c a p a b l e o f d e g r a d i n g x e n o b i oti cs h a ve
been isolated. Besides Pseudomonas,other good
examples are Mycobacterium, Alcaligenes, ano
N o c a r d i a .A s e l e c t e dl i s t o f m i c r o o r g a n i sm sa n d th e
xenobiotics degraded is given in Table 59.1 .
Consortia of microorganisms for biodegradation :
A p a r t i c u l a r s t r a i n o f m i c r o o r g a n i s mm a y d e g r a d e
o n e o r m o r e c o m p o u n d s . S o m e t i m e s , fo r th e
Chaot er59 : B IO D EC R A D A T IOANN D B IO R E ME D IA TION 719

cannot serve as a source of carbon or energy for the

Tnru 59.1 A selectedlist of microorganlsms organism. The term co-metabolism is often used to
and the pollutants(xenobiotics)that indicate the non-beneficial (to the microorganism)
are degradedby bioremediation biochemical pathways concerned with the
biodegradation of xenobiotics. However, co-
Microorganism Pollutant c!'eemiaals
m e t a b o l i s m d e p e n d s o n t h e p r e s e n c eo f a s u i t a b l e
Pseudomonas
sp Aliphatic
andaromatic s u b s t r a t ef o r t h e m i c r o o r g a n i s m .S u c h c o m p o u n d s
hycirocarbons- are referred to co-substrates.
alkylaminoxides,
alkylammonium
benzene, Factors affecting biodegradation
naphthalene,
anthracene
Several factors influence biodegradation.These
xylene,
toluene,
include the chemical nature of the xenobiotic,the
polychlorinated
biphenyls
(PCBs), parathion,c a p a b i l i t y o f t h e i n d i v i d u a l m i c r o o r g a n i s m ,n u t r i e n t
malathion,
and O2 supply, temperature, pH and redox
organophosphates.
potential. Among these, the chemical nature of the
My'cobacteriun
sp Benzene,
branched substrate that has to be degraded is very important.
hydrocarbons,
cyclcparaff
ins Some of the relevant featuresare given hereunder.
Alcaligenes
sp Polychlorinated
biphenyls,
. I n g e n e r a l ,a l i p h a t i c c o m p o u n d s a r e m o r e e a s i l y
haiogenated
alkylbenzene,
degraded than aromatic ones.
hydrocarbons.
. Presence of cyclic ring structures and length
Nocardia
sp Naphthalene,
alkylbenzenes
chains or branches decrease the efficiencv of
phenoxyacetate,
biodegradation.
Arlhrobacter
sp polycyclic
Benzene,
phenoxyacetate, r Wa t e r s o l u b l e c o m p o u n d s a r e m o r e e a s i l y
aromatics,
pentachlorophenol. degraded.

Corynebacterium
sp hydrocarbons, . M o l e c u l a r o r i e n t a t i o n o f a r o m a t i c c o m p o u n d s
Halogenated
phenoxyacetate. influences biodegradation i.e. ortho > para >
meta.
Bacillus
sp Longchainalkanes,
phenylurea. o The presence of halogens (in aromatic
compounds) inhibits biodegradation.
Jdttutua >V Polychlorinated
biphenyls
lspergillussp Phenols Besidesthe factors listed above, there are two
recent developmentsto enhance the biodegradation
,Qnthomonas
sp hydrocarbons
Polycyclic
by microbrganisms.
S:'eptomyces
sp Halogenated
hydrocarbons,
phenoxyacetate. B i o s t i m u l a t i o n : T h i s i s a p r o c e s sb y w h i c h t h e
-,sanunsp microbial activity can be enhanced by increased
Propanil
supply of nutrients or by addition of certain
sp
-^,tninghamella Polycyclic
aromatics, stimulating agenfs (electron acceptors, surfactants).
polychlorinated
biphenyls.
Bioaugmentation : lt is possible to increase
biodegradation through manipulation of genes.
'.:'ad atio n o f q s ingle c om pound, t he s y ner g e t i c M o r e d e t a i l s o n t h i s g e n e t i c m a n i p u l a t i o n i . e .
, - :: of a fe w m ic r oor ganis m s( i. e. a c ons or ti u m g e n e t i c a l l ye n g i n e e r e dm i c r o o r g a n i s m s( G E M s ) ,a r e
- rcktail of microbes) may be more efficient. For d e s c r i b e d l a t e r B i o a u g m e n t a t i o n c a n a l s o b e
- r-; -ce, the ins ec t ic ide par at hion is m o r e a c h i e v e d b y e m p l o y i n g a c o n s o r t i u m o f m i c r o -
,- - :n tly de gr aded by t he c om bined ac t ion o f o r g a n r s m s .
: .:
- --crnonas aeruginosa and Psudomonas stulzeri.
Enzynre systems for biodegradation
fo-metabolism in biodegradation : In general,
'. --:abolism (breakdown) of xenobiotics is not Several enzyme systems (with independent
, ,:eo with any advantage to the enzymes that work together)are in existence in the
- -." -:an ism. That is t he pollut ant c hem ic a l microorganisms for the degradation of xenobiotics.
 72(J B IOTE CHNO LO G Y

It takesabout 4-5 yearsfor the degradationof

D D T (75-1OO% )i n the soi l . A gr oup of
the plasmidscontaininggenes microorganisms (Aspergi
llus flavus,Mucor aternans,
Fusarium oxysporum and Trichoderma viride) are
associated with the slow biodegradation of DDT.
Xenobiotic Name of plasmid in
Pseudomonas Biomagnification : The phenomenon of
progressive increase in the concentration of a
Naphthalene NAH
xenobioticcompound,as the substanceis passed
Xylene XYL
through the food chain is referred to as
Xylene
andtoluene TOL,pWWO,XYL-K biomagnification or bioaccumulation. For instance,
Salicylate SAL the insecticideDDT is absorbedrepeatediyby
Camphor CAM plantsand microorganism. When they are eatenby
pAC25 fish and birds, this pesticide being recalcitrant,
3-Chlorobenzene
accumulates, and entersthe'foodchain.Thus,DDT
may fi nd i ts entry i nto rrari ousani mal s,including
The genes coding for the enzymes of man. DDT affectsthe nervcussystems,and it has
biodegradative pathways may be present in the been bannedin some countries.
chromosomal DNA or more frequently on the
plas m ids . I n c er t air r m ic r oor ga n i s m s ,t h e g e n e s o f TYPES OF BIOREMEDIATION
both chrornosome and plasmid contribute for the
The most important aspect of environmental
enzymes of biodegradation.
biotechnologyis the effective managententof
As already described, the microorganism hazardousand toxic pollutants(xenobiotics)by
Pseudomonas occupies a special place in bi oremedi ati on.The envi ronmenta! clean- up
biodegradation. A selected list of xenobiotics and processthroughbioremediation can be achievedin
t he plas m ids c ont aining t he g e n e s f o r t h e i r two ways-in situ and ex situ bioremediation.
degradation is given in Table 59.2.

ln situ bioremediation
RECALCITRANT XENOBIOTICS
ln situ bioremediation involves a direct
There are certain compounds that do not easily
approach for the m icrobial degradation of
undergo biodegradation and therefore persist in
xenobioticsat the sitesof pollution (soil, ground
the environment for a long period (sometimes irr
water).Additionof adequatequantitiesof nutrients
years).They are labeled as recalcitrant.There may
at the sitespromotesmicrobialgrowth.When these
be several reasons for the resistance of xenobiotics
microorganismsare exposed to xenobiotics
t o m ic r obial degr adat ion.
(pollutants),they develop metabolic ability to
. They m ay c hem ic ally and b i o l o g i c a l l y i n e r t degradethem. The growth of the microorganisms
( highly s t able) . and their ability to bring out biodegradation are
dependenton the suppl y of essenti alnut r ient s
. Lack of enzyme system in the microorganismsfor
(nitrogerr, phosphorusetc.)./n situ bioremediation
biodegradation.
has been successfully applied for clean-upof oil
. They c annot ent er t he m icr o o r g a n i s m s b e i n g spillages,beachesetc. There are two types of rn
large molecules or lack of transport systems si fu bi oremedi ati on-i ntri nsi
andc engi neer ed.
. The c om pounds m ay be highl y t o x i c o r r e s u l t r n
Intrinsic bioremediation : The inherent
the formation highly toxic products that kill metabolicabilityof the microorganisms to degrade
m ic r oor ganr s m s . certai npol l utantsi s the i ntri nsi cbi oremediat ion.
In
fact, the microorganisms can be tested in the
There are a large number of racalcitrant
laboratory for their natural capability of
xenobiotic compounds e.g. chloroform, freons,
biodegradation and appropriatelyutilized.
insecticides (DDI, lindane), herbicides (dalapon)
and synthetic polymers (plastics e.g. polystyrene, Engineered in sifu bioremediation : The inherent
polyethylene, polyvinyl chlorine). ability of the microorganisms for bioremediation is
 Chaot er59 : BIOD EC R A D A T IOAN
N D B IO R E ME D IA TION 721

gener allys l o w a n d l i m i te d . H o w e v e r,b y usi ng not always possible This is evident from the
suitable physico-chernical means (good nutrient different types of metabolic effects as shown below.
and O, supply, addition of electron acceptors,
Detoxification : This process involves the
optimal temperature), the bioremediationprocess
m i c r o b i a l c o n v e r s i o no f t o x i c c o m o o u n d t o a n o n -
can be engineered for moreefficientdegradation of
toxic one. Biodegradation involving detoxification
oollut ant s .
is highly advantageous to the environment and
Advantagesof in situ bioremediation population.

1. Cost-effective, with minimal exposurercr Activation : Certain xenobiotics which are not
pub l i c o r s i te p e rs o n n e l . toxic or less toxic may be converled to toxic or
n ma i nm i ni mal l y more toxic products. This is dangerous.
2. S ite so f b i o re me d i a ti ore
disru p te d . Degradation : The complex compounds are
'
Disadvantages
of in sifu bioremediation degraded to simpler products which are generally
harmless.
1. V eryti m e c o n s u mi n gp ro c e s s .
Conjugation : The process of conjugation may
2. Sites are directly exposed to involve the conversion of xenobiotics to more
environmentalfactors (temperature,O, complex compounds. This is however, not very
s up p l ve tc .). common.
3. M ic ro b i a l d e g ra d i n g a b i l i ty vari es
seasonally. TYPES OF REACTIONS IN
BIOBEMEDIATION
Ex situ bioremediation
Microbial degradation of organic compounds
The wasteor toxic materialscan be collected primarily involves aerobic, anaerobic and
with
from the oollutedsitesand the bioremediation sequential degradation.
the requisite microorganisms (frequently a
consortiumof organisms)can be carried out at Aerobic bioremediation
designed places. This process is certainly an
improvementover in situ bioremediation,and has Aerobic biodegradation involves the utilization
been successfullyusedat some places. of O, for the oxidation of organic pompounds.
These compounds may serve as substratesfor the
Advantagesof ex situ bioremediation supply of carbon and energy to the microorganisms.
1. Better controlled and more efficient Two types of enzymes namely monooxygenases
orocess. and dioxygenases are involved in aerobic
biodegradation. Monooxygenasescan act on both
2. Processcan be improvedby enrichment
aliphatic and aromatic compounds while
with desiredmicroorganisms.
d i o x y g e n a s e so x i d i z e a l i p h a t i c c o m p o u n d s .
3. T im e re o u i re di n s h o rt.
of ex sifu bioremediation
Disadvantages Anaerobic bioremediation

1. Very costly process. Anaerobic biodegradation does not require O,

2. S it e so f p o l l u ti o na re h i g h l yd i s tu r bed.
3. Theremay be disposalproblemafterthe
processis complete.
METABOLIC EFFECTS OF
M I CRO O RG A N IS M S O N XE N OB IO T IC S
Althoughit is the intentionof the biotechnologist
to degradethe xenobioticsby microorganisms to Hydrogenation
the advantage of environmentand ecosystem, it is benzoate, phenol, catechol.
supply. The growth of anaerobic microorganisms
(mostly found in solids and sediments), and
consequently the degradation processesare slow.
However, anaerobic biodegradation is cost-
effective, since the need for continuous O, supply
is not there. Some of the important anaerobic
reactions and examples of organic compounds
degraded are listed below.
and dehydrogenation -

3crechnology[46]
 722 B IOTECHNO LO CY

Dehalogenat ion- Poly c h l o r i n a t e d b i p h e n y l s 2. Opeiring of the benzene ring.

( PCBs ) , c hlor inat ed et h y l e n e s . T h e term
Mcst of the n o n - h a l o g e n a t ed a r o i xa ti c
dechlorinatiorr is frequentlv used fur
c o m p o u n d s u n d e r g o a s e r i e s o f r e a cti o n s to
dehalogenat ionof c hlor ir r at e dc o m p o u n d s .
produce catechol or protocatechuate. The
Carboxylation and decarboxylation- toluene, b i o r e r n e d i a i i o no f t o l u e n e , L - m a n d e l a te ,b e n zo a te ,
cresoi and benzoate. b e n z e n e , p h e n o l , a n t h r a c e n e , n a p h th a l e n e ,
p h e n a n t h r e n ea n d s a l i c y l a t et o p r o d u c e ca te ch o l i s
Sequential bioremediation showrr in Fig. 59.1 . Likernrise, Fig. 59.2, depicts the
b i o r e r r e d i a t i o n o f q u i n a t e , p - h y d r o x ym a n d e l a te ,
In the degradation of several xenobiotics, both
p-hydroxybenzoyl formate, p-toluate, benzoate
aer obic and anaer obic Dr oc e s s e sa r e i n v o l v e d . T h i s
a n d v a n i l l a t e t o p r o d u c e p r o t o c a t e c h u a teC . a te ch o l
is often an effective way of reducing the toxicity of
a n d p r o t o c a i e c h u a t e c a n u n d e r g o o xi d a ti ve
a pollut ant . For ins t anc e,t e t r a c h l o r o m e t h a n ea n d
cleavage pathways. ln artho-cleavage pathway,
tetrachloroethaneundergo sequential degradation.
catechol and protocatechuate form acetyl CoA
(Fig.59.3), w h i l e i n m e t a c l e a v a g e p a th w a y
BI O DEG RADATI O N OF HYDROCARBONS
(Fig,. 59.a), they are converted to pyruvate and
Hy dr oc ar bon ar e m ainly t h e p o l l u t a n t sf r o m o r l acetaldehyde. The degraded products of catechol
r ef iner ies and oil s pills . Tlr e s e p o l l u t a n t s c a n b e and protocatechuate are readily metabolised br
degr aded by a c ons or t i u m o r c o c k t a i i o f almost all the organisnrs.
microorganisms e.g. Pseudomonas, Coryne-
bacterium, Arthrobacter, Mycobacterium and BIODEGRADATION O F P E S T I C I DES
Nocardia. AND HERBICIDES

P e s t i c i d e sa n d h e r b i c i d e s a r e r e g u la r l y u se cito
Biodegr adat ion of aliph a t i c
c o n t a i n v a r i o u s p l a n t d i s e a s e s a n d im p r o ve th e
hy dr oc ar bons
crop yield. In fact, they are a part of the modern
The upt ak e of aliphat ic h y d r o c a r b o n s i s a s l o w a g r i c u l t u r e , a n d h a v e s i g n i f i c a n t l y c o n tr i b u te d tc,
pr oc es s due t o t heir low s o l u b i l i t y i n a q u e o u s green revolution. fhe common herbicides anc;
m edium . Bot h aer obic and a n a e r o b i c p r o c e s s e sa r e p e s t i c i d e s a r e p r o p a n i l ( a n i l i d e ) , p r o p h a n '
oper at iv e f or t he degr a d a t i o n o f a l i p h a t i c ( c a r b a m a t e )a, t r a z i n e ( t r i a z i n e ) ,p i c l o r a m ( p yr i d i n e
hy dr oc ar bons . For inst a n c e , u n s a t u r a t e d d i c h l o r o d i p h e n y l t r i c h l o r o e t h a n e ( D D T) m o n o -
hydrocarbons are degraded in both anaerobic and c h l o r o a c e t a t e ( M C A ) , monochloropropionaie
aer obic env ir onm ent s , while s a t u r a t e d o n e s a r e (MCPA) and glyphosate lorganophosphate).
degraded by aerobic process.
M o s t o f t h e p e s t i c i d e sa n d h e r b i c i d e s a r e to \ -
Som e aliphat ic hy dr o c a r b o n s w h i c h a r e a n d a r e r e c a l c i t r a n t ( r e s i s t a n tt o b i o de g r a d a ti o n
reclacitrant to aerobic process are effectively Some of them are surfactants(activeon the sui'fact
c iegr adedin anaer obicenv ir o n m e n te g . c h l o r i n a t e d and retained on the surface of leaves.
aliphat ic c om pounds ( c ar bo n t e t r a c h l o r i d e ,m e t h y l
c hlor ide, v iny l c hlor ide) . Biodegradation of halogenated
aromatic cornpounds
Biodegradation of aromatic
M o s t c o r n n r o n l y u s e d h e r b i c i d e s a n d p e sti ci c;.'.
hydrocarbons (predominar--
are arornatic halogenated
' M ic r obial
degr adat ionof a r o m a t i c h y d r o c a r b o n s c h l o r r n a t e d )c o m p o u n d s . T h e b i o d e g r a d a ti vep a - - -
oc c ur s t hr ough aer obic and a n a e r o b i c p r o c e s s e s . ways of halogenated compounds df€ corllpdrd: :
The m os t im por t ant r nic r oor g a n i s mt h a t p a r t i c i p a t e s y r i t h t h a t d e s c r i b e c f o r t h e d e g r a d a ti o n o f l c- -
in these processesis Pseudomonas. h a l o g e n a t e da r o m a t i c c o m p o u n d s ( F i g s .5 9 .1, 5 9 .)
59.3 and 59.4). The rate of degradation
The biodegr adat ion of a r o m a t i c c o m p o u n d s
h a i o g e n a t e dc o m p o u n d s i s i n v e r s e l y r e l a te d to :- .
bas ic ally inv olv es t he f oll o w i n g s e q u e n c e o f
n u m b e r o f h a l o g e n a t o m s t h a t a r e o r i g i n a l l y p r e :r - '
reactions.
o n t h e t a r g e t m o l e c u l e i . e . c o m p o u n d s w i th h r ,:'-
1 . Rem ov al of t he s ide ch a i n s . n u m b e r o f h a i o g e n s a r e l e s s r e a d i l y d eg r a cl e d
 AN D B IO R EME D IA TION
-r apt er 59 : B I O D E C R AD AT ION 723

Toluene L-Mandelate

I J
Benzoylalcohol Benzoylformate

1-------)
Benzaldehyde Anthracene

- +
I
Benzoate Salicylate{- Nephthalene

+ *- Phenanthrene
./l -.rt
\
BenzenePhenol Anthranilate

Fig. 59.1 : Bioremediation of certain arcmatic compounds by bacteria to produce catechol.

Quinate p-Hydroxy -f P-HYdroxYbenzoyl

L-mandelate formate

ll
++
Shikimate p-Hydroxybenzaldehyde
+----<------p-Toluate
ll
x "f
rJ
S-Dehydro- P-Hydroxybenzoate{- Benzoate
shikimate
\
\
\ k-
{-
PROTOCATLCHU,T,Ti:. Vanillate

Fig. 59.2 : Bioremediation ol ceftain organic compounds by bacteria to ptoduce protocatechuate.

Catechol Protocatechuate Catechol Protocatechuate

rl II II
+tl + p-carboxYcts' cts-
J I
,-s cls-Muconate 2-Hydroxy +
muconicacid 2-Hydroxysemialdehyde
tl
tl
++ II II
Vuconolactone 1-Carboxymuconolactone J J
(/ Oxopent4-enoate carboxypentenoate

\.+ I I
4-Oxoadipate
enol lactone
J
Oxovalerate
J
Oxovalerate
I II
.'-t \
I \ J
P rl rLrr,:-,te A cetal dehyde 2 Pyruvate

Fig. 59.3 : Conversion of catechol and Fig. 59.4 : Conversion of catechol and
protocatechuate to acetyl CoA and succinate prctocatechuate to pyruvate and acetalciehyde
by ortho-cleavage PathwaY. by meta-cleavage Pathway'
724
Dehalogenat ion ( i. e. r em o v a l o f a h a i o g e n
substituent from an organic compound) of
halogenated compounds is an essential step for
their detoxification. Dehalogenation is frequently
catalysed by the enzyme dioxygenase. In this
reaction, there is a replacement of halogen on
benzene with a hydroxyl group. Most of the
halogenated compounds are also converted to
catechol and protocatechuate which can be
metabolised (Fig. 59.Q.
Besides Pseudomonas, other microorganisms
such as Azotobacter, Bacilluefs and E. coli are also
inv olv ed in t he m ic r obi a l d e g r a d a t i o n o f
halogenated aromatic compounds.
Biodegradation of polychlorinated
biphenyls {PGBsl
The ar om at ic c hlor inat edc o m p o u n d s p o s s e s s i n g
bipheny l r ing ( s ubs t it ut edwi t h c h l o r i n e ) a r e t h e . M i c r o b i a l d e g r a d a t i o no f o r g a n i c c o m p o u n d s i s a
PCBs e. g. pent ac hlor obiphen y l .
PCBs are commercially synthesized,as they are
us ef ul f or v ar ious pur pos e s- a s p e s t i c i d e s , i n
electrical conductivity (in transformers),in paints
and adhesives. They are inert, very stable and
resistantto corrosion. However, PCBs have been
implicated in cancer, damage to various
organs and impaired reproductive function.
Their commercial use has been restrictedin recent
years, and are now used mostly in electrical
transformers.
PCBs ac c um ulat e in s oi l s e d i m e n t s d u e t o
hy dr ophobic nat ur e and h i g h b i o a c c u m u l a t i o n
potential. Although they are resistant to
biodegradation,some methods have been recently
developed for anaerobic and aerobic oxidation by
em ploy ing a c ons or t ium o f m i c r o o r g a n i s m s .
Pseudomonas, Alkaligenes, Corynebacterium and
Acinetobacter. For more efficient degradation of
PCBs ,t he m ic r oor ganis m sar e g r o w n o n b i p h e n y l s ,
so that the enzymes of biodegradationof PCBs are
induc ed.
BI O DEG RADATI O N OF SOME OTHER
IMPORTANT COMPOUNDS
Organo.nitro compounds
Some of the toxic organo-nitro compounds can
be degraded by microorganisms for their
detoxification.
B IOTE CH\ C_ -
2, 4, 6-Trlnilrotoluene (TNT) : Certain ca: =
a n d f u n g a l s p e c i e sb e l o n g i n g l o P s e u d om o r ., - '
Clostrium can detoxify TNT.
Nitrocellulose : Hydrolysis, followec :
anaerobic nitrification by certain bacteria,degra:=.
nitrocellulose.
S y n t h e t i c d e t e r g e n t s : T h e y c o n ta i n So - :
surfactants (surface active agents) which are -:'
r e a d i l y b i o d e g r a d a b l e . C e r t a i n b a c t e r i a l p l a sr :
can degrade surfactants.
GENETIC ENGINEERING F O R M OR E
EFFICIENT BIOBEMEDIATION
A l t h o u g h s e v e r a l m i c r o o r g a n i s m s th a t ca r
d e g r a d e a l a r g e n u m b e r o f x e n o b i o t i c s h a ve b e e -
identified, there are many limitations in bi,
remediation.
very slow process.
. N o s i n g l e m i c r o o r g a n i s m c a n d e g r a d e a l l th e '
x e n o b i o t i c s p r e s e n t i n t h e e n v i r o n m e n ta
pollution.
. The growth of the microorganisms may be
inhibited by the xenobiotics.
o Certain xenobiotics get adsorbed on to the
o a r t i c u l a t em a t t e r o f s o i l a n d b e c o m e un a va i l a b l e
for microbial degradatron.
It is never possible to address all the above
l i m i t a t i o n s a n d c a r r y o u t a n i d e a l p r o ce ss o f
bioremediation. Some attempts have been made in
recent years to create genetically engineered
microorganisms (GEMs) to enhance bio-
remediation, besides degrading xenobiotics which
are highly resistant (recalcitrant) for breakdown.
Some of these aspects are briefly described.
Genetic manipulation bY transfer
of plasmids
The majority of the genes responsible for the
synthesisof biodegradativeenzymes are located on
the plasmids. It is therefore logical to think of
g e n e t i c m a n i p u l a t i 6 n s o f p l a s m i d s . N e w str a i n so f
bacteria can be created by transferof plasmids (by
conjugation) carrying Senes for different
degradative pathways. lf the two plasmids contain
homologous regionsof DNA, recombination occurs
Chapt er59 : BIOD EC R A D A T IOANN D BIOR E ME D IA TION 725

CAMplasmid OCTplasmid XYL plasmid NAH plasmid

A \." B
\.,/

+]conlrgation

CAM_OCT XYL
plasmid plasmid

XYL plasmid
CAM-OCT
plasmid NAH plasmid

Fig. 59.5 : Creation of the superbug by transfer of plasmids (A, B, C, D, E, F and G are the different
slrains ol bacteria egn!?iningth.e.plasmidsshow1, Slrain G is the gupq{bug.,)

between them, resulting in the formation of a larger Greation of superbug by transfer

fused plasmid (with the combined functions of both of plasmids
pla smid s).In c as e of plas m idswhic h do not p o s s e s s
Superbug is a bacterial strain of Pseudomonas
homologous regions of DNA, they can coexrst
that can degrade camphor, octane, xylene and
in the bacterium (fo which plasmid transfer rvas
naphthalene. Its creation is depicted in Fig. 59.5.
do ne ).
The bacterium containing CAM (camphor-
The first successfuldevelooment of a new strain
degrading) plasmid was conjugated with another
of bacterium (Psuedomonas)by manipulations of
bacterium with OCT (octane-degrading)plasmid.
plasmid transfer was done by Chakrabartyand his
These plasmids are not cornpatibie and therefore,
co-workers in 1970s. They used different plasmids
cannot coexist in the same bacterium. However,
and constructed a new bacterium called as
due to the presence of homologous regions of
superbug, that can degrade a number of
DNA, recombination occurs between these tw'o
hydrocarbons of petroieum simultaneously.United
.l plasmids resulting in a single CAM-OCT plasnrid.
Statesgranted patent to this superbug in 981 (as
This new bacterium possesses the degradative
per the directive of American SupremeCourt). Thus,
genes for both camphor and octane
superbug became the first genetically engineered
microorganism to be patented, Superbug has Another bacterium with XYL (xylene-degrading)
played a significant role in the development of plasnrid is conjr.rgatedwith NAH (naphthaiene-
biotechnology industry, although it has not been d e g r a d i n g )p l a s m i d c o n t a i n i n g b a c t e r i u m . X Y L a n d
used for large scale degradationof oil spills. NAH plasmids are compatible and therefore c-an
726 B IOTECHNO LO G Y

c oex is t in t he s am e bac t e r i u m T h i s n e w l y ,
produced bacterium contains genes for the Trru 59.3 A selectedllst of genetical$l
degr adat ionof x y lene and n a p h t h a l e n e .
englneelednicloorganisrns(GEltlslwith the
potentlalxenobloticsthat can be degraded
The next and final step is the conjugation of
bac t er ium c ont aining CAM - OC T p l a s m i d w i t h t h e G eneticalIy engin eered Xenobiotic
ot her bac t er iumc ont aining X Y L a n d N A H p l a s m i d s . microorganism (CEMs)
The newly created strain is the superbug that
diminuta
Pseudononas Parathion
carries CAM-OCT plasmid (to degrade camphor
and octane), XYL (xylene-degrading) plasmid and P. oleovorans Alkane
NAH (naphthalene-degrading) plasmid.
P. cepacia 2, 4, 5-Trichlorophenol

Development of salicylate-toluene P.putida Mono-anddichloro-

degr ading bac t er ia by p l a s m i d t r a n s f e r c0mp0unds
aromatrc

Some attempts have been made for the creation sp

Alcaligenes 2, 4-Dichlorophenoxy
of a new strain of the bacterium Pseudomonas aceticacid
putida to simultaneously degrade toluene and
sp
Acinetobacter 4-Chlorobenzene
s alic y lat e.

Toluene- degr ading ( TOL ) plasmid was

transferred by conjugation to another bacterium xenobiotics is given in Table 59.3. Almost all these
t hat is c apable of degr adin g s a l i c y l a t e ( d u e t o t h e GEMs have been created by transferring plasmids
presence of SAL plasmid) The newly developed
strain of Pseudomonascan simultaneouslydegrade Biosurfactant producing GEM
bot h t oluene and s alic y lat e.A n d t h i s o c c u r s e v e n a t A genetically engineeredPseudomonasaeruginosa
a low temperature (0-5'C). However, the new has been created (by Chakarabarty and his group)
bacterium is not in regular use, as more researchts This new strain can produce a glycolipid emulsifier (a
being c onduc t ed on it s m er i t s a n d d e m e r i t s . biosurfactant)which can reducethe sudacetensionoi
an oil water The reduced inter{acialtension
interface.
Genetic manipulation by gene promotes biodegradation of oils.
alteration

Work is in progressto manipulate the genes for GEM for degradation o f v a n i l l a te

more efficient biodegradation.The plasmid pWWO
of Pseudomorras codes for 12 different enzymes
responsiblefor the meta-cleavagepathway (for the
conversion of catechol and orotocatechuate rcr
pyruvate and acetaldehyde, See Fig. 59.4) for
degradation of certain aromatic compounds. Some
success has been reported to alter the genes of
plasmid pWWO for more efficient degradation of
t oluene and x y lene.
G ENETI CALLY ENG I NEE R E D
M I CRO O RG ANI SM S (GEMs| lN
BI O REM EDI ATI O N
Superbug (described already) is the first
genetically engineered microorganism. Several
workers worldover have been working for the
creation of CEMs, specifically designed for the
detoxification of xenobiotics. A selected list of
CEMs with a potential for the degradation' of
and SDS
A new strain of Pseudomonassp (strain ATCC
1 9 1 5 ) h a s b e e n d e v e l o p e d f o r t h e d e g r a d a ti o n o f
vanillate (waste product from paper industry) and
sodium dodecyl sulfate (SDS, a compound used in
detergents).
GEMs and environmental safety
T h e g e n e t i c a l l y e n g i n e e r e d m i cr o o r g a n i sm s
(CEMs) have now become handy tools of
biotechnologists. The risks and health hazards
associated with the use of CEMs are highly
controversial and debatable issues.The fear of the
biotechnologists,and even the general public is
that the new organism (CEM), once it enters the
e n v i r o n m e n t , m a y d i s t u r b t h e e c o l o gi ca l b a l a n ce
and cause harm to the habitat. Some of the CEMs
may turn virulant and become genetic bombs,
causing great harm to humankind.
 Chapt er59 : B IO D EC R A D A T IOAN
N D B IO R E ME D IA TION 727

Because of the risks involved in the use of
GEMs, so lar no GEM has been allowed to enter
the environmental fields. Thus, the use of CEMs
has been confined to the laboratories, and fully
controlled processes of biodegradation (usually
employing bioreactors). Further, several pre-
ca utio na ry meas ur es ar e t ak en while c r ea t i n g
GEMs, so that the risks associatedwith their use are
min imal.
Some researchersare of the ooinion that CEMs
will create biotechnological wonders for the
environmental management of xenobiotics, in the
next few decades.This may be possible only if the
a ssociate d risk s of eac h CEM is t hor oug h l y
evaluated, and fully assured of its biosafety.
result is that there occurs biostimulationby effective
b i o r e m e d i a t i o no f p o l l u t e d s o i l o r w a s t e l a n d .
Bioaugmentation ln soil biorennediation
Addition of specific microorganisms to the
polluted soil constitutes bioaug,'nentation. The
pollutants are very complex molecules and the
native soil microorganisms alone may not be
capable of degrading them effectively. The
examples of such pollutants include
p o l y c h l o r o b i p h e n y l s ( P C B s ) ,t r i n i t r o t o l u e n e ( T N T ) ,
polyaromatic hydrocarbons (PAHs) and certain
pesticides.
Based on the researchfindings at the laboratory
level (with regard to biodegradation), it is now
possible to add a combination of microorganisms
referred to as consortium ar cocktail of
m i c r o o r g a n i s m s ,t o a c h i e v e b i o a u g m e n i a t i o n .

With the development of genetically engineered

microorganisms(CEMs), they can be also used to
bioaugment soils for very efficient bioremediation.
Due to industrialization and extensive use of But the direct use of CEMs in the soils is associated
insecticides, herbicides and pesticides, the solids with several risks and health hazards.
and waste lands worldover are getting polluted.
The most common pollutants are hydrocarbons, T E C H N I Q U E S O F S O I L
c h lorin ate d so lv ent s , poly c hlor obipheny ls a n d B I O R E M E D ! A T I O N
meta ls. The most commonly used methods for the
Bioremediation of soils and waste lands by the bioremediation are soils are in situ bioremediation,
use of micro org anis m sis gaining im por t anc e in th e land farming and slurry phase bioreactors.
'ecent years. In fact, some success has been
ln silu bioremediation of soils
.eported for the detoxification of certain pollutants
e g hydrocarbons)in the soil by microorganisms. ln situ bioremediation broadly involves the
biological clean-up of soils without excavation.
Bioremediationof soils can be done by involving
This technioue is used for the bioremediation of
nvo principles-biostimulationand bioaugmentation.
sub-surfacesof soils, buildings and road ways that
are polluted. Sometimes, water (oxygenated) is
Bio stirn ula tion in s oil bior er nediat ion
cycled through the sub-surfacesfor increasing the
Biostimulationbasically involves the stimulation efficiency of microbial degradation.There are two
of microorganisms already present in the soil, by types of in situ soil bioremediation techniques-
,,a riou smea ns. T his c an be done by m any way s . bioventing and phytoremediation.
. Add ition o f nut r ient s s uc h as nit r ogen a n d Bioventing l This is very efficient and cost-
p no sp no rus. effective technioue fof the bioremediation of
. Supplementationwith co-substratese.g. methane petroleum contaminated soils. Bioventing involves
added to degrade trichloroethylene. aerobic biodegradation of pollutants by circulating
air through sub-surfaces of soil. Although, it takes
. Addition of surfactants to disperse the
some years, bioventing can be used for tl-re
hydrophobic compounds in water.
degradation of soluble paraffins and polyaromatic
Rddition of nutrient and co-substratespromote hydrocarbons. The major limitation of this
rnicrobial growth while surfactants expose the technique is air circulation which is not always
hvdrophobic molecules. In all these situations,the practicable.
72'|
Nutrients
Pollutedsoil
Sand layerwith
mrcroorganrsms
Liner
Fig. 59.6 : A diagrammatic representation of
landfarming system.
Phytoremediation : Bioremediation by use of
planfs constitutesphytoremediation.Specific plants
are cultivated at the sites of oolluted soil. These
plants are capable of stimulatingthe biodegradatron
of pollutants in the soil adjacent to roots
(rhizosphere). although phytoremediation is a
c heap and env ir onm ent alf r i e n d l y c l e a n - u p p r o c e s s
for the biodegradation of soil pollutants, it takes
severaryears.
Landfarming in soil bioremediation
Landfarming is a technique for the
bioremediation of hydrocarbon contaminated soils.
A diagrammatic representation of land farming
system (also referred to as solid phase soil reactor)
is depicted in Fig. 59.6.
The soil is excavated, mixed with
microorganisrnsand nutrients and spread out on a
liner , jus t below t he pollut e d s o i l . T h e s o i l h a s t o b e
regularly ploughed for good mixing and aeration. lf
the soil is mixed with compost and/or temperature
is increased the efficiency of biodegradation
increases.Addition of co-substrates,and anaerobic
pretreatment of polluted soils also increases the
degradation process.
Landfarming has been successfullyused for the
bior em ediat ionof s oils pol l u t e d w i t h c h l o r o e t h a n e
benzene, foluene and xylene. The last three
compounds are often referred to as BIX aromatics.
B IOTECHNO LO CY
Slurry-phase bioleactors in soil
bioremediation
Slurry-phasebioreactorsare improved land
farming systems.In these cases,the excavated
polluted soil is subjected to bioremediation under
optimally controlled conditions in specifically
designed bioreactors.Due to a close contact
betweenthe xenobioticsand the microorganisms,
and the opti mal condi ti ons (nu t r ient supply,
temperature, aerationetc.),the degradationis very
rapid and efficient. Slurry-phasebioreactors,
however,are not suitablefor widespreadusedue to
high cost.
BIOREMEDIATION OF GROUND WATER
E nvi ronmentalpol l uti on al so re sult s in t he
contamination of ground water at several
pl aces.The commonl yfound pol l u t ant sar e t he
petroleumhydrocarbons (aliphatic,aromatic,cyclic
and substitutedmolecules). Bioremediationof
groundwater can be carriedout by two methods
pump- and -treat tecl-rniqueand biofencing
techni que.
Pump. and .treat technique for
bioremediation of ground watel
Bioremediation of undergroundwater by pump-
and -treattechnologyis mostlybasedon physico-
chemi cai pri nci pl esto remove the pollut ant s.
The treatmentunits are set up abovethe ground.
Strio columns and activatedcarbon filters can
remove most of the ground water pollutants.
Treatedwater is recycledthrough injection well
severaltimes so that the pollutantsare effectively
removeo.
For removal of certain organic pollutants,
biologicalreactors(bioreactors)
haveto be installed
(Fig.59.74).For instance,
for the biodegradationof
tetrachloroethane,a bioreactor with granular
methogenicsludge is found to be effective.In
recent years, bioreactors with both aerobic
and ana.erobicbacteriahave been developedfor
better bioremediationof highly polluted ground
waters.
It is however,not possibleto achievegoodclean
up of groundwaterby pump-and -treattechnology,
for various reasons(sub-surface heterogenecities,
stronglyadsorbedcompounds,low permeabilityof
pollutantsetc.).
 A N D B IO R EME D IA TION
Chapt er59 : B I O D EGR AD AT ION 729

Activated
carbonfilter
Nutrients
Clean
Soil sudace water

Clean
water Watertable Injection
weil
Flecovery---;, Injection
well well

I
Bioactivezone
Pollutedground
waler
Directionof waterflow

(A) (B )

Fig. 59.7 : Bioremediation of ground water (A) Pump- and lreat technique (B) Biofencing technique.

Biofencing technique for gradient edge of a contaminatedground \ /ater area.

bioremediation of ground water Nutrients are iniected through a well to the
bioactive zone (Fig. 59.78). As the ground rvater
Biofe ncing is an im pr ov ed t ec hnique f or t h e passesthrough the bioactive zone (by the impact of
lioremediation of ground water. lt consists of natural direction of flow), the pollutants are
installation of a bioactive zone at the down- biodegraded, and clean ground water comes out.
,<-"ome of the environmental issuesattract world-
- - i wic le at t ent ion,as t hey ar e t h e g l o b a l p r o b l e m s .
These include green house effect and global
warming, depletion of ozone and acid rain. rhe
global env ir onm ent al pr obl e m s a l o n g w i t h t h e
env ir onm ent al s us t ainabilit va r e b r i e f l v d e s c r i b e o
in t he f ollowing pages .
GREEN HOUSE EFFECT
AND GLOBATWARMING
;Fi€Efii t''i+U$E GegE$
The gases that cause green house effect are
collectively referred to as green house gases
(Fig. 60.1). The most important green house gases
are carbon dioxide (55'/"), methane (15n/,,) ,
chlorofluorocarbons (17o/o), nitrous oxide (6"t,,
a n d s e v e r a l o t h e r g a s e s I i k e c a r b o n m o n o xi d e
n i t r o u s o x i d e , o z o n e , s u l f u r d i o x i d e , fl u o r i n e
b r o m i n e a n d i o d i n e ( 7 " k ) . T h e p r e s e nce o f w a te r
v a p o u r i n t h e a t m o s p h e r ei n a s s o c i a t i onw i th g r e e r '
h o u s e g a s e s s i g n i f i c a n t l y c o n t r i b u t e s to g l o b a l
warmrnS

':..t)o-;iii;.;b ;i qtr,er-lr E!{rLE5*Seseii

The earth receivessolar energy from the sun in
t he f or m of s hor t wav e r adi a t i o n s ( m o s t l y v i s i b l e The important sources of major green house
light ) . This s olar ener gy is ab s o r b e d b y t h e e a r t h 's g a s e s a r e g i v e n i n t h e T a b l e 6 0.1. Fo ssi l -
surface and emitted into the space as long-wave f u e l c o m b u s t i o n , d e c o m p o s t i o n of o r g a ni c
( inf r a r ed) r adiat ions . lf t he i n c o m i n g r a d i a t i o n s wastes, deforestation and industries largelr
f r om t he s un and t he out goin g r a d i a t i o n sf r o m t h e c o n t r i b u t e t o g r e e n h o u s e g a s e s . C h l o r o fl u o r o -
earth are equal, then a good balance between the c a r b o n s ( C F C s )a r e s y n t h e t i cc h e m i c a l sw i d e l y u se c;
absorbed and emitted energy by the earth is in the preparation of refrigerants, solvents anc
m aint ained. But t his does no t h a p p e n . aerosol propellants CFCs are dangerous
environmental pollutants that significantlr
The inc om ing s hor t - wav e r a d i a t i o n s ( f r o m t h e
contribute to green house effect. The glotra
s un) c an eas ily pas s t hr ough th e a t m o s p h e r ew h i l e
w a r m i n g p o t e n t i a l o f C F C s i s a r o u n d 7 ,OOO -
the outgoing long-wave radiations (from the earth)
c o m p a r i s o n t o C O , t a k e n a s 1 , m e t h a n e l .l a n i ;
ar e par t ially abs or bed by c e r t a i n g a s e s r n t h e
nitrous oxide 260
atmosphere. Green house effect refers to the
phenomenon af retention of earth's heat by the T h e r e l a t i v e c o n t r i b u t i o n o f t o t a l g r e e n h o L r ..
atmosphere The conseguence of green house g a s e sf r o m t h e m a j o r c o n t r i b u t i n g s o u r ce s i s g i r e '
effect is global warming. in the next page.

730
 Cha ote r 60 : G LO BAL ENVI RO NM ENI AL PR O B L E N 4 S 731

2. Changes in sea level : Due to a rise in

temperature, there occurs thermal expansion of
o c e a n s a n d m e l t i n g o f g l a c i e r s , i c e s h e e t sa n d i c e
c a p s . T h e n e t r e s u l t i s a n i n c r e a s ei n s e a l e v e l . I t i s
estimatedthat during the last 100 years,the average
sea level has risen by about 20 cm. As per the
present prediction, the sea level is expected to rise
b y 2 0 3 0 c m i n t h e n e x t 2 - 3 d e c a d e s .R i s e i n s e a
l e v e l s m a y b e d i s a s t r o u st o s o m e l o w - l y i n g a r e a s
e g . N e t h e r l a n d s ,M a l d i v e s .

3 Water irnbalance : The warmer world would

have lesswater available on the earth and this may
lead to water crisis.

4. Crop yiefd : Due to a rise in temperature,the

a g r i c u l t u r a lp r o d u c t i o n m a y b e r e d u c e d .T h e r e m a y
also occur dislocation of crop lands. Some
researchershowever, predict that the crop yield
may be higher in some areas due to increased
Fig. 60.1 : Approximate contribution of green house availabilitv of COr.
gases in global warming (CFCs-Chlorofluorocarbons)
5 Ecosystem : Some disturbances in tne
ecosystem and the existence of living organisms
l-ossil-tuelcombustion - 57% may not be ruled out.
a nd a na er obic dec om pos it ion
o f org an ic s
Ch loro fluor oc ar bons - 17% Tlrrs 60.1 lnportant sources of major
Agriculture - 1 5 ' /" ' green house gases
Deforestation -B% Crecn house gas Sources
Others _3%
Carbondioxide (CO2) Fossil-fuel
burnihg
Deforestation
Effects of green house g a s e s a n d Respiration
global war m i n g
fromindustries
Emissions
The natural occurrence of green house effect is (CH+) Anaerobicdecomposition
of
rssential for the existence of life on earth. For organic
wastes
:astance,it is only due to the presenceof CO, and Gas,oil andcoalproduction
,\ater vapour, the mean temperature of the earth's Burningof biomass
is a rou nd 17"C. ln t heir t ot al abs enc e ,t n e Wetland cultivation
'u rface
:emperaturewould have been around -1 7oC,where
Chlorofluorocarbons Refrigerants
,fe can no t exis t !
(CFCs) Solvents
The ma in pr oblem is t he ev er inc r ea s i n g propellants
Aerosol
concentrations of the green house gases in the Foampackagings
a tmosph ere th at ar e r es ult ing in ir r ev er s ible a n d
Nitrousoxide(NrO) Fertilizers
rig hly da ng ero us c lim at ic c hanges . The ef f ect s o f
Naturalsoils
qlobal warming contributed by green house gases
FossilJuel
combustion
a re liste d be low .
.l Ozone(O3) Photochemical
reactions
in
. Increase in temperature : There occurs a troposphere
q en era lwarmin g up of t he at m os pher edue t o gr e e n
house gases. lt is estimated that the temperature
Carbonmonoxide(CO) FossilJuel
combustion
increasesby about 0 3'C per decade.
Biomassburning
732 B IOTE CHNO LO CY

6 Hum an healt h: The c h a n g i n g c l i m a t i c 5. Other energy sources : Ceneration of energy

c ondit ions , due t o global war m i n g , m a y a d v e r s e l y from different sourcesthat do not produce CO, are
af f ec t hum an healt h For in s t a n c e , t h e r i s k o f d e s i r a b l e T h e s e i n c l u d e h y d r o e l e c t r i ce n e r g y,so l a r
s pr eadingm ajor t r opic al dis ease s( m a l a r i a ,f i l a r i a s i s , energy, wind energy, geothermal energy and tidal
s c his t os om ias isdengue
, f ev er , y e l l o w f e v e r ) w o u l d ener8,y.
be higher .
5 Minimal use of CFCs : As already stated
M EASURES TO CO NTBO L G R E E N c h l o r o f l u r o c a r b o n s s i g n i f i c a n t l y c o n t r i b ute to th e
HO USE EFFECT green house effect.They contribute to about 17% oi
green house gases. CFCs are about 4,000-7,000
The major contribution of green house gases t i m e s m o r e p o t e n t t h a n C O , i n c a u s i n gg r e e n h o u se
comes from the developed countries. Thus, with a effect. For these reasons, manv environmentalists
wor ld' s populat ion of about 1 5 %, t h e s e c o u n t n e s
advocate a halt for the production of CFCs. This is
contribute to as much as 65'k of green house gases not practicable,since CFCs have become a part of
This is m os t ly bec aus e of in d u s t r i a l i z a t i o n a n d t h e l i f e s y s t e m o f a m o d e r n m a n . H e nce , th e tr
c hanging lif e s t y lesof people. lt i s t h e r e f o r e ,n a t u r a l
minimal use is advocated.
to expect that the developed countries significantly
c ont r ibut e ( f inanc es and r e s o u r c e s ) f o r t h e A s i s e v i d e n t f r o m t h e a b o v e d i s c u ssi o n , th e
c ont ainm ent of global war m ing . green house effect can be minimized by reducing
the addition of CO, CFCs, methane and nitrous
Ther e is a wor ldwide c onc e r n o n t h e a l a r m i n g
oxide to the environment.
im pac t of global war m ing du e t o g r e e n h o u s e
effect Several steps are being taken at the
int er nat ionaland nat ional I ev e l s f o r t h e p r o t e c t i o n
of env ir onm ent wit h par t ic ula r r e f e r e n c et o g r e e n
house effect.
O z o n e ( O r ) i s a b l u e c o l o u r e d g as w i th a
Some of the important approaches for the p u n g e n t s m e l l . l t f o r m s a t h i c k l a y e r i n th e
management of green house effect with special stratosphereof the atmosphere(16 to 40 km). The
referenceto biotechnology are briefly describeo concentration of ozone is in the range of 2-B ppm
I . Renewable forms of energy : The various ( p a r t s p b r m i l l i o n b y v o l u m e ) d e p e n d i n g o n th e
forms of energy generation (fossil-fuelburning, fuel d i s t a n c e .
wood c om bus t ion) ar e int im a t e l y l i n k e d t o t h e
pr oduc t ion of gr een hous e gas e s .S w i t c h i n g o v e r t o Generation of ozone
renewable froms of energy (the best being solar O z o n e i s p r o d u c e d n a t u r a l l y w h e n oxyg e n i s
s our c e) is highly des ir able. d i s s o c i a t e db y s o l a r u l t r a v i o l e t r a d i a t i o n s ( 8 0 - 2 4 0
2 Reforestation : Deforestation is major nm).
c onc er n. Cr owing of plant s wh e r e v e r p o s s i b l e a n d UV radiation
reforestationare the need of the hour. Plants will (80-240 nm)
O r 4 O +O
take up atmospheric CO., and generate Or, and
t hus s ignif ic ant ly help t o r edu c e t h e g r e e n h o u s e O + O, + M --------------)O, + M
effect. ( M r e p r e s e n t sa t h i r d b o d y m o l e c u l e l i ke N ,
3. Development of energy-efficient industries : or O2).
At t ent ion s hould be dir ec t ed f o r t h e t e c h n o l o g i c a l
advancement of industries rvith low energy Beneficial affects of ozone
consumption. And u'herever possible, renewattle
O z o n e i s c a p a b l e o f a b s o r b i n g ul tr a vi o l e t
s our c esof ener gv s hould be u s e d .
radiations frorn the sun (200-300 nm) so that they
4. Nuclear power industries : Cost-effective do not reach the earth and cause health hazards
ins t allat ionof nuc lear power s ta t i o n si s a d v o c a t e d . ( d e s c r i b e dl a t e r ) .I n t h i s p r o c e s s ,o z o n e d isso ci a ti o n
This will c er t ainly help t o s o l v e e n e r g y c r i s r s , o c c u r s a s f o l l o w s .
bes idesm inim iz ing t he gr een h o u s e e f f e c t . B u t c a r e UV radiation
s hould be t ak en f or a s af e d i s p o s a l o f n u c l e a r (200-100nm)
^ r-O2-O
O
wastes
 Chaot er60 : C L O B ALEN VIR O N M EN T AL
P R OB LE MS 733

Thu s, o zo ne is c ons t ant ly being gener at e da n d Other systems

destroyed in the stratosphere.
Besides the major systems described above,
there are severalother compounds that can destroy
DEPL ETION O F O ZO NE
o z o n e A g r o u p o f c h e m i c a l s n a m e l y h a l o n s ,w i d e l y
The re are a num ber of pollut ant gas es i n t h e r - t s e di n f i r e e x t i n g u i s h e r s ,a r e i m p o r t a n t i n t h i s
ox ide ( NO ) , nit r ousox ide ( N r O ) , r e g a r d T h e m a j o r h a l o n s a r e b r o m o c h l o r o f l u r o -
stra tosph ere - nit r ic
ch lorin e (Cl) and c hlor of luor oc ar bons( CFC s l i l <e c a r b o n s a n d b r o m o f l u o r o c a r b o n s .
ch loro fluo rom et haneor f r eon) t hat c an r eac t w r t h
ozone to produce oxygen. The net result is that the O Z O N E H O L E
ozone gets depleted in the stratosphere of the
atmo sp he re lt was in l9B7, the first evidence for the
depletion of ozone over the entire Antarctic content
The re ar e t hr ee m ajor m ec hanis m s f o r t h e c a n r e t o l i g h t . T h i s i s r e g a r d e da s o z o n e h o l e , a n d
destruction of ozone-nitrogen system, hydrogen the depletion of O, was observed between .l2 to
system and chlorine systern. 24 km altitude. Later evidence indicated that the
occurrenceof ozone hole over Antarctic continent
Nitrogen system is an annual phenomenon, during August and
Ab ou t 60 % of t he oz one des t r uc t ionoc c u r s b y Seotember The factors for the causaticn of ozone
niro ge n system . Nit r or - r sox ide ( N, O ) , pr oduc e d by h o l e a r e n o t clearly known.
the micro bia i ac t ion in s oils and oc eans ent e r s t h e T h e p r e s e n tb e l i e f t h a t t h e c h l o r i n e a n d c h l o r r n e
atmosphereand reachesthe stratosphere Here, in r a d i c a l sa r e m a i n l v r e s o o n s i b l ef o r o z o n e h o l e . l t i s
the presence of light, NrO reacts with nascent feared that more ozone holes mav develoo which
oxygen (O) to form nitric oxide (NO). The latter is is highly dangerous.
a powerful destroyer of ozone.

UV r adialion EFFECTS OF OZONE DEPLETION

N,O + O -_---------+ 2NO
Ozone acts as a filter of ultraviolet radiations
UV r adiat ion from the sun with a result that the hazardous UV
NrO ---------------- NO + O
rays do not reach the earth. fhe biologically
NO + O, ----) NOr + Ot
active UV radiations (UV-B) are in the range of
2.9 x 10-7 nnr to 3.2 x 1O-7nm They are highly
Hydrogen system s e n s t i v ef o r o z o n e d e o l e t i o n .
Approximately 10% of tlre ozone is destroyed UV-B radiationscause several harmful effectsro
by hydrogen system.The hydroxyl (OH) group that t h e l i v i n g ( h u m a n s ,a n i m a l s , p l a n t s ) a n d n o n - l i v i n g
reactswith ozone is mostly derived from water, and (materials) systems on earth. Some of them are
to a lesser extent from methane briefly described.
The re act ions inv olv ing Hr O ar e s hown b e l o w .
Effects on human health
UV radiation
HrO + O 2OH .l
. T h e i n c i d e n c e o f s k i n c a n c e r ( m e l a n o m a )r s
OH + O, -------+ HrO + O, very high in the population exposed to UV-B
radiations. Melanoma is associated with
Ghlorine system o v e r e x p o s u r e t o s u n l i g h t - m o s t l yf o u n d i n p e o p l e
who spend more tinre outdoors. lt is estimatedthar
Chlo roflu or oc ar bons( C. FCsand
) nat ur al c h l o r i n e
with every 17odecreasein ozone layer,there would
ca n a lso de str oyoz one t o a s ignif ic antex t ent .C F C s
b e a n i n c r e a s eo f a b o u t 3 % i n t h e s k i n c a n c e r s o f
on d issociation f or m Cl whic h ac t s on oz o n e , a s
people.
shown below.
UV r adiat ion 2. UV-B radiations may damage DNA and
CFC|2 , CF2CI2+ Cl, + C ! c a u s e m u t a t i o n st h a t m a v r e s u l t i n v a r i o u s t v o e s o f
Cl +Or - ) ClO + O , cancers.
734 B IOTE CHNO LO CY

3. Exposureto UV-B radiation is also associated sulfuric acid and nitric acid, and to a lesser extent
with several other health complications-damageto b y h y d r o c h l o r i c a c i d a n d o r g a n i c a c i d s.
eyes, decreased immune response, increased
incidence of several infections. DEVELOPMENT OF ACID RAIN

As the industrialization worldover increases,

Effects on terrestrial plants
t h e r e i s a n i n c r e a s ei n t h e e n v i r o n m e n t alp o l l u ti o n
1 . Exposureto UV-B radiations in some plants by oxides of sulfur and nitrogen. These gaseous
may result in reduced growth and smaller leaves, pollutants are light and they can be carried away
with a reduced efficiency of photosynthesis. (from the site of production) to hundreds and

2. The qualit y and qua n t i t y o f thousands of kilometers by the winds. Thus, the
foods are
adversely affected. export of the acid forming pollutants may occur
from one region to another region, and from one
3. Retardation in the growth of forests. country to another country. For this reason, acid
rain is a global problem.
Effects on aquatic ecosystems
The following reactionssummarisethe formation
1. The aquat ic lif e is v er y v u l n e i - a b l et o U V - B of acid rain from the oxides of sulfur (SO") and
r adiat ions , par t ic ular ly up t o a d e p t h o f 2 0 m r n oxides of nitrogen (NO*)
clear waters and a depth of 5m in unclear waters.
SO* + O, + HrO ---------) H2SO3 + HrSOo
2. Harmful effectshave been observed in fishes,
{Su lfurous (Su Ifu ric
c r abs , s hr im p and z ooplank t o n s .
acid) a ci d )
3. Ther e oc c ur s a reduction in the
photosynthetic efficiency of phytoplankton NO* + Or+ HrO -------+ HNO2 + HNO,
(Nitrous ( N i tr i c
M EASURES TO CO HTRO L acid) a ci d )
O ZO NE DEPLETI O N
In the development of acid rain, there occurs
The only effective way of controlling ozone diffusion of SOr, and diffusion of HNO, gas into
depletion is the complete elimination of the factors the cloud particles/droplets.
responsible for it. As already described oxides of
nitrogen, chlorofluorocarbons and halons largely EFFECTS OF ACID RAIN
contribute to ozone destruction. These are the
A c i d r a i n d i s t u r b s t h e e n v i r o n m e n t . T h e a ctu a r
env ir onm ent al pollut ant s and r e d u c t i o n i n t h e i r
effects of acid rain depends on the degree of the
pr oduc t ion/ ut iliz at ionwill lar g e l y h e l p t o c o n t r o l
acidity and the nature of the environment-aquatic,
oz one deplet ion.
terrestrial,human population and materials. Some
of the important aspects are described.

Effects on aquatic environment

The aquatic system is very sensitiveto acid rarn.

T h e n o rma lra i n w a te r i s s l i ghtl yaci di cw i th a
With periodical falls of acid rains,the lakes become
p H i n th e ra n g eo f 6 -7 . T h i s aci di tyi s contri buted
more and more acidic
b y th e n a tu ra l l y o c c u rri ng C Oz w hi ch gets
.l
dissolvedin the rain water to form cart.onicacid. . T h e f i s h e s a n d o t h e r a q u a t i c o r g a n i sm s
i n c l u d i n g m i c r o o r g a n i s m sa r e v e r y s e n s i ti veto p H .
CO, + HrO ----------+
H2CO3
At a oH below 4. most of them die.
The lowest pH of the normal rain is around
2. The surving fishes c o n t ai n high
5 .6 .
concentrations of toxic metals such as mercury,
When the pH of the rain water is lessthan 5.5, a l u m i n i u m , l e a d a n d z i n c ( t h e s e m e t a l s fi n d th e r r
it is considered as acid rain. This acidity is e n t r y i n t o l a k e s b y l e a c h i n g o f s u r r o u n d i n g r o cks
predominantlycontributedby two acids namely b v a c i d r a i n ) .
 Chapt er60 : GL OB ALEN VIR O N M EN TAPLR OB LE MS 735

Effects on terrestrial environment Despite the occurrence of a complex and

sometimes dangerous alterations in the
1. Acid rain damagesforestsand all forms of
vegetation. environment,the life processesshould go on,
normal l y as f ar as possi bl e. E nvi ronmental
2. Soil acidification, due to acid rain, may lead sustainabilitybroadly deals with the sustenance
to necrosisof leaves. (support and maintenance)of the environment to
3. Plantsalso accumulateseveraltoxic metals continue life on earth in a normal way.
e. g. alum i n i u m c, a d m i u m,m e rc u ryl,e a d . Biotechnology significantly contributes to
environmental sustainability.There are three
Effects on human health differentapproachesin this regard.
1. Sulfuricacid of acid rain is very dangerous,
1. Pollution protection : Environmentcan be
as it causesbreathingproblems. protectedfrom pollution by using renewableraw
2. Thetoxicmetals(Cd,Zn, Hg,Cu, Al) liberated materials, recycling the processes,appropriate
from rocks and soils by acid rain may ultimately methodsfor degradationof wastes.
reachthe humanbody throughplantsand animals
2. Pollution clean-up : This can be done by
throughfood chain or drinking water. The toxic
employingbiotechnological methodsfor clean up
effectsof thesemetalson humansare well known
of oil spills,detoxificationof contaminatedsoils,
and purificationof water supplies.
Effects on materials
The acid rain causesdeterioration of buildings, 3. Pollution control : Once pollution has
marblerocks,limestones etc. Thus,acid rainshave occurred, biotechnological methodscan be used
becomea big threatto preservation of monuments for control. For instance,heavy metals can be
and culturalheritagesthroughoutthe world. recoveredfrom pollutedwater.
It is a fact that the variousactivitiesof humans
M E A S URES T O C O N T R OL A C ID R A IN are exceedingthe sustainable capacityof the earth.
The most effectivemeasureto control acid rain Increasedindustrializationand urbanisationare
is to drasticallyreduce the productionof acid- adverselyaffecting the environment (with heavy
pol l uti on).
forming gasesi.e. the industrial emissionof oxides
of sulfur and nitrogen should be minimized. The biotechnologists are expectedto improve
Somemeasureshave been takento reducethe the exi sti ng bi ol ogi cal , chemi cal and vari ous
SO, emissioninto atmosphere-desulfurification
of other industrial processes to make them
the fuels used in industries,recoveryof SO, as environmental friendly. They should develop
H2SO4. integratedsystemswhich are efficient,controllable
and clean. lt is the duty of the environmental
More concrete measures need to be taken biotechnologists to achieve a maximum global
globallyfor effectivecontrol of acid rain. environmental safetv.
Eco-Tech : This is a new environmental
technologyconcept.lnternational
Organisationfor
Biotechnologyand Bioengineering
in 1994,defined
Eco-Techas follows :
The various aspectsof environmentand their "Embeddingtechnology into ecosphereand
impacton earth with specialreferenceto human human culture by using the whole range of
beingshave bebn described(Chapter54). Someof biodiversityin a holisticand low-invasiveway in
the importantglobal enVironmentalproblem are order to achievebenefits for humankindobeying
describedin this chapter. ecol ogi cal pri nci pl es" .
 B i otechnol ogy- S oci ety,R i sks,
E thi cs and P atenti ng 739

ON
BIOTEGI{NOLOGY
AruDSOGIETY
 ,\ dvan ce s in biot ec hnology , and lhe i r Ii is a fact that modern technology in various
/-a .rpp lica tion s ar e nr os t f r equent ly as s oc . r a t e d f o r n r s i s w o v e n t i g h t l y i n t o t h e f a b r i c o f o u r l i v e s
' ,iith con troversies Bas ed on t heir per c ept ion t o O u r d a y - t o - d a y l i f e i s i n s e p a r a b l ef r o m t e c h n o l o g y .
: r io techn olo gy,th e people m ay be gr ouped int o l m a g i n e l i f e a b o u t l - 2 c e n t u r i e s a g o w h e r e t h e r e
' r rp e h rn :d r-rl e o nr ies w a s n o e l e c t r i c i t y ,n o r u n n i n g w a t e r , s e w a g e i n t h e
streets,unpredictablefood supply and an expectecl
1 Strong opponents who oppose the new
years Undoubtedly,
- ..clrno log y,a s it will giv e r is e t o pr oblem s , is s u e s l i f e s p a n o f l e s s t h a n 4 0
technology has largely contributed to the present
- :rcl co ncern s h um ans hav e nev er f ac ed bef or e day world we Iive in. Many pepople consider
- .e v
con sid er biot ec hnology as an unnat ur a l
biotechnology as a technology that will improve
- -,in ipu lativetechnology
the quality of life in every country, besicles
) Strong proponents who consider that the m a i n t a i n i n g I i v i n g s t a n d a r d sa t a r e a s o n a b l yh i g h e r
- :techn olo gy w ill pr ov ide unt old benef it s t o tevet.
. iie ty. The y a rgue t hat f or c ent ur ies t he s oc ie t y
- -. safe ly u se d t he pr oduc t s and pr oc es s es o f The probable positive and negative effects oi
,:e cnno log y. b i o t e c h n o l o g y ,w i t h s p e c i a l r e f e r e n c et o d e v e l o p i n g
c o u n t r i e s a r e g i v e n i n T a b l e 6 1. 1
) A neutral group ol people who [ave a
- . .:nced ap pro ac h t o biot ec hnology This gr or - r p
ELSI OF tsIOTEGHNOLOGY
e \ es th at res ear c h on biot ec hnology ( wit h
---. rtory system s ) ,and ex t ending it s f r uit s t o t h e Wh y s o m u c h u p r o a r a n d n e g a t i v i t y t o
. ...'\ sh ou ld be pur s ued wit h a c aut io u s b i o t e c h n o l o g y ?T h i s i s m a i n l y b e c a u s e t h e m a j o r
' -.rach The r is k s and benef it s of t h e p a r t o f t h e r n o d e r n b i o t e c h n o l o g y d e a l s w i t h
: r)l)nrcntsof biot ec hnology m ay not be m uc h g e n e t i c m a n i p u l a t i o n s . T h e s e u n n a t u r a l g e n e t i c
.'--':,n tfrom tha t of any ot her br anc h of s c ience . m a n i p u l a t i o n s ,a s m a n y p e o p l e f e a r , m a y l e a d t o
u n L n C r WnC O n S e q U e n C e s .
E ENEFITS OF BI O TECHNO LO G Y
ELSI is the short form to represent the ethical,
- -.' irr,rits legal and social implications of biotechnology. ELSI
o f biot ec hnology ar e benef ic ial t o t h e
- i,i he alth c ar e,agr ic uit ur e, f ood pr oduc iio n , broadly covers the relationship between bio-
-. -f,ctureof indus t r ialenz y m es and appr opr ia t e t e c h n o l o g y a n d s o c i e t y w i t h p a r t i c u l a r r e f e r e n c et o
- -me nta l managem ent . ethical and legal aspects.

739
744 B IOTECHNO LO CY

r e s e a r c hd e a l i n g w i t h D N A m a n i p u l a ti o nw e r e ve r y
Tnsre61.2 The probablc positive and negative s t r i n g e n ti n t h e e a r l i e r Y e a r s
effects of biotechnology (particularly in the
So far, risk assessmentstudies have failed to
developingcountries)
demonstrateany hazardous propertiesacquired bv
Positiveeffects host cells/organismsdue to transferof DNA. Thus,
the fears of genetic manipulations may be
. lmprovedhealthand life span(throughmoreelfective
unfounded to a large extent. Consequently, there
vaccines
pharmaceuticals, etc.).
has been some r e l a x a t i o n i n t he r e g u l a to r y
. increasedcropproductionandmoreiood. g u i d e l i n e sf o r r e c o m b i n a n tD N A r e s e ar ch( Fo r m o r e
. Decreased
dependence and d e t a i l s , R e f e r C h a P t e r 6 ) .
on importof food,fertilizers
chemicals
I t i s n o w w i d e l y a c c e p t e dt h a t b i ote ch n o l o g y i s
. Enhanced production
biomass for energy. c e r t a i n l y b e n e f i c i a l t o h u r n a n s . B u t i t sh o u l d n o t
. Useo{ plants as bioreactors.
andanimals cause problems of safety to people and
environmerrta , n d c r e a t eu n a c c e p t a b leso ci a l , m o r a l
. lmproved facilities.
toodstorage
and ethical issues. The public fe a r s o f
. Increased
production
ancjhealthof ltvestock. b i o t e c h n o l o g y ,b e s i d e s s o m e o f t h e r i sks,so ci a l a n cl
ethical issuescan be better understoodwith special
Negativeeffects
reference to the follorving
. More.dependence cn developedcounlriesand private
lor and
technology resources 1 T h e r a p e u t i c p r o d u c t s f o r u s e i n h e a l th ca r e
companies
. Increase
in unemployment is
as the labourrequirement 2 . C e n e t i c m o d i f i c a t i o n s o f f o od s a n d fo o d
less i n g r a d i e n t sa n d t h e i r c o n s u m p t i o n .
. Reduction
in natural ecosyslems. 3 R e l e a s eo f g e n e t i c a l l y e n g i n e e r e do r g a n i sm s
andnatural
biodiversity
. Morelegalandfinancial
complications. into the environment.

. Increased o{cashcropsattnecostof foodcrops

growing 4 A p p l i c a t i o n s o f h u m a n g e n e t i c r e se a r ch .
. Production
of moreancimoreherbicide
resistantplants.
RECOMBINANT THERAPEUTIC
PRODUCTS FOR HUMAN HEALTHCARE
Ris k s and et hic s of b i o t e c h n o l o g Y lt is fortunate that there is no serious criticism
a b o u t t h e u s e r e c o m b i n a n t p r o d u c ts fo r m e d i ca l
The m oder n biot ec hno l o g y d e a l s w i t h g e n e t i c
a p p l i c a t i o n s .T h i s i s m o s t l y b e c a u s et h e th e r a p e u ti c
m anipulat ionsof v ir us es ,b a c t e r r a ,p l a n t s , a n i m a l s ,
products and strategies are designed to cure
f is h and bir ds . lnt r oduc t io n o f f o r e i g n g e n e s i n t o
d i s e a s e s , a l l e v i a t e s u f f e r i n g s a n d i m p r o ve th e
v ar ious or ganis m sr ais esc o n c e r n s a b o u t t h e s a f e t y ,
q u a l i t y o f l i f e . F u r t h e r ,t h e p r o d u c t s a r e u se d u n d e r
et hic s and' unf or es eenc on s e q u e n c e s .S o m e o f t h e
the m e d i c a l s u p e r v i s i o n . I n g e n e r a l , th e
popular phr as esus ed in t h e m e d i a w h i l e r e f e r i - i n g
r e c o m b i n a n t p r o d u c t s d e s i g n e d fo r h u m a n
t o ex per im ent s on r ec om b i n a n t D N A t e c h n o l o g y
h e a l t h c a r e a r e m o r e r e a d i i y a c c e p ta b l e b y th e
ar e lis t ed.
p u b l i c C o o d e x a m p l e s a r e t h e u se o f i n su l i n ,
. M anipulat ion of lif e i n t e r f e r o n s , t i s s u e p l a s m i n o g e n acti va to r a n d
. Playing v'rith Cod various vaccines (for more details, Refer Chapters
15 and 16).
. M an- m ade ev olut ion

The m ajor appr ehens i o no f g e n e t i c e n g i n e e r i n g GENETIC MODIFICATIONS A ND

is t hat t hr ough r ec om bi n a n t D N A e x p e r i m e n t s FOOD CONSUMPTION
unique m ic r oor ganis m s o r viruses (either
The overall objectives of genetic modifications
inadv er t ent ly or s om et im e s d e l i b e r a t e l y f o r t h e with referenceto foods are listed
purpose of war) may be developed that wbuld
c aus e epidem ic s and env i r o n m e n i a l c a t a s t r o p h e s . . T o i n c r e a s e t h e q u a l i t y a n d q u a n ti ty o f e xi sti n g
Due t o t hes e f ear s , t he r e g u l a t o r y g u i d e l i n e s i o r foods.
 R IS K SE, TH IC SP, A TE N TIN C
Chapt er61 : B IO T E C H N OL OC Y-S O C I E TY 741

. To producenew products. the commercialpreparations revealedthe presence

. T o im pr o v eth e fi n a n c i a re
l tu rn s . of certainmetabolicderivativeof tryptophan.The
mosti mportant amongthemw as 1-1' ethyl ene
- bi s
Each country has its own regulations for (tryptophan) which was responsible for EMS(based
introducingfoods into the market.For instance,rn on the resul tsof ani mal experi ments). It w as
USA, the Food and Drug Administration(FDA) concludedthatthe pharmaceutical companydid not
regulates the introduction of foods, drugs, take adequatecare for purificationof tryptophan.
phar r nac e u ti c a lasn, d m e d i c a l d e v i c e s i nto tne Consequently, recombinant tryptophan (even
marketplace. w i thout the i mpuri ti es!)
w as bannedfor human
As regards the foods and food ingradients consumpti on
i n U S A .
developed by genetic engineering,the FDA
Bovine somatotropin story
believesthat no new regulationsare needed.Tne
existingregulationsfor the assessment foods for Bovinesomatotropin (BSl alsoknownas bovine
safetyby toxicity,allergenicity and impuritytesting growth hormone),when injectedto dairy cattle,
are adequate.lf the chemicalcompositionof the i ncreasesthe mi l k producti onsi gni fi cantl y.B y
existingfood is alteredby geneticmodification,it recombinantDNA technology,the gene for BST
s houldbe s p e c i fi e da, n d th e n e w p ro d u ctshoul d was cloned and exoressed in E. coli. The
be ac c or di n g l yl a b e l e d . recombinant BST (rBST) so produced, when
To highlightthe public perceptionof genetically iniected to cows was found to increasemilk
productionby 20-25%.
modified foods, some selectedexamplesof food
ingr adient sa n d th e i r a c c e p ta n c e ,o r certatn The effect of rBST was consideredfrom tw<,r
controversiesrelated to their use, are briefly aspects - on the ani mal sand consumers.
des c r ibed. .l
. Effectof rBSTon animals: AdministrationoI
rBSTcan producelocalizedswellingat the site of
Chymosin
injection.Theremay be someotheradverseeffects
Chy m os i ni s m i l k c l o tti n gp ro te o l y ti cenzyme like increasedsusceptibility to infection,decreased
that hydrolyses the milk proteincaseinto produce reproductivecapability.The proponentsof rBST
curd, which in turn is processedinto cheese. argue that these problem could occur even in
Traditionally,chymosinis derivedfrom the stomach normalani mal s.
of calvesin the form of rennet.
2. Effecton humanhealth: The naturalBSTor
B y gen e ti c e n g i n e e ri n g te c h n i q u e s, the even rBSTincreases the body levelsof insulinJike
chymosin gene was cloned and expressedin growth factor-l(lCF-l)which in turn enhancesmilk
E. coli. This resultedin a large scale and cost- producti on. Therei s evi dencethat IC F-lsti mul ates
effectiveproductiohof chymosin.The chemical grow thof cancercel l s.Thi scausesconcernamong
composition,structureand biological activity of the consumers of mi l k producedby usi ngrB S T.
The
recombinant chymosin were identical to the counterargumentis that rBSTinjection,afterabout
chymosinof rennet.FDA gavelicbnseto chymosin 100 daysof lactationbegins,is not associated with
for its commercialuse,and it is now widely used increasedlevelsof ICF-|. The opponentsof rBST
in c hees em a k i n g . strongl yfeel that si ncemi l k i s consumedby most
peopl e, any i nherent ri sk, how ever smal l rs
Tryptophan unacceptable.
I n 1989 -9 0 ,a n u n u s u a l l yh i g h i n c i d e n ceof the , Recombinant BSTwas licensedin USA,for use
disease eosinophilia-myalgiasyndrome (EMS) was i n dai rl ycattl ei n 1994.S omecountri esi n facthave
reportedin USA.EMSis a raredisease with muscular bannedthe use of rBST.There are some people
pain and respiratorycomplications,and may be (particularly amongthe scientists) who believethat
fatal.lnvestigations revealedthatthe victimsof EMS the hue and cry raisedagainstrBSTis more due to
wereconsuminglargequantities of food supplement economicand politicalreasons. lt is fearedthat by
tryptophan (obtained from one company). rBSTuse,the dairy industrymay be controlledby
This tryptophan was produced by genetically large industrialgroupsand the small dairy farms
engineeredmicroorganisms. Chemicalanalysisof may becomeunprofitable.
742 B IOTE CHNO LO G Y

The rBSTstorv is an interestingillustrationof the against the consumptionof CM foods. These

or oblem s s ur r ounding t he u s e o f g e n e t i c people insist that the CM foods should be
en8r neef ln8. speci fi cal llyabel ed.
As regardsthe safetyof CM foods, opinions
RECOMBINANT FOODS AND
rangefrom one extremethat they are absolutely
REL'GIOUS BEL'EFS
safe,and improvehumannutritionto the otherthat
Some of the ethical concerns of the use of they should not be consumedat all. Most of the
recombinani foods are related to religious beliefs, people have opinions somewherebetweenthese
besides food habits. For instance two extremes.

. Transferof pig genes into sheep may offend the

R E LE A S E OF GE N E TIC A LLY
s ent im ent sof lews and M us l i m s . E N GIN E E R E D OR GA N IS MS
o lntroduction of animal genes into fgod plants The releaseof geneticallyengineered organisms
m ay inv it e oppos it ion by s t r i c t v e g e t a r i a n s . (GEOs), also called as genetically manipulated
o Transferof human genes to food animals may be organisms(GMOs)into the environmenthasbeena
unacceptable to some people. controversialissue;and continuesto be so. lt rs
fearedthatthe releaseof CEOsintothe environment
o Feeding of hum an gene- c o n t a i n i n g o r g a n i s m s coul d have far-reachi ng consequences. This is
to animals sounds in bad taste (at present, the due to the fact that the living CEOs proliferate,
genetically modified yeast that produce persist, disperse,and sometimesmay transfer
recombinant proteins after their use, are fed to their DNA into otherorganisms. lt is furtherfeared
anim als ) . that there existsa possibilityof CEOs displacing'
The r eligious gr oups , in ge n e r a l , a r e s e l e c t i v e the exi sti ng
organi sms,besi des crea t ing new
about the foods to be eaten. However, they are not
species. This may lead to severeenvironmental
so rigid when it comes to the use of medically-
oamaS e.
der iv ed pr oduc t s .For ins t anc e - J e w s a r r d M u s l i m s For the reasonsstated above, the regulatory
m ay ac c ept pig- der iv ed ins ulin f o r u s e i n d i a b e t i c authoriesare very careful in permitingthe field
patients. This is due to the fact that all religious the releaseof CEOsinto the
trailsof CEOs.Further,
faiths consider human life is the most valuable, environmenthas to be carefully monitoredand
and its preservation the first priority. Further, the recorded.
general belief is that the human body is violated
only by oral consumption and nclt by injection or lce.minus Pseudomonas syringae
surgical interventions.
A geneticallymodified strain of Pseudomonas
syringae was the first CEO that was given
Eating genes everyday! .l
for fi el d trai l s(i n 987).Thi sor ganism
permi ssi on
Everyday,we eat plants and animals and various i s a geneti cal lengi
y neered i ce-mi nusstrain,when
products derived from them, besides a large sprayedonto the leavescould preventfrostdamage
number of bacteria. ln other words, we regularry to the plants(ReferChapter50).
consume genetic material,the DNA, organized into
genes in various organisms! So far no one has Field trails with other GEOs
attempted to categorize genes as vegetarian and
non-vegetarian,as we cio for foods!!
A number of open-field trails have been
conductedwith severalCEOsduringthe pasttwo
decadesor so. The studiesconcluded that the
ARE GM FOODS SAFE?
geneticallymodifiedmicororganisms do not persist
The production of transgenicplants and anima-rs in environmentfor long, do not transferthe genes
by genetic engineeringtechniqueshas now become into other organism and do not exhibit any
routine. These organismswill enter the food chain abnormal biological functions.Thus, the inifial
in the form of genetically modified foods (CM apprehensionson the use of GEOs appear to be
foods). Some social and environmental groups are unfounded.
 Y IS K SE, TH IC SP, A TE N TIN C
Chapt er6t : B IOT E C H N OL OC Y-S O C IET R 743

Release of transgenic o Cenetic testing and screening tbr diseases

plants and animals . Cenetic portifolios
The transgenicplantsdevelopedfor higher quality . Human gene therapy.
and quantity of foods, in general,are more liberallv
permittedto go to fields. lt is generallybelievedthat Genetic testing and screening
the transgenicplants do not significantlydiffer from
the natural cultivars (traditionalplants) obtained by Techniquesare now availablefor prenataltesting
plant breeding experiments to specificallydetect whether a fetus carriesgenetic
defects. This will help the parents to be better
Ihe transgenic Bt-plants such as cotton, corn,
prepared for the future baby.
soybean and potato were approved for cultivation
in IJSA. However, some countries did not allow Bt- The negativeaspectof prenataltesting is that the
p lan ts in the ir fie lds e. g. Bt - r ic ewas not allowed r n couple may opt for abortion even for a minor
Ph ilipp ine s,Bt-c ot t on in Fr anc e. genetic defect or sometimes for gender bias
Th e tran sg en icanim als hav e not pos ed as big a
pro ble m as th e t r ans genicplant s .This is due t o th e Genetic portifolios
fa clth at a nima ls c an be m uc h eas ily ident if ied a n d
The elucidation of the entire human genome
co nta ine d (sin cec opulat ion is anir nalsc an be do n e
sequence and identification of genes has no'rrr
as d erised , u nli k e in plant s wher e pollinat ion i s
become a reality. lt may soon be possible to have
difficult to con t r ol) . A m ajor it y of t r ans ge n i c
individual genetic portifolios that will diagnose
an imals a re u se d f or m edic al pur pos es ,henc e t h e y
future health complications e.g. risk for cancer,
are generally appreciated. But the major problem
heart disease. The genetic portifolios (based on
fo r tra nsge nica nim als c om es f r om anim al ac t iv is t s .
the genes) will fortell lhe individuals' future
Many people, and also some Covernments, are which is now,being predicted through stars
su so icio us of the us e of CEO s due t o v ar io u s (astrology).
reasons-risks, societal beliefs and economic
concerns. Cenetic portifolios of individual may pose
certain problems with regard to marriages,
Biological warfare i n s u r a n c e s .Wh o w o u l d l i k e t o b e a s p o u s e o f
someone who will soon be a victim of cancer or
The most serious hazard associatedwith genetrc
h e a r t a t t a c k ? Wh i c h i n s u r a n c e c o m p a n y w o u l d
en gin ee ring is t he c ons t r uc t ion of har m f uI
insure a person with a very high risk of diseases?
b iolo gical ag en t s ( v ir us es , m ic r oor ganis m s )eit h e r
Many ethical committes are of the opinion that
deliberately or otherwise. However, so far there
i n s u r a n c ec o m p a n i e s s h o u l d n o t r e q u i r e , o r s h o u l d
have been no records of any new infectious agents
not be allowed to have access to individuals'
created by recombinant DNA technology.
genetic portifolios.
Most of the countriesof the world are signatories
to the Bio log ica lW eapons Conv ent ionso{ 1972. A s Human gene therapy
a signatory, a nation pledges 'never to produce
micro bia l o r o t her biologic al agent s , or t ox i n s , Theoretically, correction of genetic defects is
whatever may be their method of production, for possible by gene therapy. The present status of
use in wars'. Many people are, however concerned h u m a n g e n e t h e r a p y h a s b e e n d e s c r i b e d ( C h a p t e r
a bo ut the po ssible us e of gene m anipulat ions f o r 13). From the ethical perspective,gene therapy
milita ry pu rpo se s . involving introduction of genes into a patient rs
comparable to the practice of transplationof organs
APPL ICATIONS O F HUM AN (e.g., heart, liver, lungs). Therefore, there is not
GENETIC TDNA RESEARCH much controversy over gene therapy, as long as it
The ultimate goal of advances in biotechnology is intended to be used to alleviate serious medical
is for the be ne f it of m ank ind ( eit her dir ec t o r disorders. However, the gene therapy must lle
indirect). Biotecl.rnology largely contributes to u n d e r a c l o s e s u p e r v i s i o nt o s a t i s f ym e d i c a l , l e g a t ,
hu man ge ne tic r es ear c h inv olv ing t he f ollowi n g ethical and safety implications, besides addressing
t h e public concerns
areas
744 B IOTECHNO LO CY

Germ 'line gene therapy : This is not being body. B y thi s approach,i t mi ght be one day
c ar r ied out at pr es entdue t o t e c h n i c a l , e t h i c a l a n d possi bl eto repai rti ssuedamagesan d cur e m anv
s oc ial r eas ons .M anipulat io n o f g e r m c e l l s w i l l l e a d di seasese.g. P arki nson'dissease, m ellit us
di ab et es
to serioi.rsproblems and complications.
At one international meeting on biotechnology
the following was the final message given to
biotechnology companies on the applications of
genet ic engineer ingt o hum a n s .

'Provide the information and listen to the Biotechnologyinvolves the production of a large
publiC. n u m b e r o f c o m m e r c i a l p r o d u c t s of e co n o m i c
importance. Broadly, biotechnology inventions can
HUMAN EMBRYONIC STEM CELL be categorized into two forms - products and
RESEARCH orocesseS.
The hum an em br y onic s t e m c e l l ( E S C ) l i n e s
BIOTECHNOLOGY PRODUCTS
wer e es t ablis hec iin Nov em b e r 1 9 9 8 . T h e s e c e l l s
are capable c-rfgiving rise to any human cell type. 1. living entities : The various forms of life-
ESC lines open t he pos s ibi l i t i e so f t r e a t i n g d i s e a s e s existing and life-supporting systems derived from
with cell therapy. f)isordersthat involve the loss of l i v i n g o r g a n i s m s a r e r e g a r d e d a s l i vi n g e n ti ti e s.
nor m al c ells s uc h as diabe t e s m e l l i t u s , P a r k i n s o n 's T h e s e i n c l u d e m i c r o o r g a n i s m ,a n i m a l s a n d p l a n ts,
dis eas e, Alz heim er ' s dis e a s e c o u l d p e r h a p s b e c e l l l i n e s , o r g a n e l l e s ,p l a s m i d s a n d g e n e s.
corrected with celltherapy. This may however, take
m or e t im e. 2. Naturally occurring products : The primary
a n d s e c o n d a r y m e t a b o l i t e s p r o d u c e d b y l i vi n g
There are many ethical and legal issuesinvolved s y s t e m se . g . a l c o h o l , a n t i b i o t i c s .
in ESC research, besides several obiections raised li

by the public. In fact, the U.S. FederalCovernment BIOTECHNOLOGY PROCESSES

has banned the use of federal funds for human
embryo researchJor over 20 years.At present,most Biotechnology involves development of several
of research on ESC is being supported by private processes-isolation, p u r i f i c a t i o n , cu l ti va ti o n ,
companres. b i o c o n v e r s i o n e t c . N o v e l , i n n o v a t i v e, si m p l e a n d
cost-effective processes are developed for the
CLO NI NG HUM ANS ? isolation and creation of the above products. Some
examplesare listed
After the cloning of the sheep Dolly (in February,
1997), some Broups of researchers naturally o Production of monoclonal antibodies
became irrterestedto eplore the possibilities of o Bioconversion of sugar to alcohol
c loning hum ans . The v e r y t h o u g h t o f h u m a n
c loning has bec om e a h i g h l y c h a r g e d a n d . M i c r o b i a l p r o d u c t i o n o f a n t i b i o t i c s.
controversial issue.
WHAT IS A PATENT?
A s uc h, c ons ider ingt he b i o m e d i c a l e t h i c s , m o s t
of the countries have banned research related to A patent is a government (or patent office) -
hum an c loning. lt c annot b e p r e d i c t e d a t p r e s e n t issued document that allows the holder (patentee)
whether some day human cloning may become t h e e x c l u s i v e r i g h t t o m a n u f a c t u r e ,use , o r se l l a n
inv it able! invention for a defined period, usually 20 years.
The patent may be regarded as a legal document
Scientists who were aw,arded Britain's first
safeguarding the previleges and rights of an
lic enc e f or hum an c loning h a v e r e c e n t l y ( 2 0 0 5 )
invention/inventor.
created an early stageof human cloned embryo by
using nuclear transfer.The researchers/as such/ are The very purpose of patenting in biotechnology
not interestedto make babies by cloning. Instead, is to ensure the iust financial returns for those who
they wish to create test-tube embryos to supply h a v e i n v e s t e d h e a v i l y - t h e f i n a n c es, i n te l l e ctu a l
stem cells that can give rise to every tissue in the a b i l i t i e s , b e s i d e st h e h a r d w o r k .
Ch APICT6 1 : B I O TECHNO LO CY- SO CI ETY R I S K S ,E T H I C S ,P A T E N T I 'N C 745

INTEL LECTUAL PRO PERTY RI G HTS t h e o n 1 1 ,i n d i v i d u a l i n t h e w o r l d w i t h m o r e t h a n

1000 patents to his credit, once said about
Patent is the most important form of intellectual
p a t e n t i n g 'a n i n v i t a t i o n t o a l a r v s u i t '.
property rights (lPRs) in biotechnology. The other
lPRs are trade secrets,copyrigl'rts,and trade marks
Patenting in different countries
'l-he prrvate
Trade secrets : information
Patenting laws are very complex and vary
p erta inin g to s pec if ic f or m ulat ions and t ec hn i c a l
between different countries. lt is interestingto note
proceduresthat a company wishes to protect from
that a patent application rejected in one country
others are regarded as trade secrets. The best
may be given patent in other country. The best
l<nown trade secret is the formuia for coca-cola.
e x a m p l e i s t h e p i 'o d u c t i o n o f r e c o m b i n a n t h u m a r r
On lv five oe rs ons in t he wor ld ar e s aid t o k n o w
tissue plasminogen activator (tPA) in E. coli.
th e fo rmula whic h has been k ept i. n a b a n k
Genentech company was awarded patent for tPA
in Atlanta, Ceorgia. The trade secret of coca-cola
p r o d u c t i o n i n l . J S A ,w h i l e i t w a s f i r s t r e j e c t e d i n
h as rema ine d a s ec r et f or m or e t han 100 y ear s !
UK After apoeals and Iegal battles, patent was
Copyrights : The protection of authc-rships f i n a l l y g r a n t e d i n U K a l s o
o f p ub lish ed w, or k c om es under c opy r ight s o f
lPRs. Patenting microorganisms

Trade marks : The specific symbols or words to A g e n e t i c a l l ye n g i n e e r e ds t r a i n o f t h e b a c t e r i u m

id en tify a pa r t ic ular pr oduc t or pr oc es s o { a Pseudomonas called superbug'was the first rnicro-
comDanv constitute trade nrarks. organism to he patented Superbug is capable of
breaking down the rnultiple impuries of crude oil.
THE PROCESS O F PATENTI NG I t t o o k a l m o s t 1 0 y e a r s o f p r o c e d u r a l c l e l a y s ,a n c l
As regards patenting of legal battles to get ihe superbug patented. Now,
biot€chnology
inventions, the products or processes(described on m a n y m i c r o r g a n i s m sa r e p a t e n t e d .
p.744) are patentable. For patenting,the following
criteria mu st be s at is f ied. Patenting multicellular organasms

. The inven tion m us t be nov el A genetically manipulated mouse namely the

oncomouse was the first and probably, to date the
o lt must be u s ef ul
only animal to be patented in both USA and UK.
. The pa ten t applic at ion m us t pr ov ide t he f u l l T h i s t r a n s g e n i cm o u s e c a r r i e s a g e n e t h a t m a k e s i t
d escrip tion o f t he inv ent ion. s u s c e p t i b l et o t u m o r f o r m a t i o n .

In con trast t o v ar ious ot her t ec hno l o g y

inventions, biotechnology inventions most often
rela te to livin g m at er ials . For t his r eas on, t h e
patenting of biotechnology inventions is often
a ssociate dwith dif f ic ult ies .Pat ent ing,in gener a l , i s
a comme rcia l dec is ion, as s is t edby legal adv ice .
Th e p ate nt applic at ion m us t be pr epar ed by a n
expert with the assistanceof a patent lar,vyer.lt
mu st co nta in t he det ailed inf or m at ion about t h e
ir.rve ntio -n b ac k gr ound,des c r ipt ionex plainingt h e
na tLrre o f th e inv ent ion wit h f igur es a n d
iilustrations,its utility etc The patent office, after a
tho rou gh scru tiny of t he applic at ion, m ay r ejec t o r
grant the patent. lf rejected, the applicant may
appeal to the Patent Appeals Board. lf again
reje cte d, the d ec is ion m ay be c hallenged leg a l l y .
Many a times, the processof patenting becomes a
fru stra ting expe r ienc e. Thom as Edis on, pr oba b l y
Patenting genes?
The human genome projects (HCPs) have
elucidated a!most the entire seouence of human
B e n o m e , b e s i d e si d e n t i f y i n gt h e g e n e s .P a t e n t i n go f
DNA sequences and genes has become a
controversialand debatable issue.Some researchers
a n d i n s t i t u t i o n si n f a c t h a v e a p p r o a c h e dt h e p a t e n t
offices for grant of patents.So far, patents have not
been granted for gehes The mosi important reason
being that the genes are natural, universal and
inherent biological functional units of all
i n di v i d u a l s .
Patenting, and celUtissue donor's rights
Wh i l e d e a l i n g w i t h h u m a n t i s s u e s / c e l l s ,t h e
involvement of the donor of source material
becomes very important. John Moore was a victim
 746 B IOTE CHNO LO CY

of hairy-cell leukemia, a rare form of cancer. His the researchers.Watson and Crick who discovereo
spleen was removed, and Mo cell lines developed. DNA structure never had a thought of patenting!
These cell lines were patented by the researchers
The situation has now changed. A good
and their associatedorganizations.Moore, however
proportion of researchis either being conducted or
was not involved in the patenting.
f i n a n c i a l l y s u p p o r t e d b y p r i v a t e co m p a n i e s/
Moore filed a suit in the court on the ground universities.lt is quite natural for these companies
that he should also be a party to the profits derived to expect returns for their investments.There is a
by us ing his t is s ue.The c our t a l l o w e d h i s c l a i m t o lot of change in the attitude of researchersalso.
share the profits. For many scientists, financial gains have become
more important than academic recognition.
PLANT BREEDERS' RI G H T S Consequently, some people carry out research
secretly and opt for patenting of their discoveries
Agriculture for the first time was included (in
ratherthan publishing.
1994) in the trade-related intellectual property
rights (TRIPS). TRIPS is a major concern for We have to accept the fact that the progressof
dev eloping c ount r ies . r e s e a r c h i s u s u a l l y m u c h f a s t e r i n p r i va te
companies compared to many government
The plant varieties in many countries (not in
o r g a n i z a t i o n s . T h i s m a y b e d u e t o th e h i g h e r
India) are protected through plant breeders' rights
f i n a n c i a l r e t u r n s a n d e f f i c i e n t a d m i n i s t ra ti o n .
(PBR) or plant variety rights (PVR). PVR provides
legal protection to the original breecier,orowner of Probably, the biotechnological research might
t he plant v ar iet y . lt is belie v e d t h a t P B R w i l l not have progressed to the same extent as it is
encourage innovative research and plant breeding today without the dreams of getting the patents by
programmes, in view of the expected financial scientistsand researchfunding organizations.

tl
ill

if
returns.

Plant breeders' rights are comparable to the

patent rights. Under TRIPSagreement,plant breeder
possesses the exclusive rights over the plant
developed. lt prevents the third parties from using
the plant without the owner's consent. A major proportion of research related to
biotechnology is carried out by the developed
Alt hough PBR will inc r ea s e c o m p e t i t i o n a n d countries. The fruits of biotechnology are probably,
development of good plant varieties, the owner's more useful and relevant to developing countries,
motto would be profit making. Therefore, PBR will a s i l l u s t r a t e dw i t h t h e f o l l o w i n g a p p l i c ati o n s
prohibit the free 6xchange of plant materials,
. I n c r e a s e d l e v e l s o f n u t r i t i o n v ; i t h i m o r o ve d
besides threatening the farmer's rights. It is feared
nutrient composition in the foods (through
that PBR will benefit rich farmers and developed
t r a n s g e n i cp l a n t s ) .
countries onlv.
o Prevention of child deaths by appropriate
PATENTI NG AND BI O TEC H N O L O G Y i m m u n i z a t i o n ( u s i n g r e c o m b i n a n t v a cci n e s) .
RESEARCH . S u p p l y o f c l e a n d r i n k i n g w a t e r , a n d i m p r o ve d
In the earlier years, researchused to be mostly sewage disposal (by appropriate bio-
for academic interest and worthwhile scientific technological treatments).
contributions. And researchers were purely Despite the known benefits, some of the
academic-oriented with not much interest in developing countries are reluctant to open doors
f inanc ial gains f r om what t hey d o . F u r t h e r ,u n l i k e for the advances made in biotechnology. This may
now, the research used to be supported mostly by be more due to political considerationsrather the
Covern rnents/Un iversiti es. economtc reasons.
There used to be no secrecy in research. The applications of biotechnology, particularly
Academic recognition and outstanding research to human healthcare,may someday be regarded as
publications (open to all) were adequate to satisfy a barometer to evaluate the progressof a nation.
 @
62 C el l -The B asi c U ni t of Li fe 719
63 Mi croorgani sms 754
64 B i oorgani cand B i ophysi cal
C hemi stry,and B i ochemi stry
Tool s 7S g
65 B i omol ecul es 771
66 E nzymol ogy 78{,
67 Metabol i sms 796
68 Immunol ogy 815
69 Genetics 822
70 B i oi nformati cs 827

EASICS TTI LEARN

BIOTEGHNOLOGY

747
-fl"he cell is t he s t r uc t ur al and f unc t ional un i t o f
I tife. tt may be also regardedas the basic unit of
biological activity.
Th e co ncept of c ell or iginat ed f r om t h e
con tribu tion s of Sc hleiden and Sc hwann ( 18 3 8 ) .
However, it was only after 1940, the complexities
of cell structure were exoosed
PROKARYOTI C AND
EUKARYOTIC CELLS
The cells o f t he liv ing k ingdom m ay be div i d e d
into two categories
1 . Prokaryotes (Creek : pro-before; karyon -
n u c l e u s )l a c k a w e l l d e f i n e d n u c l e u s a n d p o s s e s s
relatively simple structure These include the
various bacteria.
2. Eukaryotes(Creek : eu-true; karyon-nucleus)
possess a well defined nucleus and are more
c o m p l e x i n t h e i r s t r u c t u r ea n d f u n c t i o n . T h e h i g h e r
organisms (animals and plants) are composed of
eukaryotic cells
A comparison of the characteristics between
prokaryotes and eukaryotes is listed in Table
62.1.

Tlru 62.1 Comparisonbetweenprokaryotic and eukaryotic cells

(-lraracter Pr o i< lr ' . ttir t:!i '--r;1.:; lcii
': "'r,il':f

S iz e Small 1 10pm)
(generally Large
(generally pm)
10-100

Cell membrane Cellls enveloped

bya rigidcellwall Cellisenveloped plasma
bya flexible membrane
Sub-cellular
organelles Absent Distinct arefound
organelles
(eg.mitochondria,
nucleus,
lysosomes)
Nuc leus Notwelldefined;
DNAisfound Nucleus
iswelldefined.
surrounded
bya
asnucleoici,
histones
areabsent membrane;
DNAisassociated
withhistones
Energymetabolism Mitochondria
absent,
enzymesof Enzymesof energy
metabolism
arelocated
metabolism
energy to membrane inmitochondria
bound

Cell division fission

Usually andnomitosis Mitosis

Cy t oplas m Organelles
andcytoskeleton
absent Contains
organelles
andcytoskeleton
(anetwork
oftubules
andfilaments)

749
750 BIOTECHNOLOCY

Plasmamembrane
Mitochondrion

Lysosome
Vacuole
Rough- Ribosomes
endoplasmic Nucleus
reticulum
Nucleolus
Golgi
apparatus Smoothendoplasmic
reticulum

Peroxisome
Cytosol
Cytoskeleton

Fig, 62.1 : A diagrammatic represenlation of a rat liver cell.

NucleuscontainsDNA, the repository of genetic

information.EukaryoticDNA is associatedwith
basicprotein(histones) in the ratioof 1 :1, to form
The salientfeaturesof an animalceil and a plant nucleosomes.An assembly of nucleosomes
cell are briefly described constituteschromatin fibres of chromosomes (Greek:
chroma-colour; soma-body). Thus,a singlehuman
AN IM AL C EL L chromosomeis composed of about . a million
nucleosomes. The number of chromosomesis a
T h e h u ma n b o d y i s c o m p osedof about 10l a
characteristicfeatureof the species.Humanshave
c e l l s .A n e u k a ry o ti c e l l i s g e neral l y10 to 100 pm
46 chromosomes, compactlypackedin the nucleus.
in diameter.A diagrammaticrepresentation of a
typical rat liver cell is depictedin Fig. 62.1. The nucleusof the eukaryoticcell containsa
densebody known as nucleolus.lt is rich in RNA,
The cell consistsof well defined subcellular particularly
the ribosomalRNA which entersthe
organelles, envelopedby a plasmamembrane.By
cytosolthroughnuclearpores.
differentialcentrifugation of tissuehomogenate,it
i s p o s s i b l eto i s o l a tee a c h c el l ul arorganel l ei n a The ground materialof the nucleus is often
relatively pure form (Refer Chapter 64). The referredto as nucleoplasm.lt is rich in enzymes
subcellularorganellesare briefly describedin the such as DNA polymerases and RNA polymerases.
following pages. To the surpriseof biochemists,the enzymesof
giycolysis, citric acid cycle and hexose
Nucleus monophosphate shunt have also been detectedin
the nucl eopl asm.
N u c l e u s i s th e l a rg e s t cel l ul ar organel l e,
surrounded by a double membrane nuclear
Mitochondria
envelooe.The outer membraneis continuouswith
th e me mb ra n e so f e n d o p lasmi creti cul um. A t The mitochondria (Creek: mitos-thread:
certainintervals,the two nuclearmembraneshave chondros-granule) are the centresfor the cellular
nuclear oores with a diameterof about 90 nm. respiration and energy metabolism.They are
Theseporespermit the free passageof the products regarded as the power housesof the cell $'ith
synthesizedin the nucleus into the surrounding variablesize and shape.Mitochondriaare rod-like
cytoplasm. or fi l amentousbodi es,usual l yw i th di mensionsoi
 Chapter62 : CELL- THE BASICUNIT OF LIFE 751

1.0 x 3 pm. About 2, 000 m it oc hondr ia, oc c u p y i n g disrupted to form small vesicles known as
about l/uth of the total cell volume, are present in microsomes. lt may be noted that microsomes as
a typical ce ll. such do not occur in the cell.

The mitochondria are composed of a double The smooth endoplasmic reticulum does not
membrane system. The outer membrane is smooth c o n t a i n r i b o s o m e s .l t i s i n v o l v e d i n t h e s y n t h e s i so f
a nd co mple t ely env elops t he or ganelle. The i n n e r l i p i d s ( t r i a c y l g l y c e r o l s p
, h o s p h o l i p i d s , s t e r o l s )a n d
membrane is folded to form cristae (lafin-crests) metabolism of drugs, besides supplying Ca2+ for
which occupy a larger surface area. The internal t h e c e l l u l a r f u n c t i o n s .
chamber of mitochondria is referred to as matrix.
Golgi apparatus
The components of electron transportchain and
oxidative phosphorylation (flavoprotein, Eukaryotic cells contain a unique cluster of
cytochro mes b, c r , c , a and a, and c o u p l i n g membrane vesicles known as dictyosomes which,
factors) a re bur ied in t he inner m it oc ho n d r i a l i n t u r n , c o n s t i t u t e C o l g i a p p a r a t u s ( o r C o l g i
me mbra ne . The m at r ix c ont ains s ev er al en z y m e s c o m p l e x ) . T h e n e w l y s y n t h e s i z e d p r o t e i n s a r e
concerned with the energy metabolism of handed over to the Golgi apparatuswhich cataryse
ca rbo hydra t es ,lipids and am ino ac ids ( e. g., c i t r i c the addition of carbohydrates, lipids or sulfate
acid cycle, B - ox idat ion) .The m at r ix enz y m e s a l s o m o i e t i e s t o t h e p r o t e i n s . T h e s e c h e m i c a l
pa rticip ate in t he s y nt hes is of hem e and u r e a . modifications are necessary for the transport of
Mitocho nd ria ar e t he pr inc ipal pr oduc er sof A T P i n proteins across the plasma memorane.
the aerobic cells. ATP, the energy currency,
generated in mitochondria is exported to all parts Cer:tainproteins and enzymes are enclosed in
of th e cell to pr ov ide ener gy f or t he c ellular w o r k . membrane vesiclesof Colgi apparatusand secreted
from the cell after the appropriate signals. The
Th e mito c hondr ial m at r ix c ont ains a ci r c u r a r digestiveenzymes of pancreasare produced in this
do ub le-stran ded DNA ( m t DNA) , RNA a n o f a s h i o n .
ribosomes. Thus, the mitochondria are equipped
with an independent pr ot ein s y nt he s i z i n g Colgi apparatus are also involved in the
machinery. lt is estimated that about 10% of the membrane synthesis,particularly for the formation
mitochondrial proteins are produced in the o f i n t r a c e llular organelles (e.g. peroxisomes,
mitocho nd ria. lysosomes).

fhe structure and functions of mitochondria Lysosomes

closely resemble prokaryotic cells. it is
hvpothesizedthat mitochondria have evolved from Lysosomesare sphericalvesiclesenveloped by a
aerobic bacteria Further, it is believed that during single membrane. Lysosomesare regarded as the
evolution, the aerobic bacteria developed a digestive tract of the cell, since they are actively
symb iotic relat ions hip wit h pr im or dial ana e r o b i c involved in digestion of cellular substances-
eukaryotic cells that ultimately led to the arrival of n a m e l y p r o t e i n s , l i p i d s , c a r b o h y d r a t e sa n d n u c l e i c
aerobic eukaryotes. acids. Lysosomal enzymes are categorized as
hydrolases. These include the followi ng enzymes
Endoplasmic reticulum (with substratein brackets)

The network of membrane enclosed soacesthat . cr-Glucosidase(glycogen)

extends throughout the cytoplasm constitutes o Cathepsins(proteins)
endoplasmic reticulum (ER).Some of these thread-
r L i p a s e s( l i p i d s )
like structuresextend from the nuclear oores to the
pla sma memb r ane. . R i b o n u c l e a s e s( R N A )

A large portion of the ER is studded with
ribosomes to give a granular appearance which is
'eferred to as rough endoplasmic reticulum.
Ribosomesare the factoriesof protein biosynthesis.
)uring the processof cell fractionation, rough ER is
The pH of the lysosomal matrix is more acidic
(pH < 5) than the cytosol (pH-7) and this facilitates
the degradation of different compounds. The
lysosomal enzymes are responsiblefor maintaining
t h e c e l l u l a r c o m p o u n d s i n a d y n a m i c s t a t e ,b y t h e i r
752 B IOTE CHNO LO CY

Cell wall
Cell membrane

Nucleus
Smoothendoolasmicreticulum

Roughendoplasmicreticulum
Mitochondrion Nucleolus

Chloroplast
Centralvacuole

Cellsap

Tonoplast Golgiapparatus

Microfilaments

Cytoplasm

Ribosomes

Fig. 62,2 : A diagrammatic representation of a plant cell.

degradation and recycling.The degradedproducts glyoxysomes,a specializedtype of peroxisomes,

leave the lysosomes,usually by diffusion, for which are involvedin the glyoxylatepathway.
reutilization by the cell. Sometimes,however,
c e rta i n re s i d u a l p ro d u c ts , ri ch i n l i pi ds and Gytosol and cytoskeleton
proteins, collectively known as lipofuscin
The cellularmatrix is collectivelyreferredto as
a c c u m u l a tei n th e c e l l . L i pofusci ni s the age cytosol. Cytosol is basically a compartment
pigmentor wear and tear pigmentwhich hasbeen
containingseveralenzymes,metabolitesand salts
implicatedin ageingprocess. i n an aqueous gel l i ke medi um.More recentst udies
T h e d i g e s ti v ee n z y me so f cel l ul ar' compounds however, indicate that the cytoplasm actually
are confinedto the lysosomesin the best interestof contains a complex networkof proteinfilaments,
the cell. Escapeof theseenzymesinto cytosolwill spread throughout,that constitutescytoskeleton.
destroythe functionalmacromolecules of the cell The cytoplasmicfilaments are of three types-
and resultin many complications. The occurrence mi crotubul es,acti n fi l amentsand i nter m ediat e
of severaldiseases(e.g.arthritis,musclediseases, filaments.The filamentswhich are polymersof
allergicdisorders)has been partlyattributedto the proteinsare responsible shapeand
for the structure,
releaseof lysosomal enzymes. organi zati of
on the cel l .

Peroxisomes PLANT CELL

A diagrammatic of a typicalplant
representation
Peroxisomes, also known as microbodies,are
cell is depicted in Fig. 62.2. fhe cell wall,
s i n g l e me mb ra n ec e l l u l a r o r ganel l es.They are
chloroplasts and vacuolesare the most important
sphericalor oval in shapeand containthe enzyme
and di sti ngui shi ng
components of pl antcellswhen
catalase.Catalaseprotectsthe cell from the toxic
comoaredto animal cells.
effectsof H2O, by convertingit to H2O and Or.
Peroxisomes are also involvedin the oxidationof ln the lable 52.2, a comparisonbetweenplant
long chain fatty acids (>C,u) and synthesisof and ani malcel l s i s gi ven.The sal i entf eat ur esof
p l a s ma l o g e n sa n d g l y c o l i pi ds. P l ants contai n plant cell organellesare brieflydescribed.
 Chapt er62 : C EL L -T F IE B AS ICU N IT O F L I FE 753

i s i nextensi bland
e determi nes
the fi nal shaoeano
Tlorr 62.2 A comparison between plant and si zeof the pl antcel l .
animal cells
B esi descel l ul oseand l i gni n, hemi cel l ul oses,
Character Plant cell Animal cell pecti ns,and extensi ns(ol i gosacchari des)are al so
Size Generally
larger Smallerthan oresenti n the cel l w al l
plantcells
C hl oropl asts
Cellwall Sunounded by rigid Absent
cellwallof cellulose The chl oropl asts are found onl y i n the pl ant
cel l s, and are the si tes of photosynthesiThe s.
Vacuole Matureplantcells Absent
generalterm plastidsis often used to collectively
possess a rargevacuore
representchl oropl asts(greenpl asti dscontai ni ng
Plastids Chloroplasts found Absent chlorophylls) , chromoplasfs(yellow to reddish
in greenplants col our pl asti ds contai ni ng carotenoi ds)and
Celldivision Divisionof cytoplasm Division occurs leucoplasts(colourless plastids).
duringcelldivision by constriction
C hl oropl asts
have a doubl e membranesystem.
occursby theformation
Internally, chloroplasts containa systemof flattened
of a cellplate
membranousdiscs called thvlakoids. Piles of
thyl akoi dsare l ocatedi n the centralregi oncal l ed
Ge ll wall stroma.C hl orophyl lthe, green
sunl i ghtcapturi ng
pi gmenti s presenti n the thyl akoi ds.
Th e pla nt c ell wall is us ually r igid, non- liv i n g
an d p erme ab lec om ponent , s ur r oundingt he pla s m a Vacuoles
membrane. Cell walls are of two types - primary
P l antcel l shave membrane-bound, l i qui d fi l l eo
and secondary.
vesi cl escal l ed vacuol es,w hi ch may occupy as
fhe primary cell wall is the one that is formed much as 90"k ol the cells total volume. The
d urin g the co ur s e of c ell div is ion. lt is m a i n l y vacuoles may contain wide range of dissolved
comp osed o f c ellulos e. and is f lex ible in natu r e . mol ecul es suchas sal ts,sugars, pi gments and toxrc
The secondary cell wall is rigid and more complex wastes.The pigmentsof vacuolescontributeto the
in na ture . Ch em ic ally , it is m ade up of m o r e coloursof flowersand leaves.The physicalsupport
cellu lose, an d h igh c ont ent of lignin. Lignin is t h e of pl ant ti ssuescomes from the hi gh i nternal
major component of wood. The secondarycell wall pressureof water maintainedwithin the vacuoles.

3 ctechnology [48]
 ( Cr eek , m ikr o s - s m a l l ; b i o s - l i f e ) c e l l w a l l c o m p a r a b l e t o t h a t f o u n d i n p l a n ts. Th e
[ / ic r obiology
I Y I is the science of small or microscopic a v e r a g e s i z e o f a b a c t e r i u m i s a r o u n d 2 p m Th e
organisms. The most important microorganisms b a c t e r i a m a y b e s p h e r i c a l , r o d - l i k e , s p i r a l l y co i l e d
relevant to biotechnology include bacteria, fungi, o r f i l a m e n t l i k e . C e r t a i n b a c t e r i a m a y o ccu r i n
and v ir us es . M ic r oor ganis m s a r e v e r y w i d e l y more than one form. A typical structure of a rod-
distributed, and are found almost everywhere rn shaped bacterium is depicted in Fig. 63.1 .
nature. ln general, the conditions for their growth
and m ult iplic at ion( f ood,t em p e r a t u r e ,m o i s t u r ee t c . ) Gram-positive and
ar e s im ilar t o t hat of hum ans . H e n c e , t h e y a r e m o s t Gram'negative bacteria
abundantly present at places wl-rerepeople live. B a s e d o n t h e r e s p o n s e t o C r a m 's sta i n , th e
A very brief description of the microorganisms, bacteria are grouped as Cram +ve or Cram -ve. By
relevent to biotechnology is given in this chapter. C r a m 's s t a i n , s e v e r a l d i s t i n g u i s h i n g fe a tu r e s o f
bacteria can be identified. For instance,Gram +ve
bacteria possess single-layered cell wall while
Cram -ve bacteria have a double-layered one.

Bacteriology,the study of bacteria,forms a Aerobic, anaerobic

ma j o r p a rt o f mi c ro b i o l o gy.The popul ati onof
bacteriaexceedsall other organisnrs. For instance,
one kg of a soil may containmore bacteriathanthe
e n ti re h u m a n p o p u l a ti o n ! B acteri a are very
important in biotechnologyfor the following
reasons
. Diseases
they cause
r Domesticuses
. In d u s tri aal p p l i c a ti o n s
o Ag ri c u l tu rapl ro c e s s e s .
C H AR AC T ER IST IC S O F B A C TE R IA
Bacteria are prokaryotic unicellular organisms. regarded as facultative bacteria. e.g. Shigella sp,
T h e y l a c k o rg a n i z e dn u c l e u s,but possess
a ri gi d Salmonella so.
and facultative bacteria
O n t h e b a s i so f r e s p i r a t i o n( i . e . r e s p o n seto 0 2 ) ,
the bacteria are grouped into three categc.rries
1. Aerobic bacteria : l hese bacteria require
A2 for their growth e.g. Pseudomonas sp,
Mvcobacterium sp
2. Anaerobic bacteria : These bacteria do not
require O, to obtain energy, and to grow. In fact,
t h e p r e s e n c e o f 0 2 i s t o x i c t o th e m e .8 .
Peptococcus sp.
3. Facultative bacteria : The bacteria that can
grow in both aerobic and anaerobic conditions are

754
 Chapt er63 : M IC R OOR C A N IS M S 755

Cellwall
Plasmamenrbrane

Flagellum

Ribosonres
Food Cytosol
Plasmid
reserve
Pillus

Fig. 63.1 : A typical structure of a rod-shaped bacterium.

Nutritional aspects of bacteria d e s i r o y i n g t h e h o s t 's c e l l s o r r e l e a s i n gt o x i n s e . g .

Clostridium tetani.
Ba se d o n their nut r it ion, t he bac t er ia a r e
categorized as autotrophic or heterotrophic.
IMPORTANCE OF BACTERIA
1 . Autotrophic bacteria : These bacteria are
B a c t e r i ap l a y a v e r y i m p o r t a n t a n d c r u c i a l r o l e
cap ab le o f syn t hes iz ing t heir own f ood f r o m
i n t h e c o n t i n u o u s s u s t e n a n c eo f l i f e o n e a r t h . T h e y
ino rga nic sub sta nc esThey
. ar e c om pable t o high e r
are both friends and enemiesof humans Bacteria
pla nts in th is aspec t . Aut ot r ophic bac t er ia ut ili z e
are important due to the diseases they cause,
different hydrogen compounds (not HrO as in the
i n d u s t r i a l a n d a g r i c u l t u r a la p p l i c a t i o n s .
case of hig he r plant s ) . Thes e inc lude hy dr og e n ,
a mmon ia, h yd r ogen s ulf ide and m et hane e . B
Diseases caused by bacteria
Hydrogenomonas sp, Nitrosomonas sp/
Methanomonas sp. The bacteria pose a serious threat to humans,
animals and plants due to various diseases they
2. Heterotrophic bacteria : These bacteria
cause. A selected list of the imoortant diseasesand
cannot synthesizetheir own food, and are therefore
the correspondingorganismsis given in Table 63.1.
dependent on the outside source. Heterotrophsare
of two types-sporophytes and parasites.
Industrial uses of bacteria
Sporophytes obtain their food from sources of
An important aspect of biotechnology is the
anima l o r pla nt or igin. Thes e inc lude or gan i c
industrial use of bacteria for the oroduction of
r ema ins like co rps es ,anim al ex c r et a/ m eat s , f r u i t s
several compounds. Traditionally, bacteria are in
an d va riou s o ther pr oduc t s of plant and anim a l
use for the preparation of curd, vinegar, pickles,
origin. Sporophytessecrete digestive enzymes that
curing tea and tobacco leaves In recent years,
brea k th e comp lex or ganic m olec ules int o s im p l e r
Iarge-scale production of amino acids, organic
and easily absorbable forms. These heterotrophic
solvents, vitanrins and antibiotics is being carried
bacteria are useful for the disposal of sewage,
out by employing bacteria.
cle an sin g o f lea t her , and m anuf ac t ur e of c er t a i n
co mpo un ds (a lco hols , or ganic ac ids ) . Spor ophyt e s B a c t e i - i ap l a y a s i g n i f i c a n tr o l e i n t h e d e c a y a n d
can a lso spo il f oods , and dam age s oils ( b y disposal of dead plants and animals. Appropriate
d enitrificatio n). sewage disposal requires bacteria. Bacteriaare also
n e e d e d f o r t h e d i s p o s a lo f a n i m a l d u n g a n C h u m a n
Parasitesare the bacteria that obtain their food
irom iiving organisms,namely the hosts. 1'hey may excreta.

:e either harmless (non-pathogenic) or Inrmful

Agricultural applications of bacteria
oathogenic)to the hosts. E. coli is a good example
, ; no n-p ath og en icbac t er ia whic h has a s y m biot i c Nitrogen fixation by converting free niii'ngen
-:iatio nship in the hum an int es t ine.The pat hogen r c i n t o n i t r o g e n o u s c o m p o u n d s i s v e r y i m p o r t a n t i n
ba cte ria may caus e s er ious dis eas es eit her b y agriculture. This is exclusively carried out by
756 BIOIECHNOLOCY

causedby microorganisms
Mycology,a branchof microbiology, dealslvith
Diseasesof humans Pathogenic microorganism the studyof fungi. Fungiare a groupof eukaryotic,
Tuberculosis acteriun tubercuIosis
Mycob heterotrophicorganisms.They are dependenton
organi ccompoundsfor thei r nutri tion.I n gener al,
Lepr0sy M. leparae
fungi can w i thstand extreme envir onm ent al
Typhoid Salmonella
typhi conditionsbetterthan mostof the microorganisms.
Cholera Vibriocholerae
C H A R A C TE R IS TIC S OF FU N GI
Diptheria Corynebacteriu ae
n diphtheri
Syphillis pallidum
Treponema Fungicells are usuallylargerthan the bacteria.
Thesi zesmay range.l-5 pm i n w i dth and 5- 35 pm
Tetanus Clostridiuntetani i n l ength. Fungal cel l s may be elongat edor
Bacterial
dysentery Shigelladysenteriae spheri cal .
Gonorrhoea gononhoeae
Neisseria sincethey cannot
The fungi are heterotrophic,
s'ynthesizetheir own food from the inorganic
Diseasesof animals
compounds.
Anthrax Bacillusanthracis
Pneumonia Dipiococcus
oneunoniae IMP OR TA N C E OF FU N GI
Blacklegol cattle Clostridium
chauvei As is the case with bacteria,fungi are both
fri endsand enemi esof humans.
Diseasesof plants
galls
Crown Agrobacterium
tumefaciens Beneficial aspects of fungi
Pearblight Pseudomonas
amylovorus The yeasts are useful for the following
Ringrot of potato Corynebacte
riun sependonicun . Alcohol fermentation e.g. Saccharomyces
Citruscanker Xanthomonas citri cerevlslae

Blackrotof cabbage X. campestris . P roducti on of vi tanri ns e.g. A shbya gos s y pi i .

s y m b i o ti c n i tro g e n -fi x ingbacteri a. In addi ti on,
nitrifying bacteria (convert ammonia to nitrates)
and ammonifyingbacteria (convertamino acidsto
ammonia)also contributeto agriculturalpractices.
GONTBOL OF PATHOGENIC BACTERIA
As alreadystatedabove,bacteriacauseseveral
diseases.l'here are differentaooroaches
to control
pathogenicbacteria
a e.g. Candidasp.
Citric acid fermentation
a Baker'syeaste.g. S. cerevisiae.
fhe application of molds are listed
a Productionof enzymese.g. Aspergillussp.
o Citric acid fermentatione.g. Aspergillusniger.
a Penicillinproductione.g. Penicilliumnotatum.
o e.g. Rhizopussp.
Steroidtransformation
a Cluconic acid productione.g. Aspergillusniger.

. Use of antibiotics
Harmful aspects of fungi
. Treatment
with antiseptics
and disinfectants. . Spoilage of foods e.g. moldy bread, rot of fruits
. Sterilization and autoclaving and vegetables.
. Radiation treatment o Deterioration of textiles made up of cotton.
. Biologic al c ont r ol. . Dama8e to paper.

In addition to the above, vaccines have been . Diseasescaused by fungi e.g. ring worm of the
developed against certain pathogens to control scalp in children caused by Microsporum
dis eas esin hum ans and a n i m a l s . audouinii.
 Chapt er63 : MIC R OOR C A N IS M S 757

CONTROL O F FUNG I
TffiLr63.2 A selectedlist of conrmon
Th e fu ng al gr owt h c an be c ont r olled by us i n g
viral diseases
p he no l a nd its der iv at iv ese. g. c r es ol, et hy lphe n o l ,
pro pylph en ol, b ut y lphenol. Chlor ine and c hlo r r n e Diseasesof humans Virus
co mpo un ds are als o us ef ul in t his r egar d.
Common cold Rhinoviruses
VegetativecelJsof yeastsand other fungi can be pox
Sntall Pox virus
destroyedby moist heat at 50-60'C for about 5-10
Rabies Rabiesvirus
min ute s. Sp or es , howev er r equir e hig h e r
temperature 20-80"C.
Infectious
hepatitis Hepatitisvirus
Measles Measlesvirus
Poliomyelitls Poliovirus
Infiuenza lnfluenzavirus
A virus m ay be r egar ded as a s n r a l l
Encaphalitis Encephalitis
virus
rricrocrga nisrnw it h a nuc leopr ot ein ent it y whi c h MY'p1 Mumpsvirus
lives an d multi plies in t he liv ing c ells of ot h e r
Diseasesof plants
o rga nisms.Th us , v ir us es as ar e unable t o liv e in a
cell-free environment, ar-rd become active and Tobacco
mosaic Tobacco
mosaicvirus
nru ltiply a s the y ent er iiv ing c ells . Potatoleafroll Potatoleaf roll virus
leafcurl
Tomato Tonatoleafcurl virus
CHARACTERIS TI CS O F VI RUSES

Virusesare the s m alles t liv ing ent it ies They d o

Molecular theory : Some people consider
n ot p osse ssthe us ual c ellular s t r uc t ur esVir
. us esa r e
viruses are non-living chemical molecules rather
conrposed of either DNA or RNA, and not both of
t h a n l i v i n g b e i n g s . T h e y a r g u e t h a t v i r u s e sd o n o t
:hem together. Retrovirusesis the name given to
p o s s e s sa n y c e l l u l a r s t r u c t L r e s ,c a n n o t r e s p i r e a n d
', iru se sco nta ini ng RNA as t he genet ic m at er ial .
cannot live independently.
Virusesdo not possessindependentmetabolisms,
:n d fu rthe r the y als o lac k r es pir at or ym ac hiner y . IMPORTANCE OF VIRUSES

Viruses are associatedwith several diseasesin

BIOL OGICAL STATUS O F VI RUSES
humans and plants (Table 63.2). Besides tne
Organismal theory : Viruses are considered as c o m m o n d i s e a s e s s u c h a s c o m m o n c o l d , a n d
::'in itive living m ic r oor ganis m s as t hey pos s e s s r a b i e s , v i r u s e s c a u s e d i s e a s e ss u c h a s A I D S . i . e .
::1 €tiC ma teria l, bios y nt het ic m ac hiner y an d h u m a n i m m u n o d e f i c i e n c yv i r u s ( H l V ) i s r e s p o n s i b l e
-.ta in e nzyme s , bes ides being inf ec t iv e. for AIDS, the dreaded and incr-rrabledisease.
 f undam ent al pr in c i p l e s o f b i o o r g a n i c a n d e&gV!rd{,FNFU NCTIO?,IAL GROI'jF$
J he
! biophy s ic al c hem is tr y , b e s i d e s t h e c o m m o n IN bICIEFCEME$TRV
t ools em ploy ec l in b i o c h e m i s t r y a r e b r i e f l y
M o s t o f t h e p h y s i c a l a n d c h e m i ca l p r o p e r ti e so t
des c r ibed in t his c hapt e r .
o r g a n i c c o m p o u n d s a r e d e t e r m i n e d b y th e r r
f u n c t i o n a l g r o u p s . B i o m o l e c u l e s p o sse ss ce r ta l n
INTRODUCTIONTO f u n c t i o n a l g r o u p s w h i c h a r e t h e i r re a cti ve ce n tr e s
BIOORGANICCHEMISTRY T h e c o m m o n f u n c t i o n a l g r o u p s o f i m p o r ta n ce tn
biomolecrrlesare presented in Table 64'1
As lif e c om es f r om pr e v i o u s l i f e , i t w a s b e l i e v e d
for a long that the carbon compour.rdsof organisms d;$tUtM{}fii mlruG STFUCTURES
( henc e t he nam e or ganic ) a r o s e f r o m l i f e o n l y . T h i s r [ € B I O C I {E M I S T R Y
is referredto as vital force theory FriedrichWohler
( 1825) f ir s t dis c ov er edt h a t u r e a ( N H 2 - C O - N H 2 ) , T h e r e a r e m a n y h o m o c y c l i c an d h e te r o cycl i c
t he or ganic c om pound , c o u l d b e p r e p a r e d b y r i n g s , c o m m o n l y e n c o u n t e r e d i n bi o m o l e cu l e s. A
heat ing am m onium c y a n a t e ( N H o N C O ) , i n t h e selected list of them is given in Fig. 64.1 .
laboratory.Thereafter,thousandsand thoushandsof
or ganic c om pounds have b e e n s y n t h e s i z e do u t s i d e f l *n r o +y c l i c rings
t he liv ing s y s t em .
P h e n y l r i n g d e r i v e d f r o m b e n ze n e i s fo u n d i n
Organic chemistry broadly deals with the s e v e r a l b i o m o l e c u l e s ( p h e n y l a la n i n e , tyr o si n e ,
chemistry of carbon comitbunds, regardless of c a t e c h o l a m i n e s ) .P h e n a n t h r e n ea nd cycl o p e n ta n e
their origin. Biochemistry, however, is concerned form the backbone of steroids (cholesterol,
with the carbon chemistry of life only. The general a l d o s t e r o n e ) . C o e n z y m e a i s an e xa m p l e o i
pr inc iplesof or ganic c he m i s t r yp r o v i d e s t r o n gf o u n - b e n z o q u i n o n ew h i l e v i t a m i n K i s a na p h th o q u i n o n e
dat ions f or under s t and i n gb i o c h e m i s t r y H o w e v e r ,
bioc hem is t r v ex c lus iv el v d e a l s w i t h t h e r e a c t i o n s rings
i *e t e r *c y e l i c
t hat oc c ur in t he liv ing s y s t e mi n a q u e o u s m e d i u m .
F u r a n i s t h e r i n g s t r u c t u r e f o un d i n p e n to se s
M O ST CO M M O N O R G A T {I C e O r d P d }| . }N S S P v r r o l e i s t h e b a s i c u n i t o f p o r ph yr i n s fo u n d In
FO UND I N I - I VI NG S Y S T E A S m a n y b i o m o l e c u l e s ( h e m e ) w h i l e p yr o l i d i n e i s th e
The or ganic c om poun d s , n a m e l y c a r b o h y d r a t e s , r i n g p r e s e n t i n t h e a m i n o a c i d , p ro l i n e . Th i o p h e n
lipids , pr ot eins , nuc leic a c i d s a n d v i t a m i n s a r e r i n g i s a p a r t o f t h e v i t a m i n , b i o t i n . Th e a m i n o a ci d
t he m os t c om m on or ga n i c c o m p o u n d s o f l i f e . histidine contains imidazole.
ChA P t CT
64 : B IOOR C A N ICA N D BIOP H Y SIC ACLH E MIS TR A
Y N D B IOC H E MIS TRTOOLS
Y 759

Trsrr 64.1 Commonfunctional groups of importancein biomolecules

Functional growp General structural '{ype of Examples of

Name Group formula cantpountl biamoleculeis)

Hydroxyl -oH R-OH Alcohol Glycerol,

ethanol

t--,

I ii l
NU
Aldehyde R-C-H Aldehyde glucose
Glyceraldehyde,

o Lr
I lI
,C -I
Keto R1-C-R2 Ketone Fructose.
sedoheptulose

o
I I
Carboxyl -C-OH R-C -.ali--l Carboxylic
acid Aceticacid.oalmitic
acid

Amino - NHz R- NH2 Aminoacid Alanine,

serine
t-l
H
I
lmino -N- R-N- lminoacid Proline,
hydroxyproline

Sulfhydryl -SH Cysleine, A

coenzyme
Ether -O- R1-C--R2 Ether Thromboxane
A,

o
-c-o-R1
i
Ester R2-C--O---R1 Ester Cholesterol

U o
i lt -
J ,R , li . fl l
Amido -C-N1 .R , R"-C-N( Amide N-Acetylglucosamine
',2

Pyran structure is found in hexoses. Pyridine and chemical properties. Isomerism is partly
ru cle us is a par t of t he v it am ins - niac in a n d responsihlefor the existenceof a large number of
c','rido xin e. Pyr im idines ( c y t os ine, t hy m ine) a n d organic molecules.
:.rrin es (ad en ine, guanine) ar e t he c ons t it uent so f
Consider the molecularformula-CrHuO.Thereare
-:cleo tide s an d nuc leic ac ids . I ndole r ing is f ou n d
two important isomers of this- ethyl alcohol
- the amin o a c id t r y pt ophan. Pur ine and indo l e
(C 2H ' OH )and di ethylether(C H TOC H T)
as show n
:.re examples of fused heterocyclic rings.
below :
ISOMERISM
H OH H H
Tre co mpo unds pos s es s ingident ic al m olec u l a r ltt I
':'r.,ulae but different structuresare referred to as H-C_G_H H_C-.O -c-
isomers. The phenomenon of existence of isomers
' ltl I
. called isomerism (Greek : isos-equal; meros-
HH F. H
::Tiri lsomers differ from each other in physical Ethylalcohol Diethylether
760 B IOTECHNO LO CY

o
(A)

o
Benzene Phenol Benzoquinone Naphthalene a-Naphthol

U
Naphthoquinone Anthracene Phenanthrene CycloPentane

(B)
[_\,
,\ //-\
\o / t*/
'*/
I I
H H
Furan Pyrrole Thiophene lmidazole

lttl
a-\
tI
NA
\o / \- t2 \,)

Pyran Pyridine Pyrimidine

Fig. 64.1 : Common ting structures found in biomolecules (A) Homocyclic rings (B) Heterocyclic rings.

ls onr er is m is br oadl y d i v i d e d i n t o t w o of structuralisomerism,occurs due to the migratron

categories - structural isomerism and stereo- of an atom or group from one position to the other
lsomeflsm. e.g. purines and pyrimidines.

Structural isomerism Stereoisomerism

The difference in the arrangementof the atoms Stereoisomerism (Creek : stereos-space

in the molecr-rle (i.e. nrolecular framework) is
r es pons iblef or s t r uc t ur al i s o m e r i s m .T h i s m a y b e
due to variation in carbon chains (chain isomerism)
or dif f er enc e in t he pos it i o n o f f u n c t i o n a l g r o u p s
(position isomerism) or difference in both molecular
chains and functional groups (functional isomerism).
occupying)is, perhaps,more relevantand important
t o b i o m o l e c u l e s .T h e d i f f e r e n t i a ls p a cea r r a n g e m e n t
of atoms or groups in molecules gives rise to
stereoisomerism. Thus, stereoisomershave the same
s t r u c t u r a l f o r m u l a b u t d i f f e r i n th e i r sp a ci a l
arranBement.

Structural isomerism,as such, is more common Stereoisomerism is of two types-geometric

in general organic molecules. Tautomerism, atype isomerism and optical isomerism.
 AN D B IO P H Y S IC ACLH E MIS TR A
Chapt er64 : B IO OR C A N IC Y N D B IOC H E MIS TRTOOLS
Y 761

Geometrical isomerism : This is also called crs-

tran s isom er is m and is ex hibit ed by c e r t a i n
mo lecule s p os s es s ingdouble bonds Ceom e t r i c a l
rso merism is due t o r es t r ic t ion of f r eedo m o f
rota tion o f g r oups ar ound a c ar bon- c ar bond o u b l e
b on d (C = C) . M aleic ac id and f r - r m ar icac i d a r e Ordinarylight Nicolprism Planeof polarized
classicalexam oles of c is - t r ansis or ner is m . wavesvibrating lightvibratingin
in all directions one directlon
H-C-COOH H-C-COOH
il ll
H-C_COOH HOOC-C-H
Mafeic acid t'crs) Fumaric acid (trans)
Whe n sim ilar gr oups lie on t he s ar ne s ide , i t i s
ca lled cls is om s r ( Lat in : c is - on t he s am e s i d e ) .
On the oth er hand, when s im ilar gr oups l i e o n Planerotatedto Plane rotatedto
the opposite sides, it is referred to as lran-s isomer the left (levorotatory) the right (dextrorotatory)
(.Latin : trans-across) As is obsened from the
a bo ve struct ur e, m aleic ac id is a c is f or m w h i l e
Fig. 64.2 : Diagrammatic illustration of optical activity.
fumaric acid is a trans lorm-
Ce on retri c is om er is m is als o obs er v ed in s t e r o r s
a nd po rph yri ns c is - t r ans is om er sdif f er in ph y s i c a l What is optical activity?
a nd che mica l pr oper t ies .
T h e o r c l i n a r y I i g h t p r o p a g a t e si n a l l d i r e c t i o n s .
Optical isomerism : Optical isonrers or H o w 'e v e r ,o n p a s s i n go r d i n a r y l i g h t t h r o u g h a N i c o l
en an tiome rs oc c ur due t o t he or es enc e o f a n p r i s m , t h e p l a n e
of polarized light vibratesin one
asymmetric carbon (a chiral carbon). Optical
direction only (Fig. 64"2)
isome rs diffe r f r om eac h ot her in t heir o p t i c a i
activity to rotate the plane of polarized light. Certain organic compounds (optical isomers)
which are said to exhibit ootical activitv rotate the
What is an asymmetric carbon? plane of polarized light either to the left or to the
right.
An o bje ct is s aid t o be s y m m et r ic al if it c a n b e
divide d into equal halv ese. g a ball. O bjec t s w h i c h The term levorotatory (indicated by 1 or
cannot be divided into equal halvesare asymmetnc, (-) sign) is used for the substanceswhich rotate the
e.g . ha nd . An as y m m et r ic objec t c annot c oi n c i d e p l a n e o f p o l a r i z e d l i g h t t o t h e l e f t . O n t h e o t h e r
with its mirror im age. For ins t anc e,lef t hand i s t h e hand, the term dextrorotatory (indicated by d or (+)
mirror image of right hand and these two can never sign) is used for substancesrotating the plane of
be superimposed.In contrast, a symmetricalobject polarized light to righr (Fig. 64.2).
like a ball superimposesits image.
The term racemic mixture represents equal
A carbon is said to be chiral (Greek : hand) or concentration of d and I forms which cannot rotate
asymmetric when it is attached to four different t h e p l a n e o f p o l a r i z e d l i g h t .
g rou ps. The ir m ir r or im ages do not s uper i m p o s e
with each other. Configuration of chiral molecules
B B \ 4 , / h i l er e p r e s e n t i n gt h e c o n f i g u r a t i o n o f c h i r a l
I I molecules, the configuration of glyceraldehyde is
A-C-D D-C-A taken as a reference standard.
I cHo CHO
E E
Mirror I I
H-C-OH HO-C-H
Th e nu mber of pos s ible opt ic al is om er s o f a I I
mo lecule de pends upon t he s pec if ic num b e r o f cH2oH cH20H
ch iral carb on ( n) . lt is giv en by 2n D-Glyceraldehyde L-Glyceraldehyde
762
It must, however, be remembered that D- and
L- do not representthe direction of the rotation of
plane of polar iz ed light .
Existence of chiral biomolecules
As you know, you can never come across
any body who is y our m ir r o r i m a g e . T h e s a m e i s
t r ue wit h biom olec ules .O nly o n e t y p e o f m o l e c u l e s
( D or L) ar e f ound in t he liv i n g s y s t e m .T h u s , t h e
nat ur ally oc c ur r ing am ino ac i d s a r e o f L - t y p e w h i l e
the carbohydratesare of D-type.
The gener al laws and pr i n c i p l e s o f c h e m i s t r y
and physics are applicable to biochemistry as well.
It is, therefore, worthwhile to have a brief under-
s t anding of s om e of t he bas i c c h e m i c a l a n d p h y s i -
cal principles that have direct relevance to life.
It rnust, however, be remembered that all these
aspectsare quite unrelated to each other.
ACIDS AND BASES
According to Low'ry and Bronsted, an acid is
defined as a surbstancethat gives off protons while
a base is a substancethat accepts protons.Thr-rs,an
acid is a proton (H+) donor and a base is a proton
acceptor.
Alk alies : The m et allic h y d r o x i d e s s u c h a s
NaOH and KOH are commonly referred to as
alkalies. These compounds do not directly satisfy
the criteria of bases. However, they dissociate to
form metallic ion and OH- ion. The latter, being a
base, accepts H+ ions.
Ampholytes : The substanceswhich can function
both as acids and bases -are referred to as
ampholytes. Water is the best example for
ampholytes.
Hydrogen ion concentration (pHf
The ac idic or bas ic na t u r e o f a s o l u t i o n i s
measuredby H+ ion concentration.The strengthof
H+ ions in t he biologic al f lui d s i s e x c e e d i n g l y l o w .
For t his r eas on, t he c onv e n t i o n a l u n i t s s u c h a s
moles/l or {l are not commonly used to expressH+
ion concentration.
B IOTECHNO LO CY
Sorenson (1909) introduced the term pH to
express H+ ion concentration . pH is defined as the
negative logarithm of H+ ion concentration.
p H = - l o g [ H +]
BUFFERS
The pH of a given solution can be easily altered
by the addition of acids or bases. Buffers are
defined as the solutions which resist change in pH
by the addition of small amounts of acids or bases.
A b u f f e r u s u a l l y c o n s i s t so f a w e a k a c i d a n d i ts sa l t
(e.9. acetic acid and sodium acetate) or a weak
b a s e a n d i t s s a l t ( e . g . a m m o r r i u m h y d r o xi d e a n d
a m m o n i u m c h l o r i d e ) . S e v e r a l b u f f er s ca n b e
prepared in the laboratory. Nature has provided
many buffers in the living system.
Mechanism of buffgr action
Let us consider the buffer pair of acetic acid and
sodium acetate. Acetic acid, being a weak acid,
feebly ionizes. On the other hand, sodium acetate
ionizes to a large extent.
CH3COOH l^ CH3COO- + H+
C H 3 C O O N a +C H 3 C O O - + Na+
When an acid (say HCI) is added, the acetate
ions of the buffer bind with H+ ions (of HCI) to
f o r m a c e t i c a c i d w h i c h i s w e a k ly i o n i zi n g .
Therefore,the pH change due to acid is resistedby
the buffer.
H+ + CHTCOO- ----+ CH3COOH
When a base (say NaOH) is added the H+ ions
of the buffer (acetic acid) combine with OH- ions
to form water, which is weakly dissociated.Thus,
the pH change due to base addition is also
prevented by the buffer.
OH- + H+ ------+HrO
Buffering capacity : The efficiency of a buffer in
m a i n t a i n i n g a c o n s t a n t p H o n t h e a d d i ti o n o f a ci d
or base is referredto as bufferinBcapacity. lt mostly
deoends on the concentration of the buffer
comDonents.
SOLUTIONS
Solutions may be regarded as- mixtures of
substances.In general, a solution is composed of
two oarts - solute and solvent. The substance that
 Chapter
64 : BlooRCANlcAND BIOPHYSICAL
cHEMlsrRY
AND BtocHEMtsrRy
rools
is d issolvedis s olut e and t he m edium t hat dis s o l v e s Colloidal state is characterizedby the particle size
the solute is referredto as solvent. The oarticle srze o f 1 t o 1 0 0 n m . Wh e n t h e p a r t i c l e s i z e i s <1 n m ,
.l
o f a solu te in s olut ion is < nm .
The rela'tiveconcentrations of substancesin a
solution can be measured by several ways
Per cent concentration : This representsparts
p er -l00 . Th e m os t f r equent ly us ed is weight p e r
volu me (w/v) e. g. 9% s aline ( 9 gl1O 0 m l s olut i o n ) .
Parts per million (ppm) : This refers to the
n umb er o f p ar t s of a s ubs t anc ein one m illion p a r t s
of the so lutio n Thus 1O ppm c hlor ine m eans 1 0 p g
of chlorine in 1 g of water.
Molarity (M) : lt is defined as the number of
mole s of so lut e per lit er s olut ion. NaCl ha s a
mole cu lar wei ght of 58. 5. To get one m olar ( 1 M )
o r o ne mo le so lut ion of NaCl, one gr am m olec u l a r
we igh t (58 .5 g ) of it s hould be dis s olv ed in t h e dispersed
solvent (HrO) to make to a final total volume of .l
lite r. For sma ller c onc ent r at ions , m illim ole a n d
micromo le are us ed
Molality : lt representsthe number of moles of
so lute p er 1,0 00 g of s olv ent O ne m olal s olu t i o n
ca n be p rep ar ed by dis s olv ing 1 m ole of s olu t e i n
1 ,00 0 g o f sol v ent .
Norma lity : M olar it y is bas ed on m olec u t a r
we igh t wh iie nor m alit y is bas ed on equiv a l e n t
we igh t. One gr am equiv alent weight of an elem e n t
or compound representsits capacity to combine or
rep lace 1 mole of hy dr ogen.
COLLOIDAL STATE
Th oma s Craham ( 1861) , r egar dedas t he ' f a t h e r
of collo ida l che m is t r v ' ,div ided s ubs t anc esint o t w o
classes-crystalloids and colloids.
Crystallo idsar e t he s ubs t anc eswhic h in. s olu t i o n
can freely pass (diffuse)through parchment"mem-
bra ne e .g. su ga r ,ur ea, NaCl. Colloids ( G r eek : g l u e -
like), on other hand, are the substancesthat are
reta ine db y p ar c hm entm em br ane e. g. gum , gel a t i n ,
a lbu min. Th e a bov e c las s if ic at ionof Cr aham is n o
lon ge r ten ab le, s inc e any s ubs t anc e c an b e
co nverte d in to a c olloid by s uit able m eans . F o r
in sta nce, sod ium c hlor ide in benz ene f or m s a
co lloid .
Colloidal state : As such, there are no group of
substancesas colloids, rather, substancescan exist
in the form of colloidal state or colloidal system.
763
i t i s i n t r u e s o l u t i o n . F o r t h e p a r t i c l e s i z e s>. 10 0 n m ,
t h e m a t t e r e x i s t s a s a v i s i b l e p r e c i p i t a t e .T h u s , t h e
colloidal state is an intermediate between true
solution and precipitate.
Phases of colloids : Colloidal state is hetero-
g e n eouswith two phases.
1. Dispersed phase (internal phase) which
c o n s t i t u t e st h e c o l l o i d a l p a r t i c l e s .
2. Dispersion medium (external phase) which
refers to the medium in which the colloidal
p a r t i c l e sa r e s u s p e n d e d .
Classification of colloids
B asedon the affi ni tyof di spersi on medi umw i th
phase,colloidsareclassified as lyophobic
and lyophilic colloids.
1. l-yophobic(Creek : solvent-hating) : These
colloids do not have any attraction towaros
di spersi onmedi um. W hen w ater i s used as
di spersi on
medi um,the col l oi dsare referredto as
hydrophobic.
2. tyophilic (Greek : solvent-loving): These
col l oi ds have di sti nctaffi ni tytow ardsdi spersron
mediunr. The term hydrophilic is used for the
col l oi dsw hen w ater i s the di spersi on
medi um.
B i ol ogi cal i mportance of col l oi ds
1 Biologicalfluids as colloids: Theseincluoe
bl ood,mi l k and cerebrospi nal
fl ui d.
2. Biologicalcompoundsas colloidalparticles:
The compl exmol ecul es of l i fe,the hi gh mol ecul ar
w ei ght protei ns, compl ex l i pi ds and
pol ysacchari des
exi sti n col l oi dalstate.
3. Fat digestionand absorption: The formation
of emul si ons,faci l i tatedby the emul si fyi ng
agents
bile salts,promotesfat digestionand absorptionrn
the intestinaltract.
4. Formationof urine : The filtrationof urine is
basedon the pri nci pl eof di al ysi s.
D IFFU S ION
The mol ecul es i n l i qui ds or gases are i n
continuousmotion. Diffusion may be regardedas
the movement of solute molecules from a higher
concentration to a lower concentration Diffusion
7fi4
i s m o re ra p i d i n g a s e sth a n i n l i qui ds.The smal l er
particlesdiffusefasterthan the largerones. The are kept in hypotonic solution(say0.4% NaCl),the
greaterthe temperature, the higher is the rate of cells bulge due to entrv of water which often
d i ffu s i o n .
Applications of diffusion
1. Ex c h a n g e o f O , a n d C O, i n l ungsand i n (0.9%)or glucose(5'k) or a suitablecombinationof
ti s s u e so c c u rsth ro u g hd i ffu s i on.
2. Certainnutrientsare absorbedby diffusionin
the gastrointestinal tract e.g. pentoses,minerals,
w a te r s o l u b l ev i ta m i n s .
3. Passage of the wasteproductsnamelyammo-
n i a , i n th e re rra tu l b u l e so c cursdue to di ffusi on.
osMosts
Osmosis (Greek : push) refersto the movement
of solvent (most frequently water) through a
semipermeable membrane.
The flow of solvent occurs from a solution of
low c onc ent r at ion t o a s o l u t i o n o f h i g h
concentration, when both are separated by a
s emioer m eable m em br ane.
Osmotic pressure
Osmoticpressure
maybe definedasthe excess referredto as fluidity.The term viscositymay be
pressure that must be applied to a solution to
prevent the passage of solvent into the solution,
when both are separated by a semipermeable
m em br ane.
The s olut ions t hat ex er t t h e s a m e o s m o t i c
oressure are said to be isoosmofic. The term
isotonic is used when a cell is in direct contact
wit h an is oos m ot ic s olut ion ( 0 . 9 % N a C l ) w h i c h
does not change the cell volume and, thus, the cell
t one is m aint ained. A s olu t i o n w i t h r e l a t i v e l y
greater osmotic pressure is referred to as
hypertonic. On the other hand, a solution with
relatively lower pressure is hypotonic.
Units of osmotic pressure : Osmotic pressureof
biologic al f luids is f r equ e n t l y e x p r e s s e d a s
milliosmoles. The osmotic pressure of plasma (of
hum an blood) is 280- 300 m i l l i o s m o l e s / l .
Applications of osmosis
1. Fluid balanc e and bloo d v o l u m e : T h e f l u i d
balance of the different compartments of the body
is m aint ained due lo os m os i s .
B IOTECHNO LO CY
2. Redblood cellsand fragility: When the RBC
causes rupture of plasma membrane of RBC
(hemolysis).
3. Transfusi on: l sotoni c sol uti o nsof NaCl
these tw,o are commonly used in transfusionin
hospitalsfor the treatmentof dehydration,burns
etc.
4. Action of purgatives: The mechanismof
acti on of purgati vesi s mai nl y due t o osm ot ic
phenomenon. epson(Mg SO oTHr O )
For i nstance,
or Gl auber' (N
s arS O*10H 2O)sal tsw i th dr aww'at er
from the body, besidespreventingthe intestinal
water absorption.
5. Edemadue to hypoalbuminemia : Disorders
such as kw ashi orkorand gl omerul onephr itar is e
associ ated w i th l ow ered pl asma album r n
concentrationand edema. Edema is caused by
reducedoncoti cpressure of pl asma,l ea dingt o t he
of excessfl ui d i n ti ssuespaces.
accumul ati on
vrscosrrY
Li qui dor fl ui d hasa tendencyto fl ow which is
defined as the internal resistanceoffered by a
liquid or a gas to flow. fhe propertyof viscosityis
due to frictionalforcesbetweenthe laverswhile
their movementoccurs.
V i scosi tyof col l oi dal sol uti ons,par t icular ly
l yophi l i c col l oi ds,i s general l yhi gher t han t r ue
sol uti ons.
Unitsof viscosity: The unit of viscosityis poise,
who firstsystematically
afterthe scientistPoiseuille,
studied the flow of liquids. A poise represents
dynes/cm2.
Applications of viscosity
1. Viscosityof blood : Blood is about 4 times
rnoreviscousthan water.The viscosityof blood is
mainly attributedto suspendedblood cells and
col l oi dalpl asmaprotei ns.
2. Viscositychangein muscle: Excitation of the
muscleis associated with increasein the viscosity
of the muscl efi bres.Thi sdel aysthe ch angein t he
tensionof the contractingmuscle.
 Chapt er64 : BIOOR C A N IC
A N D BIOP H Y SIC ACLH E MIS TR A
YN. D B IOC H E MIS TRTOOLS
Y 765

SURFACE T ENSI O N rsoToPEs

A mole cu le in t he int er ior of a liouid is at t r a c t e d lsotapes are defined as the elements with same
b y o the r mo lec ules in all dir ec t ions . I n c ont r a s t ,a atomic number but different atomic weights. Ihev
molecr:le on the surface is attracted onlv possessthe same number of protons but differ in the
do wnwa rds a nd s idewav sand not uowar ds D u e t o neutronsin their nuclei. Therefore,isotopes(Creek :
th is, the surfa c elay er behav eslik e a s t r et c he df r l m . iso-eqr-tal; tope -place) occupy the same place in
Surface tension is the force with which the the periodic table The chemical properties of
molecules on the surface are held together. lt is d i f f e r e n ti s o t o o e so f a n a r t i c u l a re l e m e n ta r e i d e n t i c a r .
expressedas dvnesrcm. Surface tension decreases Isotopes are of two types-stable and unstable.
with increa se in t em oer at ur e The latter are more commonly referred to as
radioactive isotopes and they are of particular
Applications of surface tension i n t e r e s tt o b i o c h e m i s t s
1 . Dige sti on and abs or pt ion of f at : Bile s a l t s
-fhey
reduce the surface tension. act as detergents Stable isotopes
a nd ca use e m uls if ic at ion of f at , t her eby allo w i n g They are naturally occurring and do not ernit
the formation of minute oarticles for effective r a d i a t i o n s ( n o n - r a d i o a c t i v e )e . g . d e u t e r i u m ( h e a v y
dig estrona nd abs or pt ion. h y d r o g e n ) 2 H ; 1 3 C ; l s 5 ' 1 B C ) .S t a b l e i s o t o p e sc a n
be identified and quantitated by mass spectrometry
2. Surfactants and Iung function : The lo',v
or nuclear magnetic resonance (NMR) They are
su rfaceten sio n of t he alv eoli k eepst hem apar t a n d
lessfrequently used in biochemical investigations.
allo ws a n e ffic ient ex c hange of gas es in lung s . I n
pr
fact, ce rtain sur f ac t ant s , edom inant ly dipalm i t o y l
Radioactive isotopes
p ho sp ha tidyl c holine ( dipalm it oy l lec it hin) , a r e
respo nsiblefo r m aint aining low s ur f ac e t ens i o n i n The atomic nucleus of radioactive isotooes is
the alveoli. Surfactantdeficiency causesrespiratory unstable and, therefore, undergoes decay. The
distresssyndrome in the infants. radioactivedecay gives rise to one of the following
3 ionizing radiations.
ADSORPTION
1 . cx-Rays - an c[ parlicle possessing2 protons
Ad so rptio n is a s ur f ac e phenom enon. lt i s t h e i . e h e l i u r n n u c l e i .
p rocessof a cc um ulat ion of a s ubs t anc e( ads o r b a t e ) 2. B-Rays due to the emissionof electrons.
on the surface of ancther substance (adsorbent).
3 . y - R a y s- d u e t o e m i s s i o n o f h i g h e n e r g y
Adsorption differs fron.r absorption, as the Iatter
photons
in vo lve s the dif f us ion int o t he int er ior of t h e
ma teria l. The B and y emitting radioisotopesare employed
in biochemical researcfr.
Applications of adsorption
Applications of
1 . Formation of enzyme-substrate complex : radioisotopes in biochemistry
For th e cata ly s is t o oc c ur in biologic al s y s t e m ,
forma tion o f enz y m e- s ubs t r at e c om plex i s a Radioactiveisotopeshave become indispensable
p rere qu isite . This happens by ads or pt io n o f t o o ls of biochemistry.They can be conveniently
substrateon the enzyme. u s e d a s t r a c e r s i n b i o c h e m i c a l r e s e a r c hs i n c e t h e
chemical properties of different isotopes of a
2. Action of drugs and poisons : On adsorption p a r t i c u l a r e l e m e n t a r e i d e n t i c a l . T h e r e f o r e , t h e
a t th e ce ll su r f ac e, dr ugs and pois ons ex er t t h e t r l i v i n g c e l l s c a n n o t d i s t i n g u i s h t h e r a d i o a c t i v e
a ctro n isotope from a norrnal atom. A ferv inrportant
a p p l i c a t i o n o f r a d i o i s o t o p e sa r e l i s t e d
3. Adsorption in analytical biochemistry : The
prin cip le of a ds or pt ion is widely em ploy ed in t h e 1. By the use of isotope tracers,the metabolic
chromatography technique for the separation and o r i g i n o f c o m p l e x m o l e c u l e s s u c h a s h e m e ,
purification of comoounds (enzymes, immuno- c h o l e s t e r o l , p u r i n e s a n d p h o s p h o l i p i d s c a n b e
glo bulin s). determined.
766 B IOTE C HNO LO CY

Partition Adsorption lon-exchange Gelfiltration AffinitY HPLC

ll
I tic
"iu.n
---|> Paper chromatography

{---]r
SingledimensionalTwodimensional

I nscenoing
IL
Descending

Thin layerchromatography

Gas-liquidchromatography

Fig. 64.3 : lmporiant types of chromatography

(HPLC-High performance liquid chromatography; TLC-Thin layer chromatography)

2. The precursor-product relationshipin several related compounds from a mixture. These include
metabolic pathways has been investigatedby proteins,peptides,amino acids, lipids, carbohy-
radioisotopes. e.g. Krebscycle, p-oxidationof fatty drates,vitaminsand drugs.
acids,urea cycle, fatty acid synthesis.
Principles and classification
3. Radioisotopes are convenientlyused in the
studyof metabolicpools (e.g.aminoacid pool)and Chromatography (Creek : chroma- colour; gra-
metabolicturnovers(e.g.proteinturnover). phein- to write),usuallyconsistsof a mobile phase
a stationaryphase.The mobile phaserefersto
4 . C e rta i n e n d o c ri n e a nd i mmunol ogi cal and
(to dissoved
studiesalsodependon the useof radioisotopes e.g. the mixtureof substances be separated),
in a phaseis a porous
!iquidor a gas.The stationary
radioimmunoassay.
solidmatrixthroughwhich the samplecontainedin
5. Radioisotopes are employed in elucidating the mobile phase percolates.The interaction
d ru g m e ta b o l i s m. betweenthe mobileand stationary phasesresultsin
the separation of the compoundsfrom the mixture.
These interactions include the physico-
chemical principlessuch as adsorption,partition,
ion-exchange,molecular sieving and affinity.
The foundations for the present(and the future,
The interactionbetweenstationaryphase and
of course!)knowledgeof biochemistry are basedon
mobilephaseis oftenemployedin the classification
the laboratorytools employed for biochemical
chromatographye.g. partition, adsorption,ion-
experimentation. The basic principlesof some of of chromato-
exchange.Further,the classification
the commonlyemployedtools are describedhere. graphy is also basedeither on the natureof the
stationaryphase(paper,thin layer,column),or on
CHROMATOGRAPHY the nature of both mobile and stationaryphases
Chromatography is one of the most usefuland (gas-liquidchromatography). n summary of the
popular tools of biochemistry.lt is an analytical differentmethods(classes) of chromatographyis
technique dealing with the separationof closely given in Fig. 54.3.
 64 : B I OOR C A N IC
C hA P I CT A N D BIOP H Y SIC AL
C H E MIS TR A
Y N D B IOC H E MIS TRTOOLS
Y 767

1. Partition chromatography: The molecules 2. l soel ectri cfocussi ng: Thi s techni que i s
of a mixtureget partitionedbetweenthe stationary primarily based on the immobilization of the
p has eand m obilep h a s ed e p e n d i n o g n th e i rre l a ti v e moleculesat isoelectricpH during electrophoresis.
aninityto each one of the phases. StablepH gradientsare set up (usuallyin a gel)
coveringthe pH rangeto include the isoelectric
2. Adsorption column chromatography . pointsof the components
in a mixture.lsoelectric
Th e ads or bent ssu c h a s s i l i c a g e l , a l u mi n a , focussing
can be conveniently used for the
charcoal powder and calcium hydroxyapatite puri fi cati on of protei ns.
a r e pac k edint o a c o l u m n i n a g l a s stu b e . T h i s
servesas the stationaryphase.The samplemixture 3. lmmunoelectrophoresis: This technique
i n a s olv ent is l o a d e d o n th i s c o l u mn . T h e i nvol ves combi nati on of the pri nci pl es of
individualcomponentsget differentiallyadsorbed el ectrophoresi sand i mmunol ogi cal reacti ons.
on to the adsorbent. The elutionis carriedout by a lmmunoelectrophoresis is usefulfor the analysisof
b uf f er s y s t em ( m o b i l e p h a s e ). T h e i n d i v i d u al compl exmi xturesof anti gensand anti bodi es.
compoundscome out of the column at different
rates which may be separatelycollected and PHOTOMETRY-COLORIMETER
i d e nt if ied.F or in s ta n c e ,a mi n o a c i d s c a n b e AND SPECTROPHOTOMETER
i d e nt if iedbv ninh v d ri nc o l o ri me tri cme th o d .A n
Photometrybroadlydealswith the studyof tne
automatedcolumn chromatographyapparatus -
phenomenonof l i ght absorpti onby mol ecul esi n
fraction collector- is frequentlyused nowadays.
solution.The specificityof a compoundto absorb
3. High performanceliquid chromatography light at a particularwavelength(monochromatic
(HP t C) : I n g e n e ra l , th e c h ro ma to g ra p hi cligh! is exploitedin the laboratoryfor quantitative
te c hniquesar e s l o w a n d ti me c o n s u mi n g .T h e measurements. From the biochemist's perspective,
separationcan be greatly improvedby applying photometryforms an importantlaboratorytool for
h i g h pr es s ur ein th e ra n g e o f 5 ,0 0 0 -1 0 ,0 0 0p si accurateestimationof a wide varietyof compounds
(poundsper s qua rei n c h ),h e n c eth i s te c h n i q u ei s i n bi ol ogi calsampl es.C ol ori meterand spectro-
also referred(lessfrequently)to as high pressure photometerare the laboratoryinstruments usedfor
liquid chromatography. this purpose.T[ey work on the principlesdiscussed
below.
Moreinformation on chromatography with special
reference to gel-filtration chromatography, ion- W hen a l i ght at a parti cul arw avel engthi s
exchange chromatography, affinity chromatography passed through a solution (incident light), some
and hydrophobic interaction chromatography is amount of it is absorbedand, therefore,the light
givenunderdownstream processing (Chapter20). that comes out (transmittedligh0 is diminished.
The nature of light absorptionin a solution is
ELE CT RO P HO B ES IS governedby Beer-Lambertlaw.
that the amount of transmitted
rhe movement of charged particles (ions) in an ,,_,?"::': 11_states
light decreases.exponentially with an increase in
electric field resulting in their migration towaJs
the concentration of absorbing material (i'e' the
the oppositely charged electrode is known as
amount ligtrt absorbed depends on the
electrophoresis. Molecules with a net positiv; :f
conceltrlti3n of the absorbing molecules)' And
charge (cations) move towards the negative cathoie ,
--, according to Lambert's law, the transmitted light
\\'nile tnose wrtn net neEailve cnarSe (anronS/
exponentially with increase in the
migrate towards positive anode. Electrophoresisis a ilSases
\ltoety useo anatyncat tecnnrque ror Ine separalron
. :- amount of light absorbed is dependent on the
ot Dro tog rca tmo tec ut es s uc n as pr as m a pr oler ns' , , .:
t h i c k n e s so t t h e m e d i u m ) .
ttp op rote rnsa no rm m uno8t oouilns .
The ratio of transmittedlight (l) to that of
Different types of electrophoresis incidentlight (Io)is referredto as transmittance
(T).
I
1. Zone electrophoresis: An inert supporting T=
materialsuch as paperor gel are used. Io
768 B IOTECHNO LO CY

f F*"|---*[,-';l-* w-l -----

@ -----+ F;; l-----
F;;-]
Fig. 64.4 : Diagrammatic representation of the components in a colorimeter.

Absorbance (A) or optical densitl, (OD) is very ULTRACENTRIFUGATION

c om m only us ed in labor a t o r i e s . T h e r e l a t i o n U l t r a c e n t r i f u g a t i o ni s a n i n d i s p e n sa b l eto o l fo r
between absorbanceand transmittanceis expresseo t h e i s o l a t i o n o f s u b c e l l u l a ro r g a n e l l e s ,p r o te i n s a n d
by t he f ollowing equat ion. n u c l e i c a c i d s I n a d d i t i o n , t h i s t e c h n i q u e i s a l so
A = 2 _ log, o T e r n p l o y e d f o r t h e d e t e r n r i n a t i o n o f m o l e cu l a r
weights of macromolecules.
= 2- loe"hT
T h e r a t e a t w h i c h t h e s e d i m e n t a t i o no ccu r s tn
u l t r a c e n t r i f u g a t i o np r i m a r i l y d e p e n d s o n th e si ze
Golorimeter
a r r d s h a p e o f t h e p a r t i c l e s o r m a c r o m o l e cu l e s( i .e .
Colorimeter (or photoelectric colorinreted is the o n t h e m o l e c u l a r w e i g h t ) . l t i s e x p r e s s e di n te r m s o f
instrument used lor the measurement of colouretr sed i me ntat i on co effi c ie nt(s).
s ubs t anc es .This ins t r unr e n t i s o o e r a t i v e i n t h e T h e s e d i m e n t a t i o nc o e f f i c i e n t h a s th e u n i ts o f
visible range (400-800 nnr) of the electrcmagnetic s e c o n d s .l t w a s u s u a l l ye x p r e s s e di n u n i ts o f 1 0 1 3 s
s pec t r um of light . The wor k i n g o f c o l o r i m e t e r i s ( s i n c e s e v e r a l b i o l o g i c a l m a c r o m o l e c ul e so ccu r i n
based on the principle of Beer-Lambertlaw'. this range), which is designated as one Svedberg
The c olor im et er , in gen e r a l c o n s i s t s o f l i g h t unit. For instance,the sedimentationcoefficient of
h e m o g l o b i n i s 4 x 1 0 - 1 3 s o r 4 5 ; r i bo n u cl e a se i s
s our c e. f ilt er s am ole holde r a n d d e t e c t o r w i t h .l
2 x 0 1 3 s o r 2 5 . C o n v e n t i o n a l l y ,t h e su b ce l l u l a r
dis play ( m et er or digit al) . A f i l a m e n t l a m p u s u a l l y
serves as a light source. The filters allow the organellesare often referredto by their S value e.g.
passageof a small range of wave length as incident 70S ribosome.
light . The s am ple holder is a s p e c i a l g l a s s c u v e t t e
lsolation of subcellular
wit h a f ix ed t hic k nes s .The p h o t o e l e c t r i c s e l e n i u n r
organelles by centrifugation
cells are the most common detectors used in
colorimeter. The diagrarnmatic representationof a T h e c e l l s a r e s u b j e c t e d t o d i sr u p ti o n b y
colorimeter is depicted in Fig. 64.4 sonication or osmotic shock or by use of
homogenizer. This is usually carried out in an
Spectrophotometer isotonic (0.25 M) sucrose. The advantage with
s u c r o s e m e d i u m i s t h a t i t d o e s n o t ca u se th e
The spectrophotometer primarily differs from o r g a n e l l e st o s w e l l . T h e s u b c e l l u l a rp a rti cl e sca n b e
colorimeter by covering the ultraviolet region (200- separated by differential centrifugation. The most
400 nm) of the electromagneticspectrum- Further, commonly employed laboratory method separates
the spectrophotometeris more sophisticated with s u b c e l l u l a r o r g a n e l l e s i n t o 3 m a j o r fr a cti o n s-
several additional devices that ultimately increase nuclear, mitochondrial and microsomal (Fig. 64.5).
the sensitivitv of its operation severalfold when
When the homogenate is centrifuged aI 700 g
compared to a colorimeter. A precisely selected
f o r a b o u t 1 0 m i n , t h e n u c l e a r f r a c t i o n ( i n cl u d e s
wavelength (say 234 nm or 6.10 nm) in botl.rultra
p l a s m a m e m b r a n e ) g e t s s e d i m e n te d . On
v iolet and v is ible r ange c a n b e u s e d f o r
c e n t r i f u g i n g t h e s u p e r n a t a n t ( l ) a t 1 5 ,0 0 0 g fo r
m eas ur em ent s . ln plac e o f g l a s s c u v e t t e s ( i n
a b o u t 5 m i n n r i t o c h o n d r i a l f r a c t i o n ( th a t i n cl u d e s
colorimeter), quartz cells are used in a spectro-
lysosonres, peroxisomes) is pelleied. Further
photometer.
c e n t r i f u g a t i o no f t h e s u p e r n a t a r r (t l l ) at 1 0 0 ,0 0 0 g
The spectrophotometei' has similar basic for about 60 min separates microsomal fraction
c om ponent s as des c r ibed f o r a c o l o r i m e r e r ( t h a t i n c l u d e s r i b o s o m e s a n d en d o p l a sm i c
Gig. 6a.4 and its operation is also based on the r e t i c u l u m ) . T h e s u p e r n a t a n t ( l l l ) t h e n o b ta i n e d
Beer-Lambertlarv. corresoondsto the cvtosol.
 C FIE MIS TRAYN D B IOC H E MIS TRTOOLS
A N D B IO P H Y SIC AL
C hapt er64 : B I O OR C A N IC Y 769

the estimation of insulin in human serum. This

technique has revolutionized the estimation of
several compounds in biological fluids that are
found in exceedinglylow concentrations(nanogram
o r p i c o g r a m ) . R I A i s a h i g h l y s e n s i t i v ea n d s p e c i f i c
analyticaltool.

Principle
I zooo * to min
J R a d i o i m m u n o a s s a yc o m b i n e s t h e p r i n c i p l e s o f
radioactivity of isotopes and immunological
reactionsof antigen and antibody; hence the name.

SupernatantI The principle of RIA is primarily based on the

competition between the labeled and unlabeled
antigens to bind with antibody to form antigen-
Nuclear antibody complexes(either labelled or unlabeleo).
fraction
T h e u n l a b e l e d a n t i g e n i s t h e s u b s t a n c e( s a y i n s u l i n )
I15, 000g x 15 m in to be determined.The arrtibodyto it is produced by
|
injecting the antigen to a goat or a rabbit. The
specific antibody (Ab) is then subjected to react
with unlabeled antigen in the presenceof excess
a m o u n t s o f i s o t o p i c a l l y l a b e l e d ( 1 3 1 1a) n t i g e n ( A g +)
with known radioactivity. There occurs a
conrpetition betrveen the antigens (Ag+ and Ag) to
- Mitochondrial b i n d t h e a n t i b o d y . C e r t a i n l y ;t h e l a b e l e d A g + w i l l
fraction h a v e a n u p p e r h a n d d u e t o i t s e x c e s sp r e s e n c e
Ag++Ab;{.i,
Jt,o o ,o o o sx6 0 mi n
]---+Aq-
J
Ag-Ab
lll
Supernatant
As the concentration of unlabeled antigen (Ag)
i n c r e a s e st h e a m o u n t o f l a b e l l e d a n t i g e n - a n t i b o d y
Microsomal complex (Ag+-Ab) decreases. Thus, the
fraction
concentration of Ag+-Ab is inversely related to the
c o n c e n t r a t i o no f u n l a b e l e dA g i . e . t h e s u b s t a n c et o
Fig. 64.5 : Separation of subcellular fractions by
ditfe rential centrif ugation, be determined. This relation is almost linear. A
standard curve can be drawn by using different
c o n c e n t r a t i o n so f u n l a b e l e d a n t i g e n a n d t h e s a m e
q u a n t i t i e so f a n t i b o d y a n d l a b e l e d a n t i g e n .
Th e p urity (or c ont am inat ion)of t he s ubc ellula r
fractionation can be checked by the use of marker The labeled antigen-antibody(Ag+-Ab)complex
enzymes. DNA polymerase is the marker enzyme is separated by precipitation. The radioactivity of
for nucleus, lvhi!e glutamate dehydrogenase and l 3 l l p r e s e n t i s A g +- A b i s d e t e r m i n e d .
glucose 6-phosphatase are the markers for
mito ch on dria an d r ibos om es , r es pec t iv el y . Applications
Hexokinase is the marker enzvme for cvtosol.
RIA is no more limited to estimating of
hormones and proteins that exhibit antigenic
RADIOIMMUNOASSAY properties. By the use of haptens (small molecules
Ra dio immun oa s s ay ( RlA) was c iev eloped in s u c h a s d i n i t r o p h e n o l , w h i c h , b y t h e m s e l v e s ,a i 'e
1959 by Solomon, Benson and Rosalyn Yalow for not antigenic), several substances can be made

Siotechnology [49]
77(, B IOTECHNO LO CY

antigenicto elicit specificantibody responses.In

this way, a wide varietyof compoundshave been
brought under the net of RIA estimation.These
include peptides, steroid hormones, vitamins,
drugs,antibiotics,nucleicacids,structuralproteins
and hormonereceptorproteins.
Radioimmunoassay has tremendousapplication
in the diagnosis cancersand
of hormonaldisorders,
therapeuticmonitoring of drugs, besides being
usefulin biomedicalresearch.

E N Z Y M E.L IN KE D
IMMUNOSORBANT ASSAY
Enzyme-linked immunosorbant assay(ELISA)is
a non-isotopicimmunoassay. An enzymeis usedas
a label in ELISAin place of radioactiveisotope
employedin RlA. ELISA is as sensitiveas or even
more sensitivethan RlA. In addition,there is no
riskof radiationhazards(asis the casewith RIA)in
E L ISA .

Principle
E L ISA i s b a s e d o n t he i mmunochemi car F.d,fi]
principlesof antigen-antibody reaction.The stages
of ELISA,depictedin Fig. 64.6, are summarized. Fig. 64.6 : Diagrammatic representation of enzyme-
linked immunosorbant assay (ELISA).
1. The antibody against the protein to be
determinedis fixed on an inert solid such as
polystyrene. 5. The enzyme activity is determined by its
action on a substrateto form a product (usually
the protei n
2 . T h e b i o l o g i c asl a mp l econtai ni ng
coloured). This is related to the concentration of
to be estimatedis appliedon the antibodycoated
the protein being estimated.
surface.
3. The proteinantibodycomplexis then reacted Applications
with a secondproteinspecificantibodyto which
E L I S A i s w i d e l y u s e d f o r t h e d e t e r m i n a ti o n o f
an enzyme is covalentlylinked. Theseenzymes
small quantities of proteins (hormones, antigens,
must be easily assayableand produce preferably
antibodies) and other biological substances.The
coloured products. Peroxidase,amylase and
most commonly used pregnancy test for the
a l k a l i n ep h o s p h a ta saere c o mmonl yused.
d e t e c t i o n o f h u m a n c h o r i o n i c g o n a d o tr o p i n ( h C C
4 . A fte rw a s h i n gth e u n b oundanti bodyl i nked i n u r i n e i s b a s e d o n E L I S A .B y t h i s t e st, p r e g n a n c\
enzyme,the enzymeboundto the secondantibody can be detected within few days after conception
complex is assayed. E L I S Ai s a l s o u s e f u l f o r t h e d i a g n o s i sof AID S.
 Iiving ma tler is t om pos c d of m ainly s ix CHEMICAL MOLECULES OF LIFE
Jhe
I elenrents- carbon, hydrogen, oxygen,
Life is composed of lifeless chemical molecules.
nitrogen, phosphorus and sulfur. These elements
A single cell of the bacterium, Escherichia coli
together constitute about 90% of the dry weight of
c o n t a i n s a b o u t 6 , 0 0 0 d i f f e r e n to r g a n i c c o m p o u n d s
ihe h uma n bo dy . Sev er al ot her f unc t ionally .1
It is believedthat man mav contain about 00.000
, mpo rtan t ele rne nts ar e als o f ound in t he c ells .
different types of molecules although only a few of
T hese includ e Ca, K, Na, Cl, M g, Fe, Cu, Co, 1, 7n,
them have been characterized.
F. N1o and Se.

Carbon-a unique element of life Gornplex biornolecules

Carb on is th e m os t pr edom inant and v er s at ile
element of life. lt possessesa unique property to
iorm in finite nu m ber of c om pounds . This is
attributed to the ability of carbon to form stabte
cova len t b on ds a nd C- C c hains of unlim it eo
ength. lt is estimated that about 90% of
:ompounds found in living system invariably
: cnta in ca rbo n.
The organic compounds such as amino acids,
nucleotides and monosaccharides serve as the
monomeric units or building blocks of comptex
biomolecules-proteins, nucleic acids (DNA ano
RNA) and polysaccharides, respectively. The
i m p o r t a n t b i o m o l e c u l e s ( m a c r o m o l e c u l e s )w i t h
their respectivebuilding blocks and major functions
are given in Table 65.1. As regardslipids, it may be

Tlsre65.1 The major conplex biomoleculesof cells

Biomolecule Building block (repeatingunit) hlajor functiotts

Pr ot ein Amino
acids Fundamentalbasisofstructure
and
function
ofcell(static
anddynamic
lunctions).
acid (DNA)
Deoxyribonucleic Deoxyribon
ucleotides Repository
of hereditary
information
acid (RNA)
Ribonucleic Ribonucleotides Essentially forprotein
required biosynthesis.
(glycogen)
Polysaccharide (Elucose)
Monosaccharides Storage
formofenergy
to meetshortterm
0eman0s.
L i pids glycerol
Fattyacids, Storage
formofenergylo meetlongterm
demands:structural
components ofmembranes.

771
772 B IOTE CHNO LO CY

Functions of carbohYdrates
participatein a wide range of
Carbohydrates
functi ons
Constituent Percent (%o) Weight (kg) 1. Theyarethe mostabundantdietarysourceof
Waler o t.o 40 energy(4 Cal/g)for all organisms.
Protein 17.0 11 2. Carbohydratesare precursorsfor many
Lipid 1 3 .8 9 organiccompounds(fats,amino acids).
Carbohydrate ' 1 .5 1 (as glycoproteinsand glyco-
3. Carbohydrates
Minerals o. l 4 lipids)participatein the structureof cell membrane
suchascel l grow th,adhesion
andcel l ul arfuncti ons
and ferti l i zati on.
noted that they are not biopolymersin a strict
sense,but majorityof them containfatty acids. 4. Carbohydratesalsoserveas the storageform
of energy(glycogen)
to meetthe immediateenergy
Structural heirarchy of an organism demandsof the body.

The macromolecules (proteins,lipids, nucleic CLASSIFICATION OF CARBOHYDRATES

acids and polysaccharides) form supramolecular Carbohydrates are often referred to as
assemblies (e.g.membranes) which in turn organize saccharides(Greek : sakcharon-sugar).They are
into organelles, cells,tissues,organsand finallythe broadlyclassifiedinto 3 groups-monosaccharides,
w h o l e o rg a n i s m. oligosaccharides and polysaccharides. This
categorizationis basedon the number of sugar
Chemical composition of man
units. Mono- and oligosaccharides are sweet to
T h e c h e m i c a lc o mp o s i ti onof a normal man, taste,crystallinein characterand solublein water,
weighing65 kg, is givenin Table65.2.Wateris the hencethey are commonlyknown as sugars.
solventof life and contributes to morethan 60% of
the weight.This is followed by protein(mostlyin Monosaccharides
mu s c l e )a n d l i p i d (m o s tl yi n adi poseti ssue).The Monosaccharides (Creek : mono-one)are the
carbohydrate contentis ratherlow which is in the simplest group of carbohydratesand are often
form of glycogen. referredto as simplesugars.They havethe general
The basic information on the various formula Cn(H2O)n,and they cannot be further
hydrolysed.
biomolecules is essential for a betterunderstanding
of the conceptsof biotechnology. An overviewof Based on the number of carbon atoms,
the chemistryof carbohydrates, lipidsand proteins the monosaccharides are regardedas trioses(3C),
(amino acids) is describedin this Chapter.The tetroses(4C), pentoses(5C), hexoses(6C) and
biomolecules namely nucleic acids (DNA and heptoses(7C).Theseterms along with functional
RNA) which are directly relevant to biotechnology groups are used while naming monosaccharides.
are described in Chapter 11. For instance, glucose is a aldohexose while
fructose is a ketohexose.

Oligosaccharides
OJigosaccharides (Greek : oligo-few) contain
Carbohydrates are the most abundantorganic
2-10 monosacchari demol ecul es which ar e
moleculesin nature.They are primarilycomposed
liberatedon hydrolysis.Basedon the number of
of the elemenrscarbon,hydrogenand oxygen.fhe
monosaccharide unitspresent,the oligosaccharides
n a me c a rb o h y d ra tel i te ra l l y means' hvdratesof
are further subdivided to disaccharides, tri-
carbon.'
saccharidesetc.
Carbohydratesmay be defined as polyhydroxy-
aldehydes or ketonesor compounds which produce Polysaccharides
them on hydrolysk. The term 'sugar' is applied to (Creek : poly-many)are poly-
Polysaccharides
carbohydrates unitswith high molecular
solublein water and sweetto taste. mersof monosaccharide
C h a pt er65 : B I O M O L E C U L ES 773

iveight (u p to a million) . They ar e us ually t as t eles s

H -C = O H -C = O
{ non-sug ars)an d fo r m c olloids wit h wat er . Poly -
saccharides are of two types-homopoly-
I I
H-C-oH HO-C-H
saccharides and heieropolysaccharides.
I I
cH2oH cH2oH
MONOSACCHARIDES D-Glyceraldehyde L-Glyceraldehyde
Stereoisomerismis an imoortant character of
monosaccharides. Stereoisomersare the compounds H -C = O H -C = O
that have the sa,'nestructuralformulae but differ rn I I
: heir spa tial con figu r at ion. H-C-OH HO-C-H
I I
A carbon is said to be asymmetric when it is HO-C-H H-C-OH
:ttached to four different atoms or groups. The I I
rumber of asymmetriccarbon atoms (n) determines H-C-OH HO-C-H
: re p ossib leiso mersof a giv en c om pound whic h is I I
H-C-oH HO-C-H
: qual to 2 n. Clu c os e c ont ains 4 as y m m et r ic
:arbo ns a nd th us ha s 16 is om er s .
I I
cH20H cH2oH
D-Glucose L-Glucose
Glyceraldehyde-
the reference carbohydrate Fig. 65.1 : D- and L-forms of glucose compareC
with D- and L-glyceraldehydes (the reference
Clyce rald eh yd e ( t r ios e) is t he s im ples t m ono-
carbohydrate).
.acch arid e with o ne as y m m et r ic c ar bon at om . lt
:\ists as two stereoisomers,and has been chosen
;s the reference carbohvdrate to represent the
of an ootical isomer. it will be rotated either to the
: : ructure o f all o the r c ar bohv dr at es .
right or left. The Ierm dextrorotatory (+) and
levorotatory (-) are used to compounds that
D. and L-isomers
respectively rotate the plane of polarized Iight to
T he D- an d L-isom er sar e m ir r or im agesof eac h the right or to the left.
,,:her The sp acial o r ient at ion of - H and - O H
:.oups on the carbon atom (C, for glucose) that is GLYCOSIDES
. djace nt to th e term inal pr im ar y alc ohol c ar bon
Clycosides are formed when the hemiacetal or
: ete rmin eswh eth er t he s ugar is D- or L- is om er .lf
-re -OH gro up is o n t he r ight s ide, t he s ugar is of hemiketal hydroxyl group (of anomeric carbon) of a
carbohydrate reacts with a hydroxyl group of
l-series, and if on the left side, it belongs to
another carbohydrate or a non-carbohydrate (e g.
--.eries The structuresof D- and L-glucose basecl
^ the re fere nce m onos ac c har ide, D- and methyl alcohol, phenol, glycerol). The bond so
formed is known as glycosidic bond and th'e non-
--:ivc era lde hyde (g ly c er os e) ar e depic t ed in
carbohydrate moiety (when present) is referred to
Fig. 65.1
as aglycone.

It may be rroted that the naturally occurring

rrono sa ccha ride s in t he m am m alian t is s ues ar e
rost ly o f D-co nfig ur at ion.The enz y m e m ac hiner y
ri cells is soe cific t o m et abolis e D- s er ies of
non osacch a rid es.
Optical activity of sugars
Ootical activitv is a characteristic feature of
:ompo un ds with a sym m et r icc ar bon at om . W hen a
:reamof po larized light is pas s edt hr ough a s olut ion
DERIVATIVES OF MONOSACCHARIDES
There are severalderivativesof nronosaccharides,
s o m e o f w h i c h a r e p h y s i o l o g i c a l l yi m p o r t a n t
1 . A m i n o s u g a r s : Wh e n o n e o r m o r e h y d r o x y l
groups of the monosaccharidesare replaced by
amino groups, the products formed are amino
sugars e.g. D-glucosamine, D-galactosamine.
They are present as constituents of heteropoly-
saccharides
774 B IOTECHNO LO G Y

2. Deoxysugars: These are the sugarsthat bonds. lt is

D-glucoseunits held by cr-glycosidic
contain one oxygen less than that presentin the knov,rnas glucosanor glucan.
parent molecule. The groups -CHOH uld Starch consists of two polysaccharide
-CH2OH -CHr and -CH, due to the components-water soluble amylose (15-20%)and a
_become
absenceof oxygen.D-2-Deoxyribose is the most water insoluble amylopectin (80-85%). Chemically,
important deoxysugarsince it is a structural amylose is a long unbranched chain with
c o n s ti tu e n toDf N A (i n c o n trasttoD -ri bose i n R N A ). uni ts hel d by a ( 1 - r 4)
ZOO_f ,OOOD _gl ucose
3. L-Ascorbicacid (vitamin C) : This is a water- glycosidic linkages.Amylopectin, on the otherhand,
soluble vitamin, the structureof which closely is a branchedchainwith cr (1 -+ 6) glycosidicbonds
resembles that of a monosaccharide. at the branchingpoints and cr (1 -+ 4) linkages
everywhere else.Amylopectinmoleculecontaining
DISACCHARIDES a few thousand glucoseunitslookslike a branched
tree (20-30 glucoseunitsper branch).
Amo n g th e o l i g o s a c c hari des, di sacchari des are
the most common.As is evidentfrom the name,a
Glycogen
disaccharide consistsof two monosaccharide units
(similaror dissimilar)held together'oy a glycosidic Clycogenis the carbohydrate reservein animals,
bond.fhey are crystalline, water-soluble and sweet hence often referred to as animal sfarch. lt ls
to taste.The disaccharides are ci two types present in high concentration in liver, followed
by muscl e,brai n etc. Gl ycogeni s also f ound in
w i th free al dehyde plantsthat do not possess
1 . R e d u c i n gd i s a c c h ari des chlorophyll(e.9.yeast,
ol keto group e.g. maltose,lactose. fungi ).
2 . N o n -re d u c i n gd i s acchari des w i th no free The structureof glycogenis sinrilarto that of
aldehydeor keto group e.g. sucrose,trehalose. amyl opecti nw i th more number of br anches.
C l ucosei s the repeati nguni t i n g lycogenjoined
POLYSAGCHARIDES togetherby o (1 -+ 4) glycosidicbonds, and o
PolySaccharides simply glycans)consist5f (1 + 6) gl ycosi di cbondsat branch ingpoint s.
(or
re p e a t u n i ts o f m o n osacchari desor thei r
derivatives, heldtogetherby glycosidicbonds They Gellulose
are primarily concerned with two important
C el l ul oseoccursexcl usi vel yi n p lant sand it is
functions-structural, and storageof energy.
the most abundantorgani c subs t ancein plant
Polysaccharides are of two types ki ngdom.l t i s a predomi nant of plant
cons t it uent
cel l w al l .C el l ul ose i s total l yabsentin anim albody'
1. Homopolysaccharides which on hydrolysis
y i e l d o n l y a s i n g l ety p e o f monosacchari de.They C el l ul osei s composedof B -D- glucoseunit s
are named based on the nature of the linked by 9 (t --> 4) glycosidic bonds. Cellulose
monosaccharide unit. Thus,glucansare polymers cannotbe di gested by mammal s-i ncluding m an-
of glucose whereas fructosans are polymers of due to lack of the enzymethat cleavesp-glycosidic
fructose. bonds (o amylasebreakscr bonds only). Certain
rumi nants and herbi vorous anim als cont ain
2. Heteropolysaccharides on hydroiysisyield a
mi croorgani sms i n the gut w hi ch produceenzym es
mixture of a few monosaccharidesor therr
that can cleaveB-glycosidicbonds. Hydrolysisof
derivatives.
celluloseyieldsa disaccharide cellobiose, followed
by p-D-glucose.
HOMOPOLYSACCHABIDES
Cellulose, though not digested, has Sreat
Starch
i mportancei n human nutri ti on.lt is a m ajor
Starch is the carbohydratereserveof plants constituent of fiber, the non-digestable
which is the most importantdietary source for carbohydrate.The functionsof dietaryfiber include
h i g h e r a n i ma l s ,i n c l u d i ngman. H i gh contentof decreasing the absorption of glucoseand cholesterol
starchis found in cereals,roots,iubers,vegetables from the intesting,besidesincreasingthe bulk of
etc. Starch-is a homopolymer composed of feces.
Chaot er65 : B IOMOL E C U L ES 775

H ET E R O PO LYSACC H A R' D E S 1. Simple lipids : Esters of fatty acids with

alcohols.These are mainly of two types
When the polysaccharides are composed
of different types of sugars or their derivative5, (a) Fats and oils (triacylglycerols): These are
they are referred to as heteropolysaccharidesor esters of fatty acids with glycerol. The
heteroglycans. difference between fat and oil is only
physical.Thus, oil is a liquid while fat is a
Mucopolysaccharides solid at room ternperature.
These are heteroglycansmade up of repeating (b) Waxes: Estersof fatty acids (usually iong
un its of su ga rder iv at iv es ,nam ely am ino s ugar sa n d chain) lvith alcohols other than glycerol
uro nic a cid s. M uc opoly s ac c har ides ar e m o r e Cetyl alcohol is most commonly found rn
commonly known as glycosaminoglycans (GAG). waxes.
Acetylated amino groups, besides sulfate and
carboxyl groups are generally present in GAC 2. Complex (or compound) lipids: Esters of
structu re. fatty acids with alcohols containing additional
groups such as phosphate, nitrogenous base,
Some o f th e m uc opoly s ac c har idesar e f ound r n carbohydrate, protein etc. They are further
combination with prote!nsto form mucoproteins or divided:
mucoids or proteoglycans Mucoproteins ntay
(a) Phospholipids: Lipids cbntaining phosphoric
contain up to 95% carbohydrate and 5"h protein
acid and frequently a nitrogenous base.
Mucopolysaccharidesare essentialcomponents of This is in addition to alcohol and fatty
tissue structure. The extracellular spaces of tissue ac icls.
{particularlyconnective tissue-cartilage,skin, blood
vessels,tendons) consist of collagen and elastin fibers (b) Glvcolipids: lhese lipids contain a fatty
embedded in a matrix or grourrdsubstance.The ground acid, carbohydrate and nitrogenous base.
substanceis predominantly composed of CAC. T h e a l c o h o l i s s p h i n g o s i n e ,h e n c e t h e y a r e
a l s o c a l l e d a s g l y c o s p h i n g o l i p i d s .C l y c e r o l
Th e imp ortant m uc opoly s ac c har ides inc l u d e
and phosphateare absente.g., cerebrosides,
h ya luro nic a ci d, c hondr oit in 4- s ulf at e, hepa r i n , gangliosides.
dermatan sulfate and keratan sulfate.
(c) Lipoproteins : Macromolecular complexes
of lipids wilh proteins.
(d) Other complex lipids: Suliolipids, amino-
l i p i d s a n d l i p o p o l y s a c c h a r i d e sa r e a m o n g
Lipids(Greek: lipos-fat)are of greatimportance
the other complex lipids.
to the body as the chief concentrated storageform
of energy,besidestheir role in cellular structure 3. Derived lipids : These are the derivatives
and v ar iousot h e rb i o c h e m i c afu
l n c ti o n sA. s such, obtained on the hydrolysis of group I and group 2
'l i p i d s .
lipids are a heterogeneous group of compounds. l i p i d s w h i c h p o s s e s st h e c h a r a c t e r i s t i c so f
These include glycerol and other alcohols, fatty
Lipids may be regarded as organic substances
acids, mono- and diacylglycerols, lipid soluble
relatively insoluble in water, soluble in organic
vitamins, steroid hormones, hydr:ocarbons and
solvents (alcohol, ether etc.), actually or
ketone bodies.
potentially related to fatty acids and utilized by
the living cells. 4 . M i s c e l l a n e o u sl i p i d s : T h e s e i n c l u d e a l a r g e
number of compounds possessing the
Unlik et he p o l y s a c c h a ri d epsro, te i n sa n d n u c l ei c
squalene,
ac ids , lipids a re n o t p o l y me rs T . h e y a re m o stl y c h a r a c t e r i s t i c so f l i p i d s e . g . , c a r o t e n o i d s ,
hydrocarbons such as pentacosane(in bees wax),
s m allm olec ul e s .
terDenesetc.
CLA S S I F I CA T ION O F L IPID S 5. Neutral lipids: The lipids which are
(modifiedfrom Bloor) uncharged are referred to as neutral lipids. These
Lipidsarebroadlyclassified
;nto simple, complex,derivedand miscellaneous are mono-, di-, and triacylglycerols, cholesterol
ioids,which are furthersubdivided. and cholesteryl esters.
776 B IOTECHNO LO CY

Functions of lipids a\

Lipidsperformseveralimportantfunctions
cH2-o- c-R1
fuel reserveof the
1. Theyare the concentrated
I
body (triacylglycerols). R'-c-o-?H
2. Lioids are tlre constituentsof membrane ll
structureand regulatethe membranepermeability CH2-O-C-R3
(p h o s p h o l i p i dasn d c h o l e sterol ).
Triacylglycerol
3 . T h e ys e rv ea s a s o u rceof fat sol ubl evi tami ns
(A, D, E and K). Fig, 65.2 : General structure of triacylglycerol.
4 . L i p i d s a re i mp o rtantas cel l ul ar metabol i c
regulators(steroidhormonesand prostaglandins)
Essential fatty acids
FATTY ACIDS The fattyacidsthatcannotbe synthesized by the
body and,therefore,shouldbe suppliedin the diet
Fatty acids are carboxylic acids with
are knownas essential fattyacids(EFA). Cfremically,
hydrocarbon sidechain.Theyarethe simplestform
they are polyunsaturatedfatty acids, namely
of lipids.
Iinoleic acid (18 : 2; 9, 12) and linolenic acid
Even and odd carbon fatty acids (18 : 3; 9,12, 15).A rachi doni caci d ( 20: 4; 5, B,
11, 14) becomesessenti ali f, i ts precur sorlinoleic
Most of the fatty acids that occur in natural acid is not orovided in the diet in sufficient
l i p i d sa re o f e v e nc a rb o n s(usual l y14C -20C )Thi s amounts.
is due to the fact that biosvnthesis of fattv acids
m a i n l y o c c u rsw i th th e s equenti aladdi ti onof 2 TRIACYLGLYCEROLS
c a rb o nu n i ts .P a l mi ti ca c i d (16C )and steari caci d
(18 C )a reth e m o s tc o mmon.A mongthe odd chai n Triacylglycerols (formerlytriglycerides) are the
fatty acids, propionic acid (3C) and valeric acid esters.ofglycerol with fatty acids.The fats and oils
(5 C )a re w e l l k n o w n . that are widely distributedin both plants and
ani mal sare chemi cal l ytri acyl gl yc er ols.
They ar e
Saturated and unsaturated i nsol ubl ei n w aterand non-pol ari n char act erand
fatty acids commonlyknown as neutralfats.
Saturatedfatty acids do not contain double Fatsas storedfuel : Triacylglycerols
arethe most
bonds,while unsaturated fattyacidscontainone or abundantgroupof l i pi dsthat pri ma r ilyf unct ionas
moredoublebonds.Bothsaturated and unsaturated fuel reserves of animals.The fat reserveof normat
fatty acids almost equally occur in the natural humans (men 20oh, women 25% by weight) rs
l i p i d s .F a ttya c i d sw i th o n e doubl ebondare know n sufficientto meetthe body caloricrequirements for
as monounsaturated and those with 2 or more 2-3 months.
d o u b l e b o n d s a re c ol l ecti vel y know n as
Structuresof acylglycerols: Monoacylglycerols,
polyunsaturated fatty acids (PUFA).
diacylglycero_lsand triacylglycerols,respectively'
Shorthand representation of fatty acids consistingof one, two and three moleculesof fatti'
acids esterifiedto a molecule of glycerol, are
Insteadof writingthe full structures, biochemists known.Amongthese,triacylglycerols are the most
employ shorthand notations (by numbers) to i mportantbi ochemi cal l y.
representfatty acids.The generalrule is that the
Triacylglycerolsof plants have higher content
total number of carbon atoms is written first,
of unsaturatedfatty acids comparedto that of
followed by the number of double bonds and
ani mal s.
fi n a l l yth e (fi rs tc a rb o n )p osi ti onof doubl ebonds,
startingfrom the carboxylend.Thus,saturated fatty
a c i d , p a l m i ti ca c i d i s w ri t tenas 16:0, ol ei c aci d P H OS P H OLIP ID S
a s 1 8 : 1 ; 9 , a ra c h i d o n i aci c d as 2O : 4:5, B , 11, These are compl ex or compound lipids
14. contai ni ngphosphori caci d, i n addit iont o f at t r
 l r apt er 65 : B I O MOL E C U L ES 777

ca
20 zo
1B 25
17
27

Steroid nucleus Cholesterol

Fig. 65.3 : Structures of stetoids (A, B, C-Perhydrophenanthrene;D-Cyclopentane).

. - : = . nit r ogeno ubsa s ea n d a l c o h o l T

. h e rea retw o
- - . + . of phos ph o l i p i d s
C lycerophosphol ipids(or phosphoglycerides) P r o t e i n s a r e i h e m o s t a b u n d a n t o r g a n i c
'-= : c ont aingly c e ro a
l s th e a l c o h o l .e .g . l e c i th i ns, molecules of the living system.They occur in every
.-: - alins ,phos ph a ti d y l i n o s i p
tohl o
, s p h a ti d y l s e ri ne,
part of the cell and constituteabout 50% of cellular
: ::inalogens- dry weight. Proteins form the fundamental basis of
structure and function of life
i S phingoph o s p h o l i p i d (osr s p h i n g o m y e l ins)
-,: c ont ain s ph i n g o s i n ea s th e a l c o h o l . e .g.
Functions of proteins
=- : n ide.
Proteins perform a great variety of specialized
L IP O P RO T E I NS a n d e s s e n t i a lf u n c t i o n s i n t h e l i v i n g c e l l s . T h e s e
functions may be broadly grouped as static
Lipopr ot eins
ar e mo l e c u l a cr o m p l e x e so f l i p i ds (structuraD and dynamic.
.', : n pr ot eins .T h e y a re th e tra n s p o rtv e h i c l es
'-' I ipids in t h e c i rc u l a ti o n .T h e re a re fi v e Structural functions : Certain proteins perform
'brick and mortar' roles and are primarily
,pes of lipoproteins,namely chylomicrons, very
responsible for structure and strength of body.
low density Iipoproteins (VLDL), Iow density
These include collagen and elastin found in bone
lipoproteins (LDL), high density lipoproteins (HDL)
matrix, vascular system and other organs and o-
and free fatty acid-albumin complexes.
keratin present in epidermal tissues.

STE RO I DS Dynamic functions : The dynamic functions of

proteins are more diversified in nature. These
S t er oids
ar e t he c o mp o u n d cs o n ta i n i n ga c y c l ic include proteins acting as enzyme+ hormones,
:i e r oid nuc leus (o r ri n B) n a me l y c y c l o p e n ta n o- blood clotting factors, immunoglobulins, membrane
cerhydrophenanthrene (CPPP).lt consistsof a receptors,storageproteins, besidestheir function in
ch enant hr ene nucl e u s(ri n g sA , B a n d C ) to w h i c h genetic control, muscle contraction, respirationetc.
a cyclopentane ring (D) is attached. Proteins perfcrming dynamic functions are appro-
T her e ar e s eve ra ls te ro i d si n th e b i o l o g i c a l priately regarded as 'the working horset of cell.
svstem.These include cholesterol, bile acids,
Proteins are polymers of amino acids
vitamin D, sex hormones and adrenocortical
h or m ones .lf t he s te ro i d c o n ta i n so n e o r mo re Proteins on complete hydrolysis (with concen-
h ydr ox y l B r oups i t i s c o m m o n l y k n o w n a s trated HCi for several hours) yield L-o-amino acids.
sfer ol ( m eans s o l i d a l c o h o l ). T h e s tru c tu reos f This is a common property of all the proteins.
steroid nucleus and cholesterolare depicted in Therefore, proteins are the polymers of L-a-amino
Fig. 65.3. acids.
780 B IOTE C HNO LO CY

Name Symbol Structure Specialgroup present

3 letters 1 letter

lll. Sulf ur c ont aining am ino ac i d s

8. Cysteine Cys cH2-cH-coo- Sulfhydryl

ll+
SH NHS

Cystine cHz-cH-coo DisulJide

tl
S NH;
I
S
I
cH2-cH-coo-
la
NHs

9. Methionine Met M cH2-cH2-cH-coo- Thioether

ll+
S-CHs NHs
l V. Ac i d i c a m i n o a c i d sa n d th e i r ami des
Bo
-ooc-cH2-cH-coo-
10. Aspartic
acid Asp D B-Carboxyl
l+
NHi
11. Asparagine Asn N H2N-C-CH2-CH-COO-
- l- l+ Amide

o NHs
lBa
-ooc-cH2-cH2-cH-coo-
12. Glutamic
acid Glu E yCarboxyl

NH;

13. Glutamine Gln H2N-C-CH 2-CH 2-cH-coo- Amide

Ll
o
.l*
NHi
V. Bas ic am ino ac ids
e6Yp0
14. Lysine Lys K cH a-cH r-cH 2-cH 2-cH-coo- e-Amino
tl a
NH; NHi

15.Arginine Arg B NH-CH Guanidino

"-CH "-CH -"-CH-COO_
I l*
C = NH, NH;
I
t\Hz
cH.--cFrcod
16. Histidine His 'l lmidazole
N NH;
Hi.r -
\r.
C hapt er65 : B I O MOL E C U L ES 781

Name Symbol Structure Specialgroup present

3 letters 1 letter

Vl. Aroma tic am ino ac ids

.----\
1 7. Ph en yla lanine
Phe (, >cH2-cH--coo- or phenyl
Benzene
\-// - |
I

NHi

'18.Tyrosine
,t ----\
lyr HO <' \,/.1
)-Cr-1.-cH-coo- Phenol
:l+
NHg

cH2--cFrcod
-l+
19.TryptophanTrp W Indole
NHi

\ ll. lmino acid

H2 C- CH2
llH
20. Proline Pro I t./ or Pynolidine
Hzc- ,c \ | |
'coo- \ * '^coo-
-N'
I I
H H
Note: R groupis shownin coloul

PRIMARY STRUCTURE OF PROTEIN

Each protein has a unique sequenceof amino

acids which is determined by the genescontained
Pro tein s a re the poly m er s of L- q, - am inoac id s .
- -e structure of proteins is rather complex in DNA. The primary structure of a protein is
..^ ich ca n be d iv ided int o 4 lev els of or ganiz at io n l a r g e l y r e s p o n s i b l ef o r i t s f u n c t i o n .

Fis. 55.5) :
Peptide bond
- Primary str uc t ur e: The linear s equenc e o f
The amino acids are held together in a protein
,- ro acids form ing t he bac k bone of pr ot ein s
by covalent peptide bonds or linkages.These bonds
:,- 1rpe ptid es).
are rather strong and serve as the cementing
I Secon da ry s t r uc t ur e: The s pac i a l material between the individual amino acids.
.'-angement of protein by twisting of the
: ,- \pe ptid e cha in. F o r m a t i o n o f a p e p t i d e b o n d : Wh e n t h e
amino group of an amino acid combines with
,r Tertiary structure : The three dimensional
the carboxyl group of another amino acid, a
---rctlrre of a fu nc t ional pr ot ein. peptide bond is formed (Fig. 65.6). Note that a
-l Quaternary structure : Some of the proteins dipeptide will have two amino acids and one
, - = co ,np osed o f t wo or m or e poly pept ide c hain s peptide (not two) bond. Peptides containing
.l
to a s su bunit s .The s pac ial ar r angem ento f more than 0 amino acids (decapeptide) are
- 'e rre,lbu nits
' -:se:u is k nown as quat er nar y s t r uc t ur e referred to as polypeptides.
742 B IOTE C HNO LO CY

Primary Secondary Tertiary Quaternary

structure structure structure structure

Fig, 65.5: Diagrammatic reryesentation of proteinstructurc

(Note : The four subunitsof two types in quatqrnarystructure).

Determination of primary structure SECONDARY STRUCTURE OF PROTEIN

The primarystructurecomprisesthe identifica- The conformation of polypeptide chain by
tion of constituentaminoacidswith regardto their twisting or folding is referredto as secondary
q u a l i ty , q u a n ti ty a n d s e q u e nce i n a protei n structure.The amino acids are locatedclose to
structure.A pure sample of a protein or a each other in their sequence.Two types of
polypeptideis essentialfor the determinationof secondary structures, g.-helix and p-sheet, are
primarystructurewhich involves3 stages. mai nl y i denti fi ed.
1 . D e te rmi n a ti oonf a mi n o aci d composi ti on. cr'Helix
2. Degradationof protein or polypeptideinto a-Helix is the mosf common spiral structureof
smallerfragments. protein. lt has a rigid arrangementof polypeptide
3 . D e te rmi n a ti oonf th e a mi no aci d seouence. chain.q,-Helicalstructurewas proposedby Pauling
and C orey(1951)w hi ch i s regardedas on e of t he
milestones in the biochemistry research.The salient
H H featuresof a right-handed cr-helixwhich is a stable
*l *l and more commonlyfound structure,in the living
H3t!--c-coo + H3N-C-COO- system(Fig, 65.V are given below
I I 1. The cr-hel i x i s a ti ghtl y packe d coiled
R1 R2
structurew i th ami no aci d si de chai nse xt ending
Aminoacid 1 Aminoacid2
outwardfrom the centralaxis.
Hzo 2. The cr-helix is stabilized by extensive
hydrogen bonding. lt is formed between H atom
HH attached to peptide N, and O atom attached to
*ll peptideC.
H3N-C-CO-NH-C-COO- 3. All the peptidebondsexceptthe firstand last
tl in a polypeptidechain participatein hydrogen
R1 R2 bondi ng.
Dipeptide
4. E ach turn of cr-hel i xcontai ns3. 6 am ino
Fig. 65.6 : Formation of a peptide bond. acids and travels a distanceof 0.54 nm. The
spaci ngof each ami no aci d i s 0.15 nm.
Chaot er65 : B IO MOL E C U L ES 743

sheets(or simplyp-sheets) are composedof two or

more segments of fully extendedpeptidechains.In
the B-sheets,the hydrogen bonds are formed
betweenthe neighbouring segmentsof polypeptide
chai n(s).

TE R TIA R Y S TR U C TU R E OF P R OTE IN
fhe three-dimensionalarrangement of protein
structureis referredto as tertiarystructure.lt is a
compact structurewith hydrophobicside chains
hel d i nteri orw hi l e the hydrophi l i cgroupsare on
the surfaceof the protein molecule.This type of
arrangement ensuresstabi l i tyof the mol ecul e.
Bonds of tertiary structure: Besides the
hydrogenbonds, disulfide bonds (-S-S), ionic
interactions(electrostatic
bonds)and hydrophobic
interactions
also contributeto the tertiarystructure
of oroteins.
Domains:The term domainis usedto reoresent
/" the basic units of protein structure(tertiary)and
N functi ons.A pol ypepti dew i th 200 ami no aci ds
t\: normallyconsistsof two or more domains.

i QU A TE R N A R Y S TR U C TU R E OF P B OTE IN
I
"- r---\r^r
A great majorityof the proteinsare composed
N
of singlepolypeptidechains.Someof the proteins,
?,t'i-
6 Hi /L however, consist of two or more polypeptides
\rH which may be identicalor unrelated.Suchproteins
' No/
\o are termed is oligomers and possessquaternary
j-----=-r- c C structure.The individual polypeptidechains are
known as monomersl protomers or subunits. A
) dimer consitsoI two polypeptideswhile a tetramer
has four.
Bondsin quaternarystructure: The monomeric
subunitsare held togetherby non-convalent
bonds
Fig. 65.7 : Diagrammatic representation of secondary namelyhydrogenbonds,hydrophobicinteractions
structure of protein - a right handed a-helix and i oni c bonds.
H
I
(l-lndicate -C-R groups of amino acids;
dotted coloured shadel are hydrogen bonds; Note
that only a few hydrogen bonds shown for clarity).
Proteinsare classifiedin severalways. Three
major types of classifyingproteinsbasedon their
5. o-Helix is a stable conformation formed functi on,chemi calnatureand sol ubi l i typroperti es
.pontaneously with the lowest energy. and nutritionalimoortanceare discussedhere.

:.Pleated sheet Functional classification of proteins

This is the secondtype of structure(henceB Basedon the functionthey perform,proteinsare
rier G) proposedby Paulingand Corey.B-Pleated classifiedinto differentgroups(with examples)
|t r{

784 B IOTE C HNO LO CY

TABrr65.4 Summaryof classificationof proteins

Nucleoproteins,
Scleroproleins
Glycoproteins
Albumins Collagens Mucoproteins Coagulated Proteoses
Globulins Elastins Lipoproteins proteins Peptones
Glutelins Keratins Phosphoproteins Proteans Polypeptides
Prolamines Chromoproteins
Histones Metailoproteins l\4etaproteins Pantidaq

Globins
Protamines

1. Structuralproteins: Keratinof hair and nails, 3. Derivedproteins:Theseare the denatured

c o l l a g e no f b o n e . or degradedproductsof simple and conjugated
proteins.
I
2. Enzymesor catalyticproteins: Hexokinase,
p e p snr . The abovethreeclassesare furthersub-divioeo
into different groups. The summary of protein
3. Transport proteins: Hemoglobin, serum classification is given in the Table65.4.
albumin.
A mong the si mpl e protei ns,gl obul a rpr ot eins
4. Hormonalproteins: Insulin,groMh hormone. are sphericalin shape,soluble in water or other
5. Contractileproteins: Actin, myosin. sol ventsand di gestabl ee.g., al bumi n , globulin.
S cl eroprotei ns(fi brousprotei ns)are fi ber like in
6. Storageproteins: Ovalbumin,glutelin. shape,insolublein water and resistant to digestion
7. Geneticproteins: Nucleoproteins. e.g., col l agen;kerati n.
The conjugatedproteinsmay containprosthetic
B. Defenseproteins: Snakevenoms, lmmun-
groupssuch as nucleic acid, carbohydrate, lipid,
o g l o b ul i n s .
metal etc. The primary derived proteins are
9. Receptorproteinsfor hormones,viruses. producedby agentssuch as heat, acids, alkalies
etc., while the secondaryderived proteins are
Protein classification based on hydrolyticproductsof proteins
chemical nature and solubility
T h i s i s a mo re c o mp re hensi ve and popul ar
classification of proteins.lt is basedon the amino
acid composition,structure,shape and solubility
properties.Proteins are broadly classified into Isoprenoids and pigments are organic
compounds mostl ydi stri butedi n pl ant kingdom .
3 .majorgroups(Table65,4).
They performa wide varietyof functions.
1. Simpleproteins: Theyare composedof only
a mi n o a c i d re s i d u e s . IS OP R E N OID S
2. Conjugated proteins: Besidesthe amino lsoprenoidsare also called as terpenoidsor
acids, these proteins contain a non-protein (terpenes) as they are found in turpentineoil rn
moiety known as prostheticgroup or conjugating hi gh concentrati ons.The natural l y occur r ing
Sroup- isoprenoidsare composedo{ a five carbonisoprene
 Chapt er65 : B I O MOL E C U L ES 78'5

Tetrapyrroles
Tnru 65.5 Classificatlon of terpenes wlth
selected examples The mosi abundant coloured comoound in the
world is chlorophyll, the photosynthetic pigment.
Class Basic structure Example There are different types of chlorophylls (c, d, e, a)
tsoprene Structure with slight variation in colours-green, greenish
units b l u e , g r e e n i s hy e l l o w .
Hemiterpenes 1 csHe rs0prene Structurally, chlorophylls are composed of
Monoterpenes 2 u1on16 Limonene tetrapyrroles (pyrrole rings) with their nitrogen
Sesquiterpenes nu
v15"24 Abscisic
acid iinked to magnesium.
Diterpenes A
czoHsz Gibberellin Tetrapyrrolesare also found in heme in certain
Triterpenes a u
v30"48 Stigmasterol p r o t e i n s .T h e s e i n c l u d e h e m o g l o b i n , c y t o c h r o m e s ,
?traterpenes n nu
v40"64 Carotenes catalase and peroxidase
Pclyterpenes n (c5Hs)n Rubber
Tetraterpenes (carotenoids)

unit. A majority of the isoprenoidsare formed by The colour of carotenoids is variable, generally
' oinin g o f isop ren e unit s head t o t ail as depic t ed yellow, orange or red. A large number of
Derow carotenoids (about-600) have been identified in
ia plant kingdom e.g. B-carotene, xanthophylls,
tt
c_c_c-c-c-c-c-c
lycopene.

Head Tail Head Tail

Anthocyanins

Anthocyanins are a group of flavonoids which

Classification of terpenes
The classificationof terpenes is mainly based on
:re number of isoprene (CrHu) units present. The
-rajor classesof terpenes with selected examples
:re given in Table 65.5.
P IGMENTS
Pigmentsare cloured organic compounds found
in the livin g org an is m s ,m os t ly in plant s , and t o a
min or exten t in a nim als . Chem ic ally , pigm ent s ar e
high mo lecula r weight m olec ules , m os t ly
composed of unsaturated hydrocarbons. Some of
the pigments also contain cyclic structures. The
major groups of pigments are briefly described.
represent the natural phenolic products.
Anthocyanins are coloured compounds, mostly
found in flowers and fruits. They contain a common
ring structure called anthocyanidin.
Quinoid pigments
Being present in trace amounts, quinoid
pigments do not significantly contribute to visible
colours. They however, perform some other
functions e.g. involvement in electron transport
chain, antioxidant functions etc. The most
common quinoid pigments are benzoquinones,
naphthoquinones, anthraquinones, tannins and
l i e ni n s .

Biotechnology [50]
f nz y m es ar e bioc at aly s t s - t h e c a t a l y s t so f l i f e .
LA catalvst is defined as a subsfance that
increasesthe velocity or rate of a chemical reaction
wit hout it s elf under goingany ch a n g e i n t h e o v e r a l l
orocess.
Enzymes may be defined as biocatalysts
synthesized by living cells. They are protein in
nature, colloidal and thermolabile in character,
and specific in their action.
In recent years, certain non-protein enzymes
( c hem ic ally RNA) hav e als o be e n i d e n t i f i e d .
NO M ENCLATURE
AND CLASSI FI CATI O N
ln the early days, the enzymeswere given names
by t heir dis c ov er er s in an ar b i t r a r y m a n n e r . F o r
ex am ple, t he nam es pep s i n , t r y p s i n a n d
chymotrypsin convey no information about the
function of the enzyme or the nature of the
substrateon which they act.
Enzymes are sometimes considered under two
broad categories: (a) lntracellular enzymes-They
ar e f unc t ional wit hin c ells w h e r e t h e y a r e
synthesized. (b) Extracellular enzymes-These
enzymes are active outside the cell; all the digestive
enz y m es belong t o t his gr oup
The I nt er nat ional Union of B i o c h e m i s t r y ( l U B )
.l
appoint ed an Enz y m e Com m i s s i o n i n 9 6 1 . T h i s
committee made a thorough study of the existing
enz y m es and dev is ed s om e ba s i c p r i n c i p l e s f o r t h e
786
c l a s s i f i c a t i o na n d n o m e n c l a t u r eo f e n z y m e s. Si n ce
1964, the IUB system of enzyme classification has
been in force. Enzymes are divided into six major
c/asses (in that order). Each class on its own
represents the general type of reaction brought
about by the enzymes of that class.
1 . O x i d o r e d u c t a s e s : E n z y m e s i n vo l ve d i n
oxidation-reduction reactions.
2 . T r a n s f e r a s e s : E n z v m e s t h a t c a ta l vse th e
transfer of functional groups.
3. Hydrolases: Enzymes that bring about
h y d r o l y s i so f v a r i o u s c o m p o u n d s .
4. lyases : Enzymesspecialisedin the addition
or removal of water, ammonia, CO, etc.
5 . t s o m e r a s e s : E n z y m e s i n v o l v e d in a l l th e
isomerization reactions.
6. ligases : Enzymes catalysing the synthetic
reactions (Creek : ligate-to bind) where two
m o l e c u l e s a r e j o i n e d t o g e t h e r a n d A T P is u se d
lThe word OTHLIL (first letter in eacl.r class)
may be memorised to remeinber the six classesof
enzymes in the correct orded
E a c h c l a s s i n t u r n i s s u b d i v i d e d i n t o m a n y su b -
c l a s g e s w h i c h a r e f u r t h e r d i v i d e d . A fo u r d i g i t
Enzyme Commission (E. C.) number is assigned to
each enzyme representingthe class (firstdigit), sub-
c l a s s ( s e c o n d d i g i t ) , s u b - s u b c l a s s ( t h i r d d i g i t) a n d
the individual enzyme (fourth digit).
Chaoter66 : ENZYMOLOCY 747

ln rhe Table 66. 1, selecteclexamples for the six
cla sseso f en zym es ar e giv en.
CHEMICAL NATURE O F ENZYM ES
All the e nzy m es ar e inv ar iably pr ot eins . I n
recent years, however, a few RNA molecules have
been shown to function as enzvmes. Each enzvme
has its own tertiary structure and specific
conformation which is very essentialfor its catalytic
activity.The functional unit of the enzyme is known
as holoenzyme which is often made up of
apoenzyme (the protein part) and a coenzyme
(non-protein organic part).
Holoenzyme ----+ Apoenzyme + Coenzyme
(activeenzyme) (prot6inpad) (non-proteinpaft)
FACTORS AFFECTING
ENZYME ACTIVITY
The contact between the enzyme and substrate Enzyme kinetics and K- value : The enzyme (E)
is the most essential pre-requisite for enzyme and substrate(S) combine with each other to form
activity. The important factors th?t influence the
velocity of the enzyme reaction are discussed
hereunder
l. Goncentration of enzyme
As the concentrationof the enzyme is increased,
the velocity of the reaction proportionately
increases (Fig.66.1). In fact, this property of
e n z y m e i s m a d e u s e i n d e t e r m i n i n gt h e a c t i v i t i e so f
serum enzymes for diagnosis of diseases.
2. Concentration of substrate
Increasein the substrateconcentrationgradually
increases the velocity of enzyme reaction within
the limited range of substratelevels. A rectangular
hyperbola is obtained when velocity is plotted
against the substrate concentration (Fig. 66.2).
Three distinct ohases of the reaction are observeo
in the graph.

' : ' ' ' ' :

Tmu eO-f Cnssitication of *n 1,t".

Enzyme classwith examples* Reaction catalysed

I Oxidoreductases
Alcohol (alcohol
dehydrogenase : NAD+ E. C. 1.1.1.1.),
oxidoreductase Oxidation ----+ Reduction
cytochrome L- andD-amino
oxidase, acidoxidases A H r+ B -----+ A + B H ,
2. Transferases
(ATP: D-hexose
Hexokinase E. C. 2.7.1.1.),
O-phosphotransferase, Grouptransfer
phosphorylase
transmethylases,
transaminases, A -X + B ---+ A + B -X
Hydrolases
Lipase(triacylglycerol
acylhydrolase
E. C. 3.1.1.3),
cholirre
esterase, Hydrolysis
acidandalkalinephosphatases,
pepsin,urease A -B + H rO----+ A H + B OH
4. Lyases
(ketose
Aldolase 1-phosphate lyase,E. C. 4.1.2.7),
aldehyde Addition_+ Elimination
fumarase,
histidase A -B + X -Y -----+ A X -B Y
5. lsomerases
Triosephosphate (D-glyceraldehyde
isomerase keloisomerase,
3-phosphate Interconversion
of isomers
E.C.5.3.1.1),
retinol phosphohexose
isomerase, isomerase A -+ A '
a. Ligas es
Glutamine (L-glutamate
synthetase ligase,
ammonia E. C. 6.3.1.2), (usually
Condensation onATP)
dependent
acetylCoAcarboxylase,
succinate
thiokinase A + B ----4,-----+ A -B
ATP ADP+ Pi
* Fu oneenzynein eachclass,systenatic
namealongwithE.C.numberis givenin the brackets.
7a8 B IOTECHNO LO CY

I Vr""

fI
o
o II
c)
E 1 I
N
J\/
2
f max -ft
c
ul >
o
c)

Substrate -+
concentration

Fig. 66.1 : Effect of enzyme concentration on Fig. 66.2 : Effect of substrate concentrationon enzyme
enzyme velocity. velocity (A-linear; B-cu rue; C-almost unchanged).

an unstableepzyme-substrate complex(ES)for the measuringthe strengthof EScomplex.A low K-

formationof product(P). value indicatesa strong affinity between enzyme
. and substrate,whereasa high K. value reflectsa
l (r k.
E + S:E S---# E +P weak affinity between them. For majority of
,K2 enzymes,the K, valuesare in the rangeof 10-5to
10-2 mol es.
Here k1, k, and k, representthe velocityconstants
for the respective
reactions,
as indicatedby arrows. Lineweaver-Burk double reciprocalplot : For
the determinationof Kn' value, the substrate
K., the Michaelis-Menten constant(or Brig's saturation curve (Fig. 66.2) is not very accurate
and Haldane'sconstant),is given by the formula sinceVr"* is approached asymptotically. By taking
k r* k, the reciprocalsof the equation (1), a straightline
o _
,.m-
k1 graphicrepresentation is obtained.
The followingequationis obtainedaftersuitable The Li new eaver-B urkpl ot i s shown in
a l g e b ra i cm a n i p ul a ti o n . Fig. 66.3. lt is much easierto calculatethe K,
.. vmax[s]
e q u a t i o n( 1 )
K. + [S]
wherev = Measured velocitv.
V.", = Maximum velocity,
S = Substrate
concentration,
,-j
Km = Michaelis-Menten
constant.
Km or the Michaelis-Menten constant is
defined as the subsfrateconcentrafion(expressed
in moles/lit) to produce half-maximum velocity in
an enzymecatalysedreaction.lt indicatesthat half
o f th e e n z y m em o l e c u l e (i
s .e.50% )are boundw i th
the substrate molecules when the substrate 1
concentration equalsthe K, value. tsl
K. value is a constantand a characteristic
Fig. 66.3 : LineweavenBurk double reciprocal plot.
featureof a givenenzyme.lt is a representative
for
 Chapt er66 : E N Z YMOL OC Y 789

a c i d p h o s p h a t a s e( 4 - 5 ) a n d a l k a l i n e p h o s p h a t a s e
(i0-11) for optimum pFr.

I
)
5. Effect of product concentration
T h e a c c u m u l a t i o no f r e a c t i o n p r o d u c t sg e n e r a l l y
decreasesthe enzyme velocity. For certain enzymes/
the products combine with the active site of
c)
enzyme and form a loose complex and, thus,
o)
E inhibit the enzyme activity. In the living system,
N
this type of inhibition is generally preventedby a
uJ
quick removal of products formed.

6. Effect of activators
Some of the enzymes require certain inorganic
20 30 40 50 60 metallic cations like Mg2+, Mn2+, Zn2+, Ca2+,
Temperature('C) C o z *, C u 2 *, N a +, K + e t c . f o r t h e i r o p t i m u m a c t i v i t y .
R a r e l y ,a n i o n s a r e a l s o n e e d e d f o r e n z y m e a c t i v i t y
Fig, 66.4 : Eftect of temperature on enzyme velocity. e.g. chloride ion (Cl-) for amylase.

iro m the inte rc ept on x - ax is whic h is - ( ' llKm ) .
Furth eri the do uble r ec ipr oc al plot is us ef ul i n
u nd erstan din g the ef f ec t of v ar ious inhibit io n s
'd iscusse d la ter ) .
3. Effect of temperature
Velocity of an enzyme reaction increaseswith
n cre asein te mper at ur eup t o a m ax im um and t h e n
cie clin es.A b ell - s haped c ur v e is us ually obs er v e d
Fig. 66.a).
The optimum temperature for most of the
enzymes is between 40"C-45"C. However, a tew
:n zymes (e .g. v enom phos phok inas es , m us c l e
.l
:denylate kinase) are active even at 00"C.
ACTIVE SITE
Enzymesare big in size compared to substrates
which are relatively smaller. Evidently, a small
portion of the huge enzyme molecule is directly
i n v o l v e d i n t h e s u b s t r a t eb i n d i n g a n d c a t a l y s i s .
The active site (or active ce:ntre) of an
enzyme is defined as the small region at which
the substrate(s) binds and participates in the
catalysis.
Salient features of active site
.l
. The existence of active site is due to the
tertiary structure of protein resulting in three-
dimensional native conformation.

ln general, when the enzymes are exposed to a

:€nrperatureabove 50oC, denaturation leading to
rerangement in the native (tertiary)structureof the t
:-otern and active site are seen Majority of the
:-zvme s be co m e inac t iv e at higher t em per atu r e
:Dove 7 0'C). .>
I
o
4, Effect of pH
E
ircre ase in the hy dr ogen ion c onc ent r at ion( pH ) N
c

-:nside rab ly in fluenc est he enz y m e ac t iv it y and a IJJ

rell-shaped curve is normally obtained (Fig. 66.5).

Each enzyme has an optimum pH at which the
relo city is maxim um .
pH
Mo st o f th e enz y m es of higher or ganis m ss ho w
op timum a ctivit y ar ound neut r al pH ( 6- 8) . The r e Fig. 66.5 : Effect of pH on enzyme velocity.
a re, ho wever, m any ex c ept ions lik e peps in ( 1- 2 ) ,
790
' I he
2. ac t iv e s it e is m ad e u p o f a m i n o a c i d s
(known as catalytic residues)which are far from
eac h ot her in t he linear s eq u e n c e o f a m i n o a c i d s
(primary structure of protein). For instance, the
enz y m e ly s oz y m e has 129 am i n o a c i d s . T h e a c t r v e
s it e is f or m ed by t he c ont r i b u t i o n o f a m i n o a c i d
r es iduesnum ber ed 35, 52, 6 2 , 6 3 a n d 1 0 1.
3 The ac t iv e s it e is not r i g l d i n s t r u c t u r e a n d
shape. lt is rather flexible to promote the specific
s ubs t r at ebinding.
4. Cenerally, the active site possesses a
substrate hinding site and a catalytic site. The
latter is for the catalysis of the specific reaction
5. O f t he 20 am ino ac ids t h a t c o u l d b e p r e s e n t
in enz y m e s t r uc t ur e, only s o m e o f t h e m a r e a c l a s s i c a ie x a m p l e o f c o m p e t i t i v e i n h i b i ti o n w i th
repeatedly found at the active sites of various s u c c i n i c a c i d a s i t s s u b s t r a t e .M a l o n i c a ci d h a s
e nzyrres. Tlrese amino acids are serine, aspartate, s t r u c t u r a ls i m i l a r i t yw i t h s u c c i n i c a c i d a n d co m p e te
his t idine, c y s t eine, ly s ine, a r g r n i n e , g l u t a m a t e , r,l,iththe substratefor bindine at the active site of
t),rosine etc. Amor.rgthese amino acids, serine rs S D H .
t he r nos t f r equent lv f our r d.
6. The substrate[5]binds the enzyme (E) at the
active site to forrn enzyme-substratecomplex (ES).
The product (P) is released after the catalysis and
t he enz y m e is av ailable f or r e u s e .
E + S.- ES-----+E + P
1l
t_ _ _ _ _ _ _ _ _ _ _ _ _ ___
__l
ENZYM E I NHI BI TI O N
Enz y m e inhibit or is defi n e d a s a s u b s t a n c e
whic h binds wit h t he enz y m e a n d b r i n g s a b o u t a
decrease in catalytic activity of tlrat enzyme. The
inhibit or m ay be or ganic or i n o r g a n i c i n n a t u r e
There are two broad categories of enzyme
inh ibit ion
1. Rev er s ibleinhibit ion.
2. lr r ev er s ibleinhibit ion.
1. Reversible inhibition
The inhibit or binds non- co v a l e n t l yw i t h e n z y r n e
and t he enz y m e inhibit ion c a n b e r e v e r s e d i f t h e
inhibit or is r em ov ed. The r e v e r s i b l e i n h i b i t i o n i s
further sub-divided into
l. Cornoetitive inhibition
ll. Non-competitive inhibition
l. Com pet it iv e inhibit ion : T h e i n h i b i t o r ( l )
which closely resembles the real substrate (S) is
B IOTECHNO LO CY
regarded as a substrate analogue. The inhibitor
comoetes with substrateand binds at the active site
of the enzyme but does not undergo any catalysis.
A s l o n g a s t h e c o m p e t i t i v e i n h i b i t o r h o l d s th e
active site, the enzyme is not available for the
substrateto bind.
The relative concentration of the substrateand
i n h i b i t o r a n d t h e i r r e s p e c t i v e a f f i n i ty w i th th e
enzyme determines the degree of competitive
i n h i b i t i o n . T h e i n h i b i t i o n c o u l d b e o v e r co m e b y a
high substrate concentration. In competitive
inhibition, the K,n value increases r,r,hereasV,,,u,
remains unchanged.
The enzyme succinate dehydrogenase(SDH) is
cooH
I
CH,COOH CHZ
r-l -
Crr.oot Coot
Succinic acid Malonic acid .
Some more examples of the enzymes with
s u b s t r a t e sa n d c o m p e t i t i v e i n h i b i t o r s o f b i o l o g i ca l
significance are given in Table 66.2.
T h e s e a r e t h e ch e m i ca l
Antimetabolites:
compounds that block the metabolic reactions by
their inhibitory action on enzymes. Antimetabolites
a r e u s u a l l y s t r u c t u r a l a n a l o g u e s o f s u b str a te sa n d
thus are competitive inhibitors (Table 66.4. fhey
are in use for the treatmentof cancer, gout etc. The
Ierm antivitamins is used for the antimetabolites
which b l o c k t h e b i o c h e m i c a l acti o n s o f
v i t a m i n s c a u s i n g d e f i c i e n c i e s .e . g . , s u l ph a n i l a m i d e ,
dicumarol.
ll. Non-competitive inhibition : The inhibitor
binds at a site other than the active site on the
e n z y m e s u r f a c e . T h i s b i n d i n g i m p a i r s th e e n zym e
f u n c t i o n . T h e i n h i b i t o r h a s n o str u ctu r a l
resemblance with the substrate. However, there
usually exists a strong affinity for the inhibitor to
bind at the second site. In fact, the inhibitor does
not interfere with the enzyme-substratebinding.
But the catalysis is prevented, possibly due to a
distortion in the enzyme conformation.
Chapter66 : ENZYMOLOCY 791

rir,r'ee.i'i*i".t"a in**. *tti ttreir ictrra iuii*"te, .oip"ritive inlribitors

"*"rpii"r'ot "na
Enzyme Substrate lnhibitor Significance of inhibitor

Xanthineoxidase Hypoxanthine Allopurinol Usedin thecontrolof goutto reduce

excess
Xanthine production
of uricacidfrcnrhypcxanihine.

Monoamineoxidase Catecholamines Eohidrene. Useful

forelevatino
catecholamine
levels
(epinephrine,
norepinephrine)
amphetamine

DihydrofolatereductaseDihydrofolic
acid Aminoplerin, Employed in thetreatment
of leukemia
and
amethopterin, olhefcancers.
methotrexate

Acetylcholineesterase Acetylcholine Succinyl

cholineUsedin surgery
for muscle
relaxatlon,in
patients.
anaesthetised
Paraaminobenzoic
acid Sulphanilamide
Preventsbacterial
synthesis
of folicacid.
(PABA)
Vitamin
K Dicumarol Actsas an anticoagulant.
(vitamin
Pyridoxine 86) lsonicotinic
acid INHis anantituberculosis
drug,itspiolonged
(lNH) useleadsto Budeficiency.
hydrazide

Th e inh ibito r gener ally binds wit h t he e n -
zyme as we ll a s t he ES c om plex .
For non-competitive inhibition, the K- value is
unchanged while V^", is lowered.
Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can
no n-comp etitively inhibit t he enz y m es by bindi n g
rvith cysteinyl sulfhydryl groups.
2. lrreversible inhibition
Th e in hib itor s bind c ov alent lv wit h t he enz v m e s
a nd in activa te them , whic h is ir r ev er s ible.The s e
inh ibito rs are usually t ox ic s ubs t anc eswhic h m a y
b e pre se nt n atu r ally or m an- m ade.
to be inhibited e.g., allopurinol, an inhibitor of
xanthine oxidase, gets conve!"tedto alloxanthine, a
more effective inhibitor of the enzvme.
ENZYME SPEGIFICITY
E n z y m e sa r e h i g h l y s p e c i f i c i n t h e i r a c t i o n w h e n
compared ivith the chem ical catalysts. The
occurrence of thousands of enzymes in the
biological system might bre due io the specific
nature of enzymes. Thiee types of enzyme
specificity are wel l-recognised
1. Stereospecificity, 2. Reaction specificity,
3. Substratespecificity,

lodoacetate is an irreversible inhibitor of the Specificity is a characteristic pioperty of the

..rzyme s like papain and gly c er aldehy de 3 - active site.
rlosphate dehydrogenase. lodoacetate combines
r, ith sulfhydryl (-SH) groups at the active site of L Stereospecificity or optical
:-ese enzymes and makes them inactive specificity

Diisopropyl fluorophosphate (DFP) is a nerve Stereoisomersare the compounds which have

gas developed by the Cermans during Second
,,\o rld Wa r. DFP ir r ev er s ibly binds wit h enz y m e s
:ontaining serine at the active site, e.g . serine
proteases, acetylcholine esterase.
Suicide inhibition : In this type of irreversible
n hib ition , the or iginal inhibit or is c onv er t ed t o a
-rore potent form by the same enzyme that ought
the same molecular formula, but differ in their
s t r u c t u r a lc o n f i g u r a t i o n .
The enzymes act only on one isomer and,
therefore, exhibit stereoisonterrsm.
e g. L-amino acid oxidase and D-amino acid
oxidase act on L- and D-amino acids
respectively.
792
Fig. 66.6 : Diagrammatic representation of stereo-
specificity (a',b',c')-three point attachment of
substrate to the enzvme (a, b, c).
Hexokinase acts on D-hexoses;
Cluc ok inas e on D- gluc o s e ;
Am y las e ac t s on u- gly c o s i d i c l i n k a g e s ;
Cellulas e c leav es p- glyc o s i d i c b o n d s '
St er eos pec if ic it y is ex plai n e d b y c o n s i d e r i n g
t hr ee dis t inc t r egions of s u b s t r a t e m o l e c u l e
s pec if ic ally binding wit h t h r e e c o m p l e m e n t a r y
regions on the surface of the enzyme (Fig. 66.6).
The class of enzymes belonging to isomerasesdo
not exhibit stereospecificity, since they are
s oec ializ ed in t he int er c onv e r s i o no f i s o m e r s .
2. Reaction specificity
The same substratecan undergo different types
of reactions,each catalysed by a separateenzyme
and this is referred to as reaction specificity. An
am ino ac id c an under go t r an s a m i n A t i o n ,o x i d a t i v e
deam inat ion, dec ar box y lat io n , r a c e r n i z a t i o n e t c .
The enzymes however, are different for each of
these reactions.
3. Substrate sPecificitY
The substratespecificity varies from enzyme to
enzvme. lt may be either absolute, relative or
broad
(a) Absolute substrate specificity : Certain
enzymes act only on one substrate e.g.
B IOTE CHNO LO CY
glucokinase acts on glucose to give glucose
5-phosphate, urease cleaves urea to
ammonia and carbon dioxide.
(b) Relative substrate specificity : Some
enzymes act on structurally related
s u b s t a n c e sT. h i s , i n t u r n , m a y b e de p e n d e n t
on the specific group or a bond present.The
action of trypsin and chymotrypsin is a good
example for Sroup specificity. Trypsin
h y d r o l y s e s p e p t i d e l i n k a g e i n vo l vi n g
a r g i n i n e o r l y s i n e . C h y m o t r y p si n cl e a ve s
peptide bonds attached to aromatic amino
a c i d s ( p h e n y l a l a n i n e , t y r o s i n e a n d tr yp to -
p h a n ) . E x a m p l e s o f b o n d sp e ci fi ci ty-
g l u c o s i d a s e sa c t i n g o n g l y c o s i d ic b o n d s o f
carbohydrates,lipases cleaving ester bonds
of lipids etc.
(c) Broad specificity : Some enzymes act on
c l o s e l y r e l a t e d s u b s t r a t e s wh i ch is
commonly known as broad substrate
specificity,e.g. hexokinaseacts on glucose,
f r u c t o s e ,m a n n o s e a n d g l u c o s a m i n ea n d n o t
on Salactose.
COENZYMES
M a n y e n z y m e s r e q u i r e c e r t a i n n o n - p ro te i nsm a l l
additional factors, collectively referred to as
cofactors for catalysis. The cofactors may be
organic or Inorgantcin nature.
The non-protein, organic, low molecular weight
and dialysable substance associated with enzyme
function is known as coenzYme'
The functional enzyme is referred to as
holoenzyme which is made up of a protein part
(apoenzyme) and a non-protein part (coenzyme).
The term activator representsthe inorganic cofactor
(like Ca2+, Mg2+, Mn2+ etc.) necessaryto enhance
enzyme activity.
Coenzymes are second substrates: Coenzymes
are often regarded as the second substrates or
cosubstrates, since they have affinity with the
enzyme comparable with that of the substrates.
C o e n z y m e s u n d e r g o a l t e r a t i o n s d u r i n g th e
enzymatic reactions, which are later regenerated.
T h i s i s i n c o n t r a s t t o t h e s u b s t r a t e w h i ch i s
converted to the product
Coenzymes participate in various reactions
i n v o l v i n g t r a n s f e ro f a t o m s o r g r o u p s I i k e h yd r o g e n ,
aldehyde, keto, amino, acyl, methyl, carbon
Chapter66 : ENZYMOLOCY 793

Trou 66.3 Coenrynesof B-conplexvltarains

Coen zyme (abb rev iati on) Derived fram Atom or Dependent enzyme
vitamin graup transferred (example)

pyrophosphate
Thiamine (TPP) Thiamine Aldehyde
or keto Transkelolase
Flavin (FMN)
mononucleotide Riboflavin Hydrogen
andelectron L-Amino
acidoxidase
Flavin
adenine (FAD)
dinucleotide Riboflavin D-Amino
acidoxidase
Nicotinamide
adenine
dinucleotide(NAD) Niacin Lactate
dehydrogenase
Nicotinamide
adenine
dinucleotide
phosphate
(NADPJ Glucose
6-phosphate
dehydrogenase
Lipoic
acid Lipoic
acid Pyruvate
dehydrogenase
complex
phosphate
Pyridoxal (PLP) Pyridoxine Amino
cr keto Alanine
transaminase
A (CoA)
Coenzyme Pantothenic
acid Acyl Thiokinase
(FH.)
Tetrahydrofolate Folicacid Onecarbon Formyl
transferase
(formyl,
methenyl
etc)
Biotin
coenzyme Biotin Pyruvate
carboxylase
Methylcobalamin
; Deoxyadenosyl
cobalamin Cobalamin Methyl/rsomerisation Methylmalonyl
CoAmutase

d ioxide etc. Coe nz y m es play a dec is iv e r ole i n
enzyme function.
Coenzymes from B-complex vitamins : Most of
the coenzymes are the derivativesof water soluble
B-co mple x vitam ins . I n f ac t , t he bioc hem ic a l
functions of B-comolex vitamins are exerted
through their respective coenzymes. The chapter
on vitamins gives the details of structure and
fun ctio n of th e c oenz v m es . ln Table. 66. 3, a
summary of the vitamin related coenzymes with
the ir fun ctio ns is giv en.
Non-vitamin coenzymes : Not all coenzymes
are vitamin derivatives. There are some other
organic substances,which have no relation with
vitamins but function as coenzymes. They may be
considered as non-vitamin coenzymes e.g. ATP,
CDP, UDP etc.
Nucleotide coenzymes : Some of the coenzymes
possess nitrogenous base, sugar and phosphate.
Such coenzymes are, therefore, regarded as
n ucleo tide s e .g. NAD+ , NADP+ , FM N, FAD ,
co en zyme A, UD PC et c .
Coenzymes do not decide enzyme specificity :
A particular coenzyme may participate in catalytic
reactions along with different enzymes. For
instance, NAD+ acts as a coenzyme for lactate
dehydrogenaseand alcohol dehydrogenase.ln both
the enzymatic reactions, NAD+ is involved in
hydrogen transfer. The specificity of the enzyme is
mostly dependent on the apoenzyme and not on
the coenzyme
MECHANISM OF ENZYME ACTION
Catalysis is the prime function of enzymes.
T h e n a t u r eo f c a t a l y s i st a k i n g p l a c e i n t h e b i o l o g i c a l
system is similar to that of non-biological
catalysis. For any chemical reaction to occur, the
reactants have to be in an activated state or a
transition state.
Enzymes lower activation energy : The energy
required by the reactantsto undergo the reaction is
known as activation energy. The reactants when
heated attain the activation energy. The catalyst (or
the enzyme in the biological system) reduces the
activation energy and this causes the reaction to
proceed at a lower temperature. Enzymes do not
a l t e r t h e e q u i l i b r i u m c o n s t a n t s ,t h e y o n l y e n h a n c e
the velocity of the reaction.
The role of a catalvst or an enzvme is
comparable with a tunnel made in a mountain to
reduce the barrier as illustrated in Fig. 66.7. fhe
enzyme lowers energy barrier of reactants,thereby
making the reaction go faster.The enzymes reduce
the activation energy of the reactantsin such a way
that all the biological systems occur at body
temperature (below 40"C).
794 B IOTE CHNO LO CY

formation. As per this model, the active site ls not

rigid and pre-shaped.The essentialfeaturesof the
su$strate binding site are present at the nascent
active site. The interaction of the substratewith the
enzyme induces a fit oi' a conformation change in
the enz1,mg,resLrltingin the formation of a strong
substratebinding site. Further, due to induced fit,
the appropfiate amino acids of the enzyme are
repositionedto form the active site and bring about
the catalysis (Fig. 66.A.

lnduced fit model has sufficient experimental

evidence from the X-ray diffraction studies.
K o s h l a n d 's m o d e l a l s o e x p l a i n s t h e a cti o n o f
a l l o s t e r i cm o d u l a t o r sa n d c o m p e t i t i v e i n hi b i ti o n o n
enzymes.

Fig. 66.7 : Effect of enzyme on activation energy of a Substrate strain theory

reaction (A is the substrcte and B is the product.
Enzyme decreases activation energy). In this model, the substrateis straineddue to the
i n d u c e d c o n f o r m a t i o n c h a n g e i n t h e e n z ym e . l t i s
also possible that when a substrate binds to the
Enzyme-substrate complex formation preformed active site, the enzyme induces a strain
to the substrate.The strained substrateleads to the
The pr im e r equis it e f or e n z y m e c a t a l y s i s i s
formation of product. The concept of substrate
t hat t he s ubs t r at e ( S) m us t c o m b i n e w i t h t n e
s t r a i n e x p l a i n s t h e r o l e o f e n z y m e i n i n c r e a si n gth e
enz y m e ( E) at t he ac t iv e s ite t o f o r m e n z y m e -
rate of reaction.
s ubs t r at ec om plex ( ES)whic h u l t i m a t e l y r e s u l t s r n
t he pr oduc t f or m at ion ( P) .

E+ Sr - - - . ES- - - - - +E +P
A few theories have been put forth to explarn
mechanism of enzyme-substratecomplex formation
(Fig. 66.8).

Lock and key model or (A)

Fischer's template theory
According to this model, the structure or
conformation of the enzyme is rigid. The substrate
fits to the binding site (now active site)just as a key (B)
fits into the proper lock or a hand into the proper
glov e. Thus t he ac t iv e s it e of a n e n z y m e i s a r i g i d
and pre-shaped template where only a specific
substratecan bind. This model does not give any
scope for the flexible nature of enzymes, hence the (c)
model totally fails to explain many facts of
enzymatic reactions,the most important being the
effect of allosteric modulators.

Induced fit theory or

Koshland's model
Koshland, in 1958, proposed a more acceptable
and realistic modei for enzyme-substratecomplex
Fig. 66.8 : Mechanism of enzyme-substnte (ES)
complex formation (A) Lock and key model;
(E) lnduced fit theory (C) Substrate strain theory.
Chaoter66 : ENZYMOLOCY 795

6i6.4 lmpoftant enzlnnesin the diagnosis 0f diseases

Serum enzyme (elevated) Drsease(most important)

Amylase Acutepancreatitis
Serum pyruvate
glutamate (SGPT)
transaminase Liverdiseases (hepatitis)
Serum glutamate
oxaloacetate (SGOT)
transaminase Heartattacks (myocardialinfarction)
phosphatase
Alkaline Rickets,
obstructive jaundice
Acidphosphatase Cancer of prostategland
Lactate (LDH)
dehydrogenase Heartattacks, liverdiseases
phosphokinase
Creatine (CPK) Myocardial infarction(earlymarker)
Aldolase Muscular
dystrophy
5'-Nucleotidase Hepatitis
y-Glutamyl (GGT)
transpeptidase Alcoholism

In fact, a combination of the induced fit model l m p a i r m e n t i n l i v e r f u n c t i o n o r g e n e t i cd i s o r d e r s

with the substrate strain is considered to be often leads to a fall in the activities of olasma
operative in the enzymatic action. functional enzymes e.g.- deficiency of
c e r u l o p l a s m i n i n Wi l s o n 's d i s e a s e .
Mechanism of enzyme catalysis
2. Non-plasma specific or plasma non-
The formation of an enzyme-substratecomplex functional enzymes : These enzymes are either
tES) is very crucial for the catalysis to occur. It is totally absent or present at a low concentration in
estimated that an enzvme catalvsed reaction plasma compared to their levels found in the
oro ce ed s 10 6 to 1012 t im es f as t er t han a n o n - tissues. The digestive enzymes of the
catalysed reaction. lt is worthwhile to briefly gastrointestinaltract (e.9. amylase, pepsin, trypsrn,
understandthe ways and means through which the lipase etc.) present in the plasma are known as
catalytic processtakes place leading to the product secretory enzymes. All the other plasma enzymes
formation. The enhancement in the rate of the associated with metabolism of the cell are
rea ctio n is ma inly due t o f our pr oc es s es: collectively referred to as consfitutive enzymes
1. Acid-base catalysis; (e.g. lactate dehydrogenase, transaminases,acid
2. Substrate strain; and alkaline phosphatases, creatine phosphokinase).

3. Covalent catalysis; Estimation of the activities of non-plasma

4. Entropy effects.
DIAGNOSTIC IMPORTANCE
OF ENZYMES
Estimation of enzyme activities in biological
flu ids (pa rticu lar lyplas m a/ s er um is
) of gr eat c lini c a l
imp orta nce.Enz y m esin t he c ir c ulat ion ar e div id e d
into two groups.
1 . Plasma specific or plasma functional
enzymes : Certain enzymes are normally present in
the plasma and they have specific functions to
perform. Cenerally, these enzyme activities are
h igh er in p lasm a t han in t he t is s ues . They a r e
mostlv svnthesized in the Iiver and enter the
circula tion e.g . lipopr ot ein lipas e, plas m i n ,
th romb in, cho line es t er as e,c er uloplas m in et c .
specific enzymes is very important for the diagnosis
and prognosis of several diseases.
A summary of the important enzymes useful for
the diagnosis of specific diseases is given in
Table 66.4.
ISOENZYMES
fhe multiple forms of an enzyme catalysing the
same reaction are isoenzymes or isozymes. They,
however, differ in their physical and chemical
properties which include the structure, electro-
phoretic and immunological properties, K. and
V . u * v a l u e s , p H o p t i m u m , r e l a t i v e s u s c e p t i b i l i t yt o
inhibitors and degree of denaturation. e.g. lactate
dehydrogenase (5 isoenzymes), creatine kinase
(3 isoenzymes).
l _ l u n d re d s o f re a c ti o n ssi mul taneousl take y Energy-rich
I I p l a c ei n a l i v i n gc e l l , i n a w el l -organi zed
and
integrated manner.The entirespectrumof chemical
reactions, occurring in the Iiving system, are
collectivelyreferredto as metabolism.
A metabolic pathway (or metabolic map)
constitutesa series of enzymatic reactionsto
producespecificproducts.The term metaboliteis
applied to a substrateor an intermediateor a
o ro d u c ti n th e me ta b o l i cre a c ti ons.
S o m e s a l i e n t fe a tu re so f metabol i smsw i th
parti cul arl y products
s p e c i a lre fe re n c to
e h i g h e ro rg ani sms,
h u ma n sa re g i v e n i n th i s c h a pter. Fig. 67.1 : An outline of catabolism and anabolism.

IN T R OD U C T IO N T O ME TA B OLIS M
Me ta b o l i s m i s b ro a d l y di vi ded i nto tw o to generatethe substances (precursors) requiredfor
categories(Fig.67.1). the synthesisof complex molecules.Catabolism
1. Catabolism : The degradativeprocesses occurs in three stages(Fig. 67.2).
concerned with the breakdown of comptex 1. Conversionof complexmoleculesinto their
m o l e c u l e sto s i m p l e ro n e s , w i th a concomi tant buildingblocks: Polysaccharides are brokendown
releaseof energy. to monosaccharides, lipids to free fatty acids and
2. Anabolism : The biosynthetic reactions gl ycerol and , protei nsto ami no aci ds.
from
i n v o l v i n gth e fo rma ti o no f c o m pl exmol ecul es
2. Formation of simple intermediates: The
s i mp l ep re c u rs o rs . bui l di ngbl ocksproducedi n stage(1)aredegr aded
A clear demarcation between catabolism to simpleintermediates suchas pyruvateand acetyl
and anabolismis ratherdifficult,since there are CoA. These intermediates are not readily
common
severalintermediates to both the processes. identifiable as carbohydrates,lipids or proteins.A
small quantity of energy(as ATP) is capturedin
C a ta b o l i s m stage2.
The very purposeof catabolismis to trap the 3. Finaloxidationof acetylCoA : Acetyl CoA
energyof the biomoleculesin the form of ATPand i s compl etel yoxi di zedto C Or, l i beratingNADH

796
 Chaot er67 : ME T AB O L IS M S 797

Polysaccharides Lipids Proteins

Stage1 +
I J I
M o n o sa cch a r id e s -

Stage 2
AcetylCoA

NADH
FADH2

Stage 3

Fig. 67.2 : The three stages of catabolism (ETC-EIectron transpori chain).

a nd FADH2 th at f inally get ox idiz ed t o r eleas el a r g e Types of metabolic reactions

quantity of energy (as ATP). Krebs cycle (or citric
The biochemicalreactionsare mainly of four types
a cid cycle) is t he c om m on m et abolic pat h w a y
involved in the f inal ox idat ion of all ener g y - r i c h 1. Oxidation-reduction.
mo lecule s. This pat hway ac c ept s t he c a r b o n
2. Crouo transfer
comp ou nd s (p y r uv at e,s uc c inat eet c . ) der iv ed f r o m
carb oh yd rate s ,lipids or pr ot eins 3 . R e a r r a n g e m e na
t nd isomerization.

4. Make and break of carbon-carbon bonds.

An ab olism
These reactions are catalysed by specific
Fo r the synt hes isof a lar ge v ar iet y of c om p l e x
enzymes-more than 2,000 known so far.
mo lecule s,the s t ar t ingm at er ialsar e r elat iv elyf e w .
These include pyruvate, acetyl CoA and the
Methods employed to study metabolism
inte rmed iate s of c it r ic ac id c y c le Bes ide s t h e
a va ilab ilityo f pr ec ur s or st,he anabolic r eac t io n sa r e The metabolic reactions do not occur In
dependent on the supply of energy (as ATP or CTP) isolation. They are interdependent and integrated
a nd red ucing equiv alent s( as NADPH + H+ ) . into specific series that constitute metabolic
pathways. lt is, therefore, not an easy task to study
The a na bo lic and c at abolic pat hway s ar e n o t
metabolisms. Fortunately, the basic metabolic
re ve rsib lean d oper at e independent ly .As s uc h , t h e
pathways in most organisms are essentially
me tab olic pa t hway s oc c ur in s pec if ic c e l l u l a r
identical.
lo ca tion s (mit oc hondr ia, m ic r os om es et c . ) and a r e
con trolle d b y dif f er ent r egulat or ys ignals Several methods are emoloved to elucidate
b i o c h e m i c a l r e a c t i o n sa n d t h e m e t a b o l i c p a t h w a y s .
The terms-intermediary metabolism and energy
These experimental approaches may be broadly
metabolism-are also in use. Intermediary metabo-
divided into 3 categories :
Iism refersto the entire range o{ catabolic and anabo-
lic re action s , not inv olv ing nuc leic a c i d s . 1 . U s e o f w h o l e o r g a n i s m so r i t s c o m p o n e n t s .
Energy metabolism deals with the metabolic
2. Utility of metabolic probes.
pathways concerned with the storageand liberation
of energy. 3. Application of isotopes.
798 B IOTE CHNO LO CY

OXIDATIVE PATHWAYS SYNTHETIC PATHWAYS outlinesof major pathwayskycles

of carbohydrate
metabolismare describeo.
Glycolysis Other carbohydrates
G (galactose,fructose)
L GLYCOLYSIS
U Glycogenesis
Hexosemono- Clvcolvsis is derived from the Greek words
phosphateshunt o (glycose-sweetor sugar;lysis-dissolution). lt is a
S Lipogenesis
E (synthesisof fat) uni versalpathw ayi n the l i vi ngcel l s.

Uronicacid Non-essential Glycolysisis definedas the sequenceof reactions

pathway aminoacids converting glucose (or glycogen) to pyruvate or
lactate, with the production of ATP (Fig. 67.4.
Fig. 67.3 : Overview of glucose metabolism.
(Note : For majority of the pathways, glucose
partieipates as glucose 6-phosphate). Salient features
1. Clycolysis(alsoknown as Embden-llleyerhof
pathway\takesplace in all cells of the body.The
The actualmethodsemployedmay be either in enzymes of this pathway are present in the
vivo (in the living system)or in vitro (in the test cytosomalfraction of the cell.
tube) or, more frequently,both.
2. Clycolysisoccursin the absenceof oxygen
1. Use of whole organismor its components: (anaerobic) or in the presenceof oxygen(aerobic).
(a) Whole organisms: Clucosetolerancetest Lactate is the end product under anaerobic
(crr). condi ti on.In the aerobi ccondi ti on,pyr uvat eis
formed,which is then oxidizedto CO, and HrO.
(b) lsolatedorgans,tissueslices,whole cells,
s u b c e l l u l a ro rg a n e l l e setc., to el uci date 3. Clycolysis is a major pathway for ATP
b i o c h e mi c a l re a c ti ons and metabol i c synthesi si n ti ssuesl acki ng mi tocho ndr ia,e. g.
pathways. erythrocytes, cornea/lensetc.

2. Utility of metabolic probes : Two types of 4. Clycolysis is very essentialfor brain which
metabolicprobesare commonlyusedto trace out is dependent on glucosefor energy.The glucosein
biochemical pathways. These are metabolic brain hasto undergo glycolysisbeforeit is oxidized
inhibitors and mutations. to C O, and H rO.

3. Applicationof isotopes. 5. Gl ycol ysi s(anaerobi c)

may be su m m ar ized
by the net reaction
Clucose+ 2ADP + 2Pi ----+ 2 Lactate+ 2ATP
6. R eversalof gl ycol ysi s al ong wit h t he
alternatearrangementsmade at the irreversible
Carbohydrates are the major sourceof energy steps will result in the synthesisof glucose
for the living cells.The monosaccharide glucoseis (gluconeogenesis).
the central molecule in carbohydrate metabolism
since all the major pathways of carbohydrate
CONVERSION OF PYRUVATE
metabolismare connected with it (Fig. 67.3).
TO ACETYL COA
C l u c o s ei s u ti l i z e d a s a s o urceof energy,i t i s
synthesized from non-carbohydrate precursors ancj Pyruvateis corrvertedto acetylCoA by oxidative
decarboxvlation. This is an irreversiblereaction,
storedas glycogento releaseglucoseas and when
catalysed by a multienzyme complex, known as
the need arises. The other monosaccharides
pyruvate dehydrogenase complex (PDH),which is
importantin carbohydrate metabolismare fructose,
galactoseand mannose. found onl y i n the mi tochondr ia. High
concentrations of PDtl are found in cardiacmuscre
The fasting blood glucose level in normal and ki dney. The enzyme P D H requir es f ive
humansis 60-100ng/dl @.5-5.5mmol/l)and it is cofactors(coenzymes), namel y-TP P , lipoam ide,
v e rv e ffi c i e n tl v ma i n ta i n e dat thi s l evel . The FAD, coenzymeA and NAD+ (lipoamidecontains
Chapter67 : METABOLISMS 799

Glycogen Glucose Phosphoenolpyruvate

I I
A IH \' H e x o k i n a s e o r
+
Glucose1- tr,,t92+Jgtucokinase
phosphate nopvI
Glucose 6-phosphate
Pyruvate (enol)
1I Phosphohexose I
isomerase
I J,spoganeou$
I
Pyruvate (keto)
Fructose 6-phosphate
I NADH + Ht
ATP
\l Lactale
FhoeP-
ho{rtratbkinase
I dehydrogenase
ADP<J NAD-

Fructose 1, 6- L-Lactate
bisphosphate
Fig. 67.4 : The reactions in the pathway of glycolysis
(The three steps calalysed by hexokinase,
Aldolase phasphofructoki nase and pyruvate
kinaoe are iriaversible).

DihvdroxvacetonePhosphotriose Glyceraldehyde
ptrdsptraie , isomerase $phosphate l i p o i c a c i d l i n k e d t o e - a m i n o g r o u p o f l y s i n e ) .T h e
- overall reaction of PDH is
PDH
Pyruvate + NAD+ + CoA
' Acetyl CoA
Pi + CO, + NADH + H+
+
NAD
CITRIC ACID CYCLE
The citric acid cycle (Krebscycle or tricarboxylic
NA DH+ H + acid-TCA cycle) is the most important metabolic
pathway for the energy supply to the body. About
1, $Bisphosphoglycerate
65-700/"of the ATP is synthesized in Krebs cycle.
Citric acid cycle essentially involves the oxidation
of acetyl CoA to CO, and HrO.
The citric acid cycle is the final common
oxidative pathway for carbohydrates, fats and
amino acids. This cycle not only supplies energy
8-Phosphoglycerate but also proVides many intermediatesrequired for
^I the synthesis of amino acids, glucose, heme etc.
I Phosphogtycerate Krebs cycle is the most important central pathway
I mutase connecting almost all the individual metabolic
+
2-Phosphoglycerate pathways (either directly or indirectly).

The enzymes of TCA cycle are located in

Fig. 67.4 contd. nexl column
mitochondrial matrix, in close proximity to the
electron transport chain.
Krebs cycle basically involves the combinatron
of a two carbon acetyl CoA with a four carbon
oxaloacetate to produce a six carbon tricarboxylic
800
acid, citrate. In the reactions that follow, the two
carbons are oxidized to CO, and oxaloacetate is
regenerated and recycled. Oxaloacetate is
considered to play a catalytic role in citric acid
cycle. fhe reactionsof Krebs cycle are depicted in
Fig. 67.5.
G LUCO NEO G ENESI S
The s y nt hes is of gluc os e o r g l y c o g e n f r o m
non- c ar bohy dr at ec om pounds i s k n o w n a s g l u c o -
neogenesis. The major substrates/precursorsfor
gluconeogenesis are lactate, pyruvate, glucogenic
amino acids, propionate and glycerol.
Location of gluconeogenesis
Gluconeogenesisoccurs mainly in the cytosol,
although some precursors are produced in the
m it oc hondr ia. Cluc oneogenes ism o s t l y t a k e s p l a c e
in liv er and, t o s om e ex t ent , i n k i d n e y m a t r i x
(about one-tenth of liver capacity).
Reactions of gluconeogenesis
G luc oneogenes isc los ely r es e m b l e st h e r e v e r s e d
pat hway of gly c oly s is , alt hou g h i t i s n o t t h e
complete reversal of glycolysis. Essentially,3 (out
of 10) r eac t ionsof gly c oly s is ar e i r r e v e r s i b l e .T h e
s ev en r eac t ions ar e c om m on f o r b o t h g l y c o l y s i s
and gluconeogenesis fhe three irreversible steps
of glycolysis are catalysed by the enzymes, namely
hexokinase, phosphofructokinase and pyruvate
kinase.
G LYCO G EN M ETABO LI SM
Clycogen is the storage form of glucose rn
anim als , as is s t ar c h in plant s . lt i s s t o r e d m o s t l y r n
liv er ( 6- B% ) and m us c le ( 1- 2 %) . D u e t o m o r e
m us c le m as s , t he quant it y of g l y c o g e n i n m u s c l e
( 250 g) is about t hr ee t im es high e r t h a n t h a t i n t h e
liv er ( 75 g) .
Functions of glycogen
The pr im e f unc t ion of liv e r g l y c o g e n i s t o
m aint ain t he blood gluc os e l e v e l s , p a r t i c u l a r l y
between meals. Liver glycogen stores increase in a
well-fed state which are depleted during fasting.
Muscle glycogen serves as a fuel reserve for the
supply of ATP dur ing m us c le c o n t r a c t i o n .
B IOTE C HNO LO CY
GLYCOGENES'S
fhe synthesis af glycogen from glucose rs
C l ycogenesi stakes pl ace in t he
gl ycogenesi s.
cytosoland requiresATPand UTP,besidesglucose.
GLYCOGENOLYS'S
The degradationof storedglycogenin liver and
muscl econsti tutes gl ycogenol ysiThe
s. pathwayf or
the synthesisand degradationof glycogenare not
reversi bl eA n i ndependent set of enzyme spr esent
i n the cytosolcarryout gl ycogenol ysiCsl ycogenr s
degraded by breaki nga-1, 4- and a-1, 6-9lycosidic
bonds.
H E X OS E MON OP H OS P H A TE S H U NT
Hexosemonophosphate pathwayor HMP shunt
is also called pentose phosphate pathway or
phosphogluconate pathway.This is an alternative
pathway to glycolysis and TCA cycle for the
oxidation of glucose.However,HMP shunt is more
anabol i ci n nature,si ncei t i s concernedwit h t he
bi osynthesiof s N A D P H and pentoses.
Location of the pathway
The enzymesof H MP shuntare l ocat edin t he
cytosol. The tissuessuch as liver, adipose tissue,
adrenal gland, erythrocytes, testes and lactating
mammarygland, are highly active in HMP shunt.
Most of these ti ssues are i nvol ved in t he
biosynthesisof fattv acids and steroidswhich are
dependenton the suppl yof N A D P H .
Beactions of HMP shunt
The sequenceof reacti onsof H MP shunt
is depictedin Fig. 67,6
Significance of HMP shunt
H M P s h u n t i s u n i q u e i n g e n e r a t i n gt w o i m p o r -
tant products-pentoses and NADPH-needed for
the biosynthetic reactions and other functions.
A. lmportance of pentoses
I n t h e H M P s h u n t , h e x o s e sa r e c o n v e r te d i n to
pentoses, the most important being ribose
5-phosphate. This pentose or its derivatives are
useful for the synfhesis of nucleic acids (RNA and
DNA) and many nucleofides such as ATR NAD+,
FAD and CoA.
 M E T AB O L IS M S

"rr-3-.oo-
pyruvate

Pyruvate
Cehydrogenase

CH.-C-S Con
AcetylCoA

o I coasH
t i-'" cHp-coo-
c-coo- -# Ho_f_coo_
]H2-COO- svnthase
) CH2_COO-
Oxatoacetate Citrate
J/r. \ JrO
/ w^l^r" nconiraseY
,1 dehydrogenase \
HO_CH_COO_ OH2_COO_
rl
CH2-COO- c-coo-
L-Malate CH-COO-
Cls-Aconitate
f
,..,^...'
, \;, Hzo
' '2" --/Frt"r"r" Aconitase{
I
l+
+ OH2-COO-
H_C_COO- I
li cH-coo-
ooc-c_H I
HO_CH_COO_
Fumarate
,socitrate
a
\/
{.\ Succinate tsocitrate l. ,
cetrydrogenase dehydrogenase
(\l A , ,. ,-,,, , ,,
.L
CH2-COO-
CH2-COO-
I
cH2-coo- cH-coo-
Succinate O = C_COO-
Succinate
A- tiriotrinase - Oxalosuccinate
coAs;\ )\ cH2-coo
r - ^o** ,
..."r?
CHz
,..o\[roo",r"
.vru ^r2-vvv
_ . . '. . l d " '''. /
O =
' 0-l(or^-'
k' "tt
J;;,:.T^-!K5L!"-- -
O = C-COO-
o-Ketoglutarate
"o'
COz CoA SH

Fig. 67.5 : The citric acid (Krebs) cycle.

Biotechnology [51]
8(J2 B IOTE C H NO LO CY

Glucose 6-phosphate &Phosphogluconolactone

l
V-Hro
_
N AD P- N A D P H+ H * [ 'Gluconolectone
I hydrolase
J
6-Phosphogluconate
Fhosphogluconate
Qehydrogenase
Mg-'

RibuloseS-pnosptrate

Riboses-phosphate
ketoisomelase

Ribose 5-
phosphate

Fructose 6-phosphate

TPP

Tran$kelolase Glyceraldehyde
Glyceraldehyde 3-phosphate
3-phosphate
t

I Fleversalof
It *. olvcolvsis
Fructose 6-phosphate
Erythrose 4-phosphate Fructose 6-phosphate

Fig, 67.6 : The hexose monophosphate shunt

B. lmportance of NADPH u n s a t u r a t e d l i p i d s , p r o t e i n s a n d D N A . T h i s i s,
however, prevented to a large extent through
1 . N A D P H i s re q u i re d fo r the reducti ve
antioxidant reactions involving NADPH. Gluta-
biosynthesis of fatty acids and sferoids, hence
thione mediated reduction of HrO, is given
H MP s h u n ti s m o rea c ti v ei n th e ti ssues concerned
hereunder
w i th l i p o g e n e s i e
s ,.g . a d i p o s eti ssue,l i ver etc.
2 . N AD PH i s u s e d i n th e s ynthesiof
s certai n
amino acids involving the enzyme glutamate
dehydrogenase.

3 . T h e rei s a c o n ti n u o u ps ro d ucti on
of H rOri n
t h e l i v i n g c e l l s w h i c h c a n c h emi cal l ydamage
C hapt er62 : M E T A BOL IS M S 803

Fattyacid

Y
I
+
AcetylCoA

6x
Aconitase

l$ocitratelyase

Fig. 67.7 : The giyoxylate cycle (represented in green colour).

Clutathione (reduced, CSH) detoxifies HrOr, photosynthesis. lt is now recognized that

pe roxida se cata ly s es t his r eac t ion. NADPH i s
responsible for the regeneration of reduced
g luta thio ne fro m t he ox idiz ed one.
GLYOXYLATE CYCLE
The a nima ls, inc luding m an, c annot c ar r y ou t
:he net synthesis of carbohydrate from tat.
However, the plants and many microorganisms are
=q uip pe d with th e m et abolic m ac hiner y - nam ely
:.e glyoxylate cycle-to convert fat into
: :i-b oh yd rate s.Th is pat hway is v er y s ignif ic ant i n
--c ge rmina ting s eeds wher e t he s t or e d
,- acylglycerol (fat) is converted to sugars to meet
^ e e ne rSy ne eo s .
The g lyo xylate c y c le is r egar dedas an anabol i c
, .aria nt of citric ac id c v c le and is deoic t ed i n
Fig. 67.7
photosynthesis primarily involves the process of
energy transduction in which light energy is
converted into chemical energy (in the form of
oxidizable carbon compounds).
It is an established fact that all the energy
consumed by the biological systemsarisesfrom the
solar energy, that is trapped in the photosynthesis.
The basic equation of photosynthesisis given below.
Co,+ H,o --lehl------ (cH,o)
c n t o r o p n y tI
In the above equation, (CH2O) represents
cai'bohydrate. Photosynthesisin the green plants
o c c u r s i n t h e c h l o r o p l a s t s ,a s p e c i a l i z e do r g a n e l l e s .
The mechanism of pl.rotosynthesisis complex,
i n v o l v i n g m a n y s t a g e s ,a n d p a r t i c i p a t i o no f v a r i o u s
m a c r o m o l e c u l e sa n d m i c r o m o l e c u l e s .

PHOTOSYNTHESI S The role of photosystems

The synthesis of carbohydrates in green plants T h e i n i t i a l s t e p i n t h e p h o t o s y n t h e s i si s t h e
cr assimilationof carbon dioxide is referred to as a b s o r p t i o no f l i g h t b y c h l o r o p h y l l m o l e c u l e s i n t h e
804 B IOTECHNO LO CY

compounds(Fig.67.9).The Calvincyclestartswith
lPhosphoglyc"r"r\t to
a reactionof CO, and ribulose1, S-bisphosphate
form two molecules 3-phosphoglycerate. This
1, 3-Bisphosphoglycerate
3-phosphoglycerate can be convertedto fructose
\
ATP./\
I l;NAoeH
6-phosphate,
comoounos.
glucose6-phosphate and othercarbon
I
Y
Glyceraldehyde
Ribulose 3-phosphate
5-phosphate .,....-.-'
'-"Fructose(
6-phosphate Li pi dsare i ndi spensablfor
e cel l st r uct ur eand
functi on.D ue to thei r hydrophobi and
c non- polar
Fig. 67.8 : An outline of the Calvin cycle ot nature, lipids differ from rest of the body
photosynthesis. compoundsand are uni quei n thei r act ion
The important pathwayskycles of lipid
metabolismare brieflydescribed.
c h i o ro p l a s tsT. h i s re s u l t si n the producti onof
excitationenerSywhich is transferredfrom one
FATTY ACID OXIDATION
l o l e c u l eto a nother,unti l i t i s trapped
c h l o rc p h y lm
by a reactior.r center.The light-activated transferof The fatiy acidsin the body are mostlyoxidized
an electronto an acceptor(photosystems) occursat by p-oxidation.B-Oxidationmay be definedas the
the reactioncenter. oxidationof fatty acidson the B-carbonatom. Thrs
resultsin the sequentialremovalof a two carbon
Pirotosynthesis primarilyrequiresthe interactions
fragment,acetyl CoA (Fig. 67.10).
of two ciistinctphotosystems (l and ll). Photosystem I
generatesa strong reductantthat resultsin the Fatty acid oxidation-
formationof NADPH. Photosystem ll producesa stages and tissues
strong<.rxidant thatformsO, from HrO. Further, the
generationof ATP occurs as electronsflow from The p-oxidationof fatty acids involvesthree
photosystemll to photosystem| (Fig. 67.6. fhus, stages
light is responsible for the flow of electronsfrom l. Activationof fatty acids occurringin the
HrO to NADPH with a concomitantgenerationof cytosol;
ATP. ll. Transportof fatty acids into mitochondria;
Il l . p-Oxi dati onproper i n the m it ochondr ial
The Calvin cycle matri x.
The dark phaseof photosynthesisis referredto as Fattyacidsareoxidizedby mostof the tissuesin
C a l v i nc y c l e . In th i s c y c l e,the A TP and N A D P H the body. However,brain, erythrocytesand adrenal
producedin the light reaction(described above)are medulla cannot utilize fatty acids for energy
utilizedto convertCO, to hexoses andotherorganic requirement,

""'-"'-'NADPH
Strongreductant
Lioht ----------+t -- ---,1
(<706nm) HnolosyslemII
|
Weak oxidant
>ATP
Weak reductant
-------J
,.o|J311', fl
Fr.'"*Y"*,'
Strongoxidant-7=+@
Hzo

Fig. 67.9 : lnteraction betuteen two photosystems in photosynthesis'

Cha pte r 6 7 : M ETABO LI SM S 805

o
R-CH2-CH2-CH2-C-O
Fattyacid

ATP CoASH
Mg-
A M P + PP i a - Thiokinase
o
tl
R-CH2-CH2-CH1-C-S CoA
Acyl CoA

- I cYrosol
ll ca;,tin"tr;l-.poItrrtut
i MlrocHoNDRloN
1t. o
Bcrll

Ftg. 67.10 : p-Oxidation of fatty acids : Palmitoyl CoA (16 carbon) undergoes seven cycles to yietd B acetyt CoA
.'-lctivation; ll-TransporT; lll-B Oxidation proper-(l) Oxidation, (2) Hydratiotl, (3) Oxidation and (4) Cleavagel
806
BIOSYNTHESIS OF FATTY ACIDS
The dietary carbohydrates and amino acids,
when consumed in excess, can be converted to
fatty acids and stored as triacylglycerols.De novo
(new) synthesisof fatty acids occurs predominantly
in liv er , k idney , adipos e t is s u e a n d l a c t a t i n g
mammary glands. fhe enzyme machinery for fatty
acid production is located in the cytosomal fraction
of the cell. Acetyl CoA is the source of carbon
at om s while NADPH pr ov i d e s t h e r e d u c i n g
equivalents and ATP supplies energy for fatty acid
formation. The fatty acid synthesismay be learnt in
2 stages
l. Conversion of acetyl CoA to malonyl CoA.
ll. Reactionsof fatty acid synthasecomplex.
l. Formation of malonyl GoA
Acetyl CoA is carboxylated to malonyl CoA by
the enzyme acetyl CoA carboxylase. This is an
ATP-dependent reaction and requires biotin for
COz fixation. Acetyl CoA carboxylase is a
regulatory enzyme in fatty acid synthesis.
ll, Reactions of fatty acid
synthase complex
The remaining reactions of fatty acid synthesis
are catalysed by a multifunctional enzyme known
as fatty acid synthase (FAS) complex. In eukaryotic
cells, including man, the fatty acid synthaseexists
as a dim er wit h t wo ident ic al u n i t s . E a c h m o n o m e f
possessesthe activities of seven different enzymes
and an acyl carrier protein (ACP) bound to
4'-phosphopantetheine. Fatty acid synthase
f unc t ions as a s ingle unit c at a l y s i n ga l l t h e s e v e n
reactions. Dissociation of the synthase complex
results in loss of the enzyme activities.
The sequence of reactions for the extra-
mitochondrial synthesisof fatty acids (palmitate) is
depicted in Fig. 67.11, and described below
1. The t wo c ar bon f r agm e n t o f a c e t y l C o A i s
t r ans f er r ed t o ACP of f at t y a c i d s y n t h a s e ,
c at aly s ed by t he enz y m e, a c e t y l C o A - A C P
t r ans ac y las e. The ac et y l unit i s t h e n t r a n s f e r r e d
f r om ACP t o c y s t eine r es idu e o f t h e e n z y m e .
Thus ACP s it e f alls v ac ant .
2. The enzyme malonyl CoA-ACP transacylase
transfers malonate from malonyi CoA to bind to
ACP.
B IOTE CHNO LO CY
3. The acetylunit attachedto cysteineis trans-
fe;'redto malonylgroup(boundto ACP).Themalonyl
moiety losesCO, which was added by acetyl CoA
carboxylase.Thus, CO, is never incorporatedinto
fatty acid carbonchain.
4. p-KetoacylACP reductasereducesketoacyl
group to hydroxyacyl group. The r educing
equi val are suppl i edby N A D P H (f r om HM P
ents
shu nt).
5. B-Hydroxyacyl ACP undergoesdehydration.
A mol ecul eof w ater i s el i mi natedand a double
bond is introducedbetweenu and B carbons.
6 A second N A D P H -dependenreduct t ion,
cataiysed by enoyl-ACP reductase occurs to
oroduceacvl-ACP. The four-carbon unit attachedto
A C P i s butyrylgroup.
The carbonchain attachedto ACP is transferreo
to cysteine residue and the reactions2-6 are
repeated6 more times. Eachtime, the fatty acid
chai ni s Iengthened by a tw o-carbonuni t ( obt ained
from malonylCoA).At the end of 7 cycles,the fatty
aci d synthesi iss compl eteand a 16-ca r bonf ully
saturatedfatty acid-namely palmitate-boundto
A C P i s produced.
7. The enzymepalmitoylthioesterase separates
palmitatefrom fatty acid synthase.
This completes
s pal mi tate.
the synthesiof
ME TA B OLIS M OF C H OLE S TE R OL
C hol esteroli s found excl usi vel yi n anim als,
hence it is often called as animal sterol.The total
body content of chol esteroli n an adult m an
w ei ghi ng70 kg i s about140 g i .e.,around2 g/ kg
body weight.Cholesterol is amphipathicin nature,
si nce i t possessesboth hydrophiiic and
hydrophobicregionsin the structure.
CHOLESTEROL B'OSYNTHESTS
About 1 g of cholesterolis synthesizeciper day
i n adul ts.A l most al l the ti ssuesof the body
participatein cholesterolbiosynthesis. The largest
contribution is made by liver (5Oo/o),intestine
(15% ),ski n,adrenalcortex,reproducti ve tissueet c.
The enzymesinvolved in cholesterolsynthesis
are found in the cyfosol and microsomal fractions
of the cell. Acetateof acetyl CoA providesall tne
carbon atoms in cholesterol. The reducing
equi val entsare suppl i edby N A D P H while AI P
provides energy. The key intermediatesof
cholesterolformationare depictedin Fig. 67.12.
Chapter67 : METABOLISMS ao7

CH3-C-SCoA

@yslsH
(
9l i
)
q97-s- c-cH3
Irans A2-EnoylACP
Acetyl-S-ACP j Transferof acetyr
tocysierne
J
o
s- c-cH3
Jt1 rrfl
Acetyl S-enzyme
FC-CH2-CH2-CH3
o
ll
-OOC-CH2-C-S
Acyl-ACP (butyryl-ACP)
CoA
MalonylCoA
t_
I r ranslerof caroon
MalonylCoA-ACF I chainfrornACPto Gys
iransaoylase +
CoASH a)
t1
s-c-cH2-cH2-cH3
SH

Acylmalonyl enzyme Acyl-S-enzyme

co,- J3),.]g-Ketoacyt-AoP
| synthase

sto
rl
FC-CH2-C-CH3
I
p-Ketoacyl-ACP HzO\ Palmitoyl
Jrnroesrerase
6GfsH
( + C H 3-C H 2-(C H 2)13-C OO-
)
\ACP}-SH
\--l PALTvITATE
Fig. 67.1 I contd. next column

Fig, 67.11 : Biosynthesis of long chain fatty acid-palmitate. (Cys-Cysteine: ACP-Acyl carrier protein; The
pathway repeats 7 times to produce palmitate; the first two carbans at the methyl end are directly from acetyl
CoA, the rest of the carbons come from malonyl CoA).
808 B IOTECHNO LO CY

Acetyl,CoA(2C) Protein metabolism is more appropriately learnt as

Y metaholism of amino acids.
HMG CoA (6C)

Mevalonate(6C)

lsoprenoidunits
(5C;buildingblocks)
T h e a m i n o a c i d s u n d e r g o c e r t ai n co m m o n
i 6 units
reactions like transamination followed by
.|i condense
Squalene(30C) deamination for the liberation of ammonia. fhe
a m i n o g r o u p o f t h e a m i n o a c i d s i s u ti l i ze d fo r th e
i
formation of urea which is an excretory end
\7
product of protein metabolism.The carbon skeleton
Lanosterol(30C)
of the amino acids is first converted to keto acids
(by transamination)which meet one or more of the
*' following fates.
(27C)
Cholesterol
1 . U i i l i z e d t o g e n e r a t ee n e r g y .
2. Llsed for the synthesisof glucose.
Fig. 67.12 : Outline of cholesterol biosynthesis.
3. Diverted for the formation of fat or ketone
bod ies.

DEGBADATION OF CHOLESTEROL 4 . I n v o l v e d i n t h e p r o d u c t i o n o f no n - e sse n ti a l

amino acids.
T h e s te ro i d n u c l e u s (ri ng structure)of the
cholesterolcannotbe degradedto CO, and HrO A g e n e r a l p i c t u r e o f a m i n o a c i d m e ta b o l i sr n i s
Cholesterol(50%) is converted to bile acids, depicted in Fig. 67.13.
excreted in feces, serves as a precursorfor the
svnthesis of steroid hormones, vitamin D,
coprostanol and cholestanol. The lattertwo are the
fecal sterols,besidescholesteroi. Dietarv Body
protein protein

\ ,1
Protein Synthesisof ,
{- Aminoacids -----)
synthesis N-compounds
Proteins are the most abundant organic
compounds and constitutea major part of the body cr-Ketoglutarate
dr y v ^r eight( 10- 12 k g in a d u l t s ) . T h e y p e r f o r m a
wicie variet'y of static (structural) and dynamic Transamination
(enzymes.hormones,clotting factors,receptorsetc.)
iunctions. About half of the body protein (predo-
m inant ly c ollagen) is pr es e n t i n t h e s u p p o r t i v e
tissue (skeleton and connective) while the other
half is int r ac ellular .
lhe proteins on clegradation(proteolysis)release
indiv idual am ino ac ids . Am i n o a c i d s a r e n o t j u s t
the structural components of proteins. Each of the
20 nat ur ally oc c ur r ing am i n o a c i d s u n d e r g c e s i t s
Energy Glucose Fat Non-essential
own metabolism and performs specific functions. aminoacids
Some of the amino acids also serve as orecursors
for the synthesis of many biologically important
Fig. 67.13 : An overuiew of amino acid metabolism.
c om pounds ( e. g. m elanin, s e r o t o n i n ,c r e a t i n e e t c . ) ,
Chapter67 : METABOLISMS 809

R,-CH-COO- R,-C-COO- UREA CYCLE

I Urea is the end product of protein metabolism
NHJ
Keto acid-l
Amino acid-l
R2-C-COO-
o NH;
Keto acid-ll Aminoacid-ll
Fig. 67.14 : Transaminationreaction.
TRANSAMINATI O N
The transferof an amino (-NH2) group from an
amino acid to a keto acid is known as transamina-
tio n (Fiq.6 7.14\ . This pr oc es s inv olv es t h e
interconversionof a pair of amino acids and a parr
of keto acids, catalysed by a group of enzymes
ca Iled fransa minases (recently, aminotransferases).
The salient featr.rres
of transamination are :
l. All transaminasesrequire pyridoxal phosphate
(PLP),a coenzyme derived from vitamin Bu.
( a m i n o a c i d m e t a b o l i s m ) .T h e n i t r o g e n o f a m i n o
acids convertedto ammonia (as describedabove) is
toxic io the body. lt is converted to urea and
detoxified.As such, urea accountsfor B0-9C%of the
n i t r o g e n c o n t a i n i n g s u b s t a n c e se x c r e t e d i n u r i n e .
Urea is synthesized in liver and transoorted to
kidneys for excretion in urine. Urea c'ycle is the
first metabolic cycle that was elucidated by Hans
Krebs and Kurt Henseleit ('l932), hence it is known
as Krebs-Henseleit cycle. The individual reactions,
however, were described in more detail later on by
Ratner and Cohen.
Urea has two amino (-NH2) groups, one
derived from NH, and the other from aspartate,
Carbon atom is supplied by COr. Urea synthesisis
a five-stepcyclic process,lvith five distinct enzymes.
The first two enzymes are present in mitochondria
while the resf are localized in cytosol.The reactions
of urea cycle are depicted in Fig. 67.15.

2. There is no free NH, liberateci, only the

transfer of amino group occurs.

3. Transamination is reversible. The metabolisms of certain individual amrno

4. lt involves both catabolism (degradation)and
an ab olism ( s y nt hes is ) of am ino acids.
Transamination is uitimately responsible for the
synthesisof non-essentialamino acids.
5 . Tran sa m inat ion div er t s t he ex c es s am r n o
acids towards energy generation.
6. The amino acids undergo trbnsamination to
finally concentrate nitrogen in glutamate.
Glutamate is the only amino acid that undergoes
oxidative deamination to a significant extent to
liberate free NH, for urea synthesis.
acids are very briefly given in the form of overviervs.
GLYCINE
G l y c i n e ( C l y , G ) i s a n o n - e s s e n t i a l ,o p t i c a l l y
inactive and glycogenrc (precursor for glucose)
a m i n o a c i d . l t i s i n d i s p e n s a b l ef o r c h i c k s . T h e
outline of glycine metabolism is depicted in
Fig.67.16. Clycine is actively involved in the
synthesis of many specialized products (heme,
purines, creatine etc.) in the body, besides its
incorporation into proteins, synthesisof serine and
glucoseand participationin one-carbonmetabolism.

7 . All a mino ac ids ex c ept ly s ine, t hr eo n i n e , PHENYLALANINE AND TYROSINE

proline and hydroxyproline participate in transamr-
Phenylalanine (Phe, F) and tyrosine (Tyr, Y)
na tion .
are structurally related aromatic amino acicls.
Phenylalanine is an essential amino acid while
DEAMINATION
tyrosine is non-essential.Besides its incorporation
The removal of amino group from the amino i n t o p r o t e i n s ,t h e o n l y f u n c t i o n o f p h e n y l a l a n i n er s
a cid s as NH, is deam inat ion. lt r es ult s in t h e its conversion to tyrosine. For this reason, ingestion
liberation of ammonia Ior urea synthesis. of tyrosine can reduce the dietary requirement of
Deamination may be either oxidative or non- phenylalanine. This phenomenon is referrecito as
oxidative. 'sparing actiont of tyrosine on phenylalanine.
 B IOTE C HNO LO CY
8to

CO2+ NHI

ZA I r=\
V I
rrn n 'l C arbamovl
N"9'/
ot'oiPhatl sYnthasel+
2A D P+ P i + ' + '
OO
ltl
H2N-
-l
c -o-P-o

d
CarbamoylPhosphate
N Hz
I

I
HN
NHT ,/ I
CHz
I
CHz

QHz
CHz
I,
H-C-NHi
I
"f-**A
I
coo-
Citrulline

ATP
o j^a\r1-
Argino-
tl succlnale
H2N- C -N I synthase
Urea NHi NH; CH..
Arginase IL ll-l- A MP+ P P i
c -NH2
I
I
HN HT'J coo
I I Aspartate
CHz CHu
I t-
cHz Argino' CHc
I succlna9e l-
CHe v11.t
lt.-
l-
H-C-NHi H-c-NHa
I
coo- I
coo I coo-
H-C Argine
Arginine
il succinate
c-H
I
coo-
Fumarate
group ts
(NAG-N-acetlrlgtutamate; (in the formati.onof urea, one amino
i,n. ur.ru: Beactionsof urea cycle carbon^is obtained from co';
derivec from freeur*on,u* ion white the otier is from aspartate;
* mitochondrialenzymes, the rest of the enzymes are cytosomal)
 Chant er67 : M E T A BOL IS M S 811

Proteins
Purines
Serine G (Cq, Cs, N7 atoms) Proteins T
R
L Glutathione
Oxalate Y Glucose P NAD-,NADP-
T (coenzymes
of niacin)
C Conjugation
o
Glucose (bileacids,detoxif
ication) Fat
I P Serotonin
H
NHs N Heme
Indoleacetic A t,Ar
E acid N Melatonin 5-Hydroxy-
Creatine indoleacelicacid
Formate
+
One-carbonpool

Fig. 67.16 : Overview of glycine metabolisrn. Fig. 67,18 : Overview of iryptophan metabclism.

The p red omin ant m et abolis m of pheny lalani n e propionic acid. Tryptophan is both glucogenic and
occurs through tyrosine.Tyrosineis incorporatedinto ketogenic in nature. lt is a precusorfor the synthesis
pro tein sa nd is in v olv ed in t he s y nt hes isof a v ar ie t y of important compounds, namely NAD+ and
of biologically important compounds-epineph rine, NADP+ (coenzymes of niacin), serotonin and
norepinephrine, dopamine (catecholamines), thyroid melatonin tFig. 67.1 B\.
hormones-and the pigment melanin (Fig. 67.1/
Durin g the cou rseof degr adat ion,pheny laianinean d SULFUB AMINO ACIDS
tyrosine are converted to metabolites which can
T h e s u l f u r - c o n t a i n i n ga m i n o a c i d s a r e m e t h i o -
serve as precursorsfor the synthesisof giLrcoseand
nine, cysteine and cystine. Among these, only
fat. Hence, these amino acids are both glucogenic
methionine is essential. lt serves as a precursor for
and ketogenic.
t h e s y n t h e s i so f c y s t e i n e a n d c y s t i n e w h i c h a r e ,
therefore, non-essential. An overview of the
TRYPTOPHAN
m e t a b o l i s mo f t h e s u l f u r a m i n o a c i d s i s d e o i c t e d i n
Tryptophan (Trp, W) was the first to be identified Fig.67./9.
as an essential amino acid. lt contains an indole
-ing a nd che mic ally it is c r - am ino p- indole GLUTAMATE AND GLUTAMINE

Clutamate and glutamine are non-essential

glycogenic amino acids. Both of them play a
predominant role in the amino acid metabolism and
Melanins
are directly involved in the final transferof arnino
(skin,hair,eye)
group for urea synthesis.ln Fig. 67.20, an outline of
T
g l u t a m a t ea n d g l u t a m i n e m e t a b o l i s m i s g i v e n .
R Dopamine(CNS)
o
e
I
Norepinephrine,
N
epinephrine
E (adrenalmedulla) After the removal of amino groups, the carbon
Thyroxine,T3 skeleton of amino acids is converted to
(thyroidgland) intermediatesof TCA cycle or their precursors.The
carbon skeleton finally has one or more of the
Fig, 67.17 : Overview of phenylalanineand tyrosine following fates :
nretabofism (CNS Cenlral nervous system;
1 . Oxidation via TCA cycle to produce energy
. ,. ,,.,,,.r,T3,.TTriigdothylgnri€1. ,.. ,, ,.: (about 10-15% of body needs).
a12 B IOTE C H NO LO CY

M
E Proterns
T Transmethylation L] y-Aminobutyric
acid
H NHs L (GABA)
I U
Proteins Cystathionine Glucose Glutathione
o T
N Histidine
I Polyamines M N-Acetylglutamate
N Proline A
T
E I
"y-Carboxyglutamate
Arginine E (clottingfactors)

Proteins C Glutathione
S Taurine
T r:
Purines(Ne,Ns)
E CoenzymeA
i

'.-J
I
Proteins (N3)
Pyrimidines
Glucose N Active sulfate
i: NHs
M
I Aminosugars

<-F!s rnl]
Proteins
I +
Urea i'l
r Detoxification

Fig. 67.19 : Overview of the metabolism Fig. 67.20 : Overview of glutamate and glutamine
of sulfur amino acids. metabolism.

2. Sy nt hes isof gluc os e.

3. Formation of lipids-fatty acids and ketone

bod ies.
4. Sy nt hes isof non- es s ent ia a
l mino acids.
The carbon skeletonsof the 20 standard amino
acids (or the amino acids of proteins)are degraded
to one of the following seven products-pyruvate,
a-ketogluta rate, succi nyl CoA, fumarate, oxaloace-
tate, acetyl CoA and acetoacetate. Some authors
use the term amphibolic (Greek: amphiboles-
uncertain) intermediatesto these compounds due
to t heir m ult iole m et abolic f unc t i o n s .
The am ino ac ids ar e c las s if iedi n t o t h r e e g r o u p s ,
based on the nature of the metabolic end products
of carbon skeleton (Table 67.1\.
M e t a b o l i s m i s a c o n t i n u o u s p r o c e ss, w i th
t h o u s a n d so f r e a c t i o n s ,s i m u l t a n e o u s l yo c c ur r i n g i n
t h e l i v i n g c e l l . H o w e v e r , b i o c h e m i s t s p r e fe r to
oresent metabolism in the form of
reactionsand metabolic pathways. This is done for
t h e s a k e o f c o n v e n i e n c e i n o r e s e n t a ti o n a n o
u n d e r s t a n dni g .
ln the preceeding pages, we have learnt the
metabolism of carbohydrates, lipids and amino
acids. We shall now consider the orga-
nism as a whole and integrate the metabolisnr
with particular referenceto energy demands of the
body organism.

E$IERSY trErvlAhitr Af.{A SL9PPLY

inhor n er r or s of annino ac id
rrretabclisrn-a summary The organismspossessvariable energy demar-ros,
h e n c e t h e s u p p l y ( i n p u t ) i s a l s o e q u a l l y va r i a b l e
Several inherited disorders are associatedwith
The consumed metabolic fuel may be burnt
amino acid metabolism. ln Tahle 67.2, a summary
(oxidized to CO, and HrO) or stored to meet the
of major diseases and the enzyme defects is
energy requirementsas per the body needs. AIP
8r v en.
C hapt er67 : M E T AB O L IS M S 813

Ot
Tlslr 67.1 Classlflcatlon of anlno acids t-
EnergV-----.5 CO2 + H2O
based on the fate of carbon skeleton supprv \/ ./ \I
/v a.i i hi a\ 'Yl /

Glycogenic Stored ADP + Pi ATP

Glycog,enic anc! Kr-'lisgettir
energy ,\/
gilucogenic) Ketogenic \_r/
Alanine Phenylalaninex L e u c i n* e +
Energydemands
Argln lne + *
lsoleucine
+
4
Lysrne (variable)
nJP4r rqrtr Tyrosine Fig. 67.21 : A summary of body's energy supply and
,{, 4'
Cysteine Iryplopnan demands. (Note : ATP serves as the energy currency).
Glutamine
Glutamate serves as the energy currency of the cell in this
Glycine process fig. 67.21).
Histidine* The humans possessenormous capacity for food
Hydroxyorollne c o n s u m p t i o n Jl t i s e s t i m a t e dt h a t o n e c a n c o n s u m e
M et hionine* as much as 100 times his/her basal reouirements!
Obesity, a disorder of overnutrition mostly
Proline
prevalent in affluent societies, is primarily a
Serine c o n s e q u e n c eo f o v e r c o n s u m p t i o n .
.+
I nr eont ne+
V aline* I N T E G R A T A O }T O F M A J O R M E T A B O L ; C
PATFfWAVS @F EiITEHGV METABOI.ISM
+ Essential aminoacids;(Helpfultips to rccall-ketogenic
anino
An overview of the interrelationshio between
acidsstartwithletter'L';PITTforgtyco-andketogenicaninoacids;
the important metabolic pathw,ays,concerned with
rcstof the 20 aminoacidsare onlyglycogenic)
fuel metabolism depicted in Fig. 67.22, is briefly

Glucose Glycogen Triacylglycerols

I
I
IL +
Fattyacids

--+Hzo
I feh"29K:"n
//)1
/ A D P +P i

Fig, 67,22 : An overview of integrction of metabolic pathways of energy metabolism

(HMP shunt-Hexose monophosphate shunt).
414 B IOTE C H NO LO CY

Tnslr 67.2 Inborn errors of amino acid netabolism

Disorder Metabolic defect (enzvme/other)

l. Phenylalanine
and tyrosine
1. Phenylketonuria hydroxylase
Phenylalanine
2. Tyrosinemia
typell Tyrosine
lransaminase
3 . N e o n a ta l ty ro s i n e mi a phenylpyruvate
p-Hydroxy dioxygenase
4. Alkaptonuria Homogentisate
oxidase
5. Tyrosinosis (tyrosinemia typel) iso,nerase
Maleylacetoacetate or fumaryl hydrolase
acetoacetate
6. Albinism Tyrosinase
ll. Sulfuramino acids (methionine,cysteineand cystine)
7. Cystinuria Defectin renalreabsorption
8. Cystinosis lmpairmenl in cystine (defect
utillzation function)
in lysosomal
9. Homocystinuria
typeI Cystathionine
synthetase
10. Homocystinuria
typell N5,N1o-Methyiene
THFreductase
11 Homocystinuria
typelll THF-homocysteine
N5-Methyl methyltransf
erase
n,,^+^+h;^^;^ ^^^
12. Cystathionuria vyJtdu iluilil tdJU

l l l . Gl y c i n e
1 3 . G l y c i n u ri a in renalreabsorption
Defect
14. Primary
hyperoxaluria Glycine
transaminase
Tryptophan
15. Hartnup's
disease intestinal
Defective absorption
V. Branchedchain amino acids (valine,leucineand isoleucine)
16. Maplesyrupurinedisease Branchedchaina-ketoaciddehydrogenase
17. Intermittent chainketonuria Variant
branched of theaboveenzvme (lesssevere)
18. Hypervalinemia Valine
transaminase
19, lsovaleric
acidemia lsovaleryl
CoAdehydrogenase
vl. Histidine
20. Histidinemia Histidase
vll. Proline
typeI
21. Hyperprolinemia Proline
oxidase

des c r ibed her e. For det ailed inf o r m a t i o n o n t h e s e

metabolic pathways, the reader must refer the
nrcrpedino nroec
1. Glycolysis : The degradation of glucose to
pyruvate (lactateunder anaerobiccondition) generates amino
B ATP. Pyruvate is converted to acetyl CoA.
2. Fatty acid oxidation : Fatty acids undergo
sequential degradation with a release of 2-carbon
fragment, namely acetyl CoA. The energy is trapped
in t he f or m of NADH and FADH T .
3. Degradation of amino acids : Amino acids,
p a r t i c u l a r l yw h e n c o n s u m e d i n e x c e s st h a n r e q u i r e d
for protein synthesis,are degraded and utilized to
meet the fuel demands of the body. The glucogenic
acids can serveas precursorsfor the svnthesis
of glucose via the formation of pyruvate or
intermediatesof citric acid cycle. The ketogenic
amino acids are the precursorsfor acetyl CoA.
4. Citric acid cycle : Acetyl CoA is the key and
common metabolite, produced from different fuer
Chapt er67 : M ET A BOL IS M S 81s

sources(carbohydrates, lipids,aminoacids).Acetyl pyruvate, glycerol, amino acids) can serve as

CoA enterscitric acid cycle and Setsoxidizedto precursorsfor gluconeogenesis.
CO r . T hus ,c i tri c a c i d c y c l e i s th e fi n a l c o mmon B. Glycogen metabolism : Clycogen is the
metabolic pathway for the oxidation of all storageform of glucose,mostlyfound in liver and
foodstuffs.Most of the energyis trappedin the form muscl e. l t i s degraded (gl ycogenol ysi s)and
of NA DH and F AD H T . synthesized (glycogenesis) by independent
5. Oxidativephosphorylation : The NADH and pathways.Clycogen effectivelyservesas a fuel
for a brief period
FADH2,producedin differentmetabolicpathways, reserveto meet body needs,
(between meals).
ar e f inallyox i d i z e di n th e e l e c tro ntra n s p o rct hai n
(ETC). The ETC is coupled with oxidative
R E GU LA TION OF
phosphorylationto generateATP.
METABOLIC PATHWAYS
6. Hexose monophosphate shunt : This The metabol i c pathw ays, i n general , are
pathwayis primarilyconcernedwith the liberation controlledby four differentmechanisms
of NA DP Han d ri b o s es u g a rN A D P Hi s u ti l i z edfor
the biosynthesis of severalcompounds,including I . The availabilitvof substrates
fatty acids. Riboseis an essentialcomponentof 2. Covalent modification of enzymes
nuc leot ides an d n u c l e i ca c i d s(n o te -D N A c o ntai ns 3. Allosteric regulation
deoxyribose). 4. Regulation of enzyme synthesis.
7. Gluconeogenesis : The synthesisof glucose The details of these regulatoryprocessesare
from non-carbohydrate sources constitutes discussed underthe individualmetabolicpathways,
gluc oneogen e s i s .Se v e ra l c o m p o u n d s (e.9. in the preceedingpages.
 m m unology deals wit h t he s t ud y o f i m m u n i t y Second line of defense
I
t and im m une s y s t em s of v er t eb r a t e s .l m m u n i t y
D e s p i t e t h e p h y s i c a l b a r r i e r s , t h e m i cr o -
(imm unis lit er ally m eans ex em pVf r e ef r o m b u r d e n )
organisms do enter the body. The body defends
broadly involves the resistance shown, and
itself and eliminates the invading organisms by
protection offered by the host org,anism against the
non-specific mechanisms such as sneezing and
infectious diseases.The immune system consistsof
s e c r e t i o n so f t h e m u c u s I n a d d i t i o n , t h e b o d y a l so
a co m plex net wor k of c ells and m ole c u l e s ,a n d t h e r r
t r i e s t o k i l l t h e p a t h o g e n s b y p h a g o cyto sr s
in ter ac t ions .lt is s pec if ic allydes ign e dt o e l i m i n a t e
( i n v o l v i n g m a c r o p h a g e sa n d c o m p l e m e n t s y ste m ) .
infectiousorganismsfrom the body. This is possible
The inflammatory response and fever responseof
since t he or ganis m is c apable of dis t i n g u i s h i n gt h e
t h e b o d y a l s o f o r m a p a r t o f i n n a t e i m m u n i t y.
self f r om non- s elf ,and elim inat e no n - s e l f

l m m unit y is br oadly div ided int o t w o t y p e s -

in nat e ( non- s pec if ic ) im m unit y an d a d a p t i v e o r
a cq uir ed ( s pec if ic )im m unit y .

IN}I ATE I M M T' iI I TY T h e i m m u n e s y s t e mr e p r e s e n t tsh e t h i r d a n d m o st

potent defense mechanism of the body. Acquired
I nnat e im m unit y is non- s pec if ic ,a n d r e p r e s e n t s
( a d a p t i v e o r s p e c i f i c ) i m m u n i t y i s c a p a bl e o f
th e inher ent c apabilit y of t he or g a n i s m t o o f f e r
s p e c i f i c a l l yr e c o g n i z i n ga n d e l i m i n a t i n gt h e i n va d i n g
resistanceagainst diseases.lt consistsof defensive
m i c r o o r g a n i s m sa n d f o r e i g r .m r o l e c u l e s( a n t i g e n s)In.
b ar r ier s .
c o r r t r a s t o i n n a t e i m n t u n i t y ,t h e a c q u i r e d i m m u n i ty
d i s p l a y sf o u r d i s t i n c t c h a r a c t e r i s t i c s
Firs t line of def en* e
o Antigen specificity
The skin is the largestorgan itr the human body,
con s t it ut ing about 15% of t he adu l t b o d y w e i g h t . . R e c o g n i t i o nd i v e r s i t y
The s k in pr ov ides m ec hanic al bar r ie rt o p r e v e n ti h e . lmmunolo8ical memory
e ntr y of m ic r oor ganis m sand v ir u s e s . T h e a c i d i c
. Discrimination between self and non-self.
(pH 3- 5) env ir onm ent on t he s k in s u r f a c e i n h i b i t s
th e gr owt h of c er t ain m ic r oor ganis m s .F u r t h e r ,t h e The body possess tremendous capability t<-,
sweat c ont ains an enz y m e ly s o z y m e t h a t c a n specifically identify various antigens (antigen is a
d es t r oy bac t er ial c ell wall. f o r e i g n s u b s t a n c e , u s u a l l y a p r o t e i n or a

at 6
 C haot er68 : I MMU N OL OC Y 417

'car bohy dr at e t h a t e l i c i ts i mmu n e re s p o nse).

Exposure to an antigenleadsto the development of Adenoids
im m unologic am l e m o ry .As a re s u l t,a s e c o nd Tonsils
encounterof the body to the sameantigenresults nodes
in a heightenedstate of immune response.The Thymus
immunesystemrecognizes and responds to foreign Lymphnodes
ant igensas it is c a p a b l eo f d i s ti n g u i s h i nsge l f a n d Spleen
non-self.Autoimmunediseases are causeddue to a
failur et o dis c r im i n a te
s e l fa n d n o n -s e laf n ti g e n s. Peyespatches
Appendix
Lymphnodes
ORG A NI Z A T I O N OF IMMU N E S YS T EM Lymphvessels
The immune systemconsistsof severalorgans marrow
distributedthroughoutthe body (Fig.68.1).These
lymphoid organsare categorizedas primary and
seconoary.

Primary lymphoid organs

These organs provide appropriate micro- Fig. 68.1 : A diagrammatic representation of
environmentfor the developmentand maturation human lymphatic system.
of antigen-sensitive lymphocytes(a type of white
blood cells).The thymus(situatedabovethe heart)
and bone marrow are the central or primary T.lymphocytes
lymphoidorgans.T-lymphocytematurationoccurs
The maturationof T-cellsoccursin the fhymus,
i n t he t hy m uswh i l e B-l y mp h o c y te
ma tu ra ti ota
n k es
hence the name. The T-cellscan identifyviruses
olace in the bone marrow.
and microorganisms from the antigensdisplayedon
their surfaces. Thereare at leastfour differenttypes
Secondary lymphoid organs
of T-cells.
Theseare the sitesfor the initiationof immune . Inducer l-cells that mediatethe developmentof
response. e.g.spleen,tonsils,lymphnodes,appendix, T-cellsin the thymus.
Peyerspatchesin the gut. Secondarylymphoid t
Cytotoxic T-cells (T), capable of recognizing
organsprovidethe microenvironment for interaction
and ki l l i ngthe i nfectedor abnormalcel l s.
beha;een antigensand maturelymphocytes.
o Helper T-cells (Ti that initiate immune
responses.
CE LLS O F T H E IM M U N E SY ST E M
. Suppressorl-cells mediate the suppressionof
Two types of lymphocytesnamely B-cellsand rmmuneresoonse.
T-cells are critical for the immune system. In
T-lymphocytesare responsiblefor the cell-
addition,severalaccessory cells and effectorcells
mediated immunity.
also participate.
IMMU N OGLOB U LIN S
B-lymphocytes
The humoralimmunityis mediatedby a special
The site of developmentand maturationof group of protei ns cal l ed i mmunogl obul i nsor
B-cellsoccursin bursafabricusin birds, and bone antibodies, produced by B-lymphocytes.
m ar r owin m am ma l sD . u ri n gth e c o u rs eo f i mmu ne
response.B-cells mature into plasma cells and STRUCTUBE OF ]MMUNOGLOBULINS
::r ' et e ant ibodi e (i
s mmu n o 8 l o biunls ).
All the immunoglobulin(lg)moleculesbasically
--e 3-<ellspossess the capabilityto specifically consistof two identicalheavy(H) chains(mol. wt.
'.: -..- re each antigen and produce antibodies 53,000to 75,000each)and two identicallight (L)
;jj -j: :. B-llmphocytesare inimatelyassociated chains (mol. wt. 23,000 each) held together by
\Mr lrg,rrcral immunitv. di sul fi del i nkagesand non-coval enti nteracti ons

a,:r+:--,oiogy[52]
818 B IOTE C HNO LO C\

TNr.r 68.1 €haracteristics of human lmmunoglobullns

Type H-Chain L-Chains Molecular ll4olecular Percentage Serumconc. Maiorfunction(s)

formula weight carbohydrate mg/dl

lgG ror]" y2r2 or y2)u2 -150,000 800-1,500Mostly for

responsible
humoral
immunity
lgA rcorl" (oqK2)1_3 0r -(160,000)1_3 150-400 Protects
thebody
(ozlz)r-s surfaces
lgM rorl. (p2rj5 or -900,000 ia
ta 50-200 Humoral immunity, ser-
(pzrz)s vesas firstlineo{
defense
lgD ror). (6212or 62)"2) -180,000 t\) 1-10 R - nol l r onontn r ?

lgE rorl, E2K2ot e2)v2 -190,000 0.02-005 Humoralsensitivity

release.
andhistamine

(Fig. 68.2) Thus, immunoglobulin is a Y-shapecl T h e c h a r a c i e r i s t i c so f t h e 5 c l a s s e sof h u m a n

t et r am er ( t l2L2) . Eac h heav y c h a i n c o n t a i n s i m m u n o g l o b u l i n s a r e g i v e n i n T a b l e 6 8 . 1
appr ox im at ell, 450 am ino ac id s w h i l e e a c h l i g l r t
c hain has 212 am ino ac ids . The h e a v y c h a i n s o f l g l m m u n o g l o b u l i n G (lgG)
ar e link ed t o c ar bohy dr at es , h e n c e i m m u n o - l g C i s t h e m o s t a b u n d a n t ( 7 5 - 8 0 %) cl a ss o f
globulins are glycoproteins. i m m u n o g l o b u i i n s . I g C i s c o m p o s e d o f a si n g l e
Cons t ant and v ar iable r eg i o n s : E a c h c h a i n Y - s h a p e d u n i t ( m o n o m e r ) . l t c a n t r a v e r se b l o o d
( L or H) of lg has t w' o r egions ( d o m a i n s ) , n a m e l y v e s s e l s r e a d i l y . l g C i s t h e o n l y i n r m u no g l o b u l i n
t he c ons t ant and t he v ar ia b l e . T h e a m i n c r that can crossthe placentaand transferthe mother's
t er r . ninal half of t he light c ha i n i s t h e v a r i a b l e i m m u n i t y t o t h e d e v e l o p i n g f e t u s . l g C tr i g g e r s
r egion ( Vr ) while t he c ar box y t e r m i n a l h a l f i s foreign cell destruction mediated by complement
t he c ons t ant r egion ( C1) As r e g a r d s h e a v y c h a i n , system.
appr ox im at ely one- quar t er of t h e a m i n o t e r m i n a l
r egion is v ar iable ( VH) whi l e t h e r e m a i n i n g lmmunoglobulin A (lgAl
t hr ee- quar t er s is c ons t ant ( Cu ' C H z , C p r ) . T h e l g A o c c u r s a s a s i n g l e ( m o n o m e r )o r d ou b l e u n i t
am ino ac id s equenc e ( wit h it s t e r t i a r y s t r u c t u r e ) ( d i m e r ) h e l d t o g e t h e r b y J c h a i n . l t i s m o stl y fo u n d
of v ar iable r egions of light an d h e a v y c h a i n s i s in the body secretionssuch as saliva, tears, sweat,
r es pons ible f or t he s pec i f i c b i n d i n g of m i l k a n d t h e w a l l s o f i n t e s t i n e . l g A i s th e m o st
im m unoglobulin ( ant ibody ) w i t h a n t i g e n . p r e d o n r i n a n ta n t i b o d y i n t h e c o l o s t r u m , th e i n i ti a l
secretion from the mother's breast after a baby rs
CI.ASSES OF'MMUNOGLOBUL'NS b o r n . T h e l g A m o l e c u l e s b i n d w i t h b a cte r i d l
Hum ans hav e f iv e c las s e s o f i m m u n o - a n t i g e n s p r e s e n t o n t h e b o d y ( o u t e r e p i th e l i a l )
globulins- namely IgG, lgA, IgM, lgD and lgE s u r f a c e s a n d r e m o v e t h e m . I n t h i s w a y, Ig A
c ont aining t he heav y c hains y , u , p , 6 a n d e , prevents the foreign substancesfrom entering the
respectively.The type of heavy chain ultimately body cells.
det er m inest he c las s and t he f un c t i o n o f a g i v e n l g .
Two t y pes of light c hains - n a m e l y k a p p a ( r ) lmmunoglobulin M (lgMl
and lam bda ( 1")- ar e f ound in i m m u n o g l o b u l i n s . l g M i s t h e l a r g e s ti m m u n o g l o b u l i n c o m p o se d o f
They dif f er in t heir s t r uc t ur e i n C , r e g i o n s . A n 5 Y-shaped units (igC type) held together by a J
im m unoglobulin ( of any c las s ) c o n t a i n s t w o K o r polypeptide chain. Thus IgM is a pentamer.Due t<-l
t wo ) , light c hains and nev e r a m i x t r - i r e . T h e i t s l a r g e s i z e , l g M c a n n o t t r a v e r s e b l o o d ve sse l s,
oc c ur r enc e of r c hains is m or e c o m m o n i n h u m a n hence it is restrictedto the blood stream. lgM is the
im m unoglobulins t han l" c hains . first antibody to be produced in response to an
Chapt er68 : I M MU N OL OC Y 819

Interchain
disulfidebonds

NH;

Fab

S*
Hingeregion Papain
S-

Fc

coo- coo-
ijg. 68.2 : Diagrammatic representation of human lgG molecule (V-VariaHe region, C-Constant region;
CHO-Carbohydrate; Each heavy chain is composed of four units-Vr, Cr,, Crr, Cr, while light
chain consists of two units-V,, C,)

-'
-=" and is the most effective against invading
-: , c roorganisms.
- mun og lob ulin D ( lgDl
-- s comp osed of a s ingle Y- s hapedunit an d
:::-i in a lo w c onc ent r at ionin t he c ir c ulat io n .
-- :cr-rle sare pr es enton t he s ur f ac eof B c ells .
'. :ttion , h owev er , is not k nown f or c er t ain .
: ---r€r-sb eliev e t hat lgD m ay f unc t ion a s
i--ttt re ce pto r
r* -u no glo bu lin E ( lgEl
-i .- . rgle Y - s hapedm onom er . I t is nor m al l y
-' - ^ ''-rte c onc ent r at ionin blood. lgE lev e l s
,-- r r individuals with allergies as it is
:^ :he body ' s aller gic r es pons es Th
. e
. :: : :' r t l\ bind wit h m as t c ells whic n
: -' ^ : . : 1t l c aus e aller gy .
SY'VTHEs'S OF'MMANOGLOBULINS
T h e r e a r e m i l l i o n s o f d i f f e r e n ta n t i g e n s l t w a s a
big puzzle for a long time how an individual can
produce so many antibodies to protect against
antigens. lt is now recognized that a gene
r e a r r a n g e m e n ti n v o l v i n g a c o m b i n a t i o n o f s e v e r a i
g e n e s i s r e s p o n s i b l ef o r g e n e r a t i n g a n e x t r e m e l y
l a r g e n u m b e r o f a n t i b o d i e s .( F o r m o r e d e t a i l s R e f e r
Chapter 5).
MAJOR HISTOCOMPATIBI LITY
COMPLEX
The major histocompatibilitycomplex (MHQ
represents a special group of proteins, present on
the cell surfacesof T-lymphocyfes. MHC is involved
i n t h e r e c o g n i t i o no f a n t i g e n so n T - c e l l s .l t m a y b e
noted here that the B-cell receptors(antibodies)can
r e c o g n i z ea n t i g e n sa n d o n t h e i r o w n , w h i l e T - c e l l s
can do so through the mediation of MHC.
82tJ B IOTE CHNO LO C\

I n hum ans , t he M HC pr ot ei n s a r e e n c o d e d b y a
cluster of genes located on chromosome 6 (it is on
c hr om os om e 17 f or m ic e) . T h e m a i o r h i s t o -
c om pat ibilit y c om plex in hum a n s i s r e f e r r e dt o a s
human leukocyte antigen (HLA). fhree classes of C l a s sI M H C
M HC m olec ules ( c hem ic ally g l y c o p r o t e i n s ) a r e
Antigenfragment
k nown in hum an. Clas s I m ol e c u l e s a r e f o u n d o n
T-cellreceptor
alm os t all t he nuc leat ed c ells o f t h e b o d y . C l a s s l l
m olec ules ar e as s oc iat ed on l v w i t h l e u k o c v t e s Ts cell
inv olv ed in c ell- m ediat ed im m u n e r e s p o n s e .C l a s s
lll m olec ules ar e t he s ec r et ed p r o t e i n s p o s s e s s i n g
im m une f unc t ions e g. c om p l e m e n t c o m p o n e n t s
(C2, C4), tumor necrosisfactor.

The immune resoonse refers to the series of

r eac t ionsc ar r ied out by t he im m u n e s y s t e m i n t h e
body againsttl.reforeign invader.When an infection
iakes place or when an antigen enters the body, it
is t r appec lby t he m ac r ophage si n l y n r p h o i d o r g a n s .
The phagoc y t ic c ells r n, hic har e g u a r d i n g t h e b o d y
by c ons t ar itpat r olling engulf a n d d i g e s tt h e f o r e i g n
s ubs t anc e Howev er , t he par t i a l l y d i g e s t e d a n t i g e n
( i. e. pr oc es s ed ant igen) wit h a n t i g e n i c e p i t o p e s T" cell B-cell
I
attachesto lymphocytes II I
+ +
T- helper c ells ( T, ) play a k e y r o l e t h e i m m u n e C el l -medi ated Humoral
r es pons e( Fig. 68. 3) .This is br o u g h t o u t t h r o u g h t h e i mmuni ty immunity
par t ic ipat ion of ant igen pr e s e n t i n g c e l l ( A P C ) ,
us ually a m ac r ophage.Rec ept o r so f T , c e l l b i n d t o
Fig. 68,3 : A diagrammatic representation of the
c las s ll M HC- ant igen c om ple x d i s p l a y e d o n t h e
central role of helper T cells (7, cells) in
s ur f ac e of APC. APC s ec r et e si n t e r l e u k i n - 1 w
, hich
immune response (APC-Antigen presenting
ac t iv at est he T" c ell. This ac t iv a t e dT n c e l l a c t i v e l y cell; T" cell-Cytotoxic T-cell).
gr ows and div ides t o pr oduc e c l o n e s o f T " c e l l s . A l l
the T" cells possessreceptorsthat are specific for
t he M HC- ant igen c om plex . T h i s f a c i l i t a t e s
w e i g h t g l y c o p r o t e i n s a n d a r e p r o d u ce d b y
t r igger ing of im m une r es pon s e i n a n e x p o n e n t i a l
l y m p h o i d a n d n o n - l y m p h o i d c e l l s d u r i n g th e co u l se
m anner The T" c ells s ec r et e i n t e r l e u k i n - 2 w h i c h
promotes the prolifiration of cytotoxic T cells (T. of immune response.Cytokines may be regardedas
s o l u b l e m e s s e n g e rm o l e c u l e s o f i m m u ne syste m .
cells) to attack the infected cells through cell-
They can act as short messengersbetween the cells
m ediat ed im m unit y . Fur t her , i n t e r l e u k i n - 2 a l s o
o r l o n g r a n g e m e s s e n g e r sb y c i r c u l a t i n g i n th e
ac t iv at es B- c ells t o pr oduc e i m m u n o g l o b u l i n s
blood and affecting cells at far off sites. The latter
whic h per f or m hum or al im m u n i t y
f u n c t i o n i s c o m p a r a b l e t o t h a t o f h o r m o n e s.

The term interleukin (lL) is frequently used to

representcytokines. There are more than a dozen
i n t e r l e u k i n s( l L - 1 . . . . . . 1 1 1 2p)r,o d u c e db y d i ffe r e n tce l l s
Cytokines are a group of proteins that bring w i t h w i d e r a n g e o f f u n c t i o n s . T h e m a in fu n cti o n
about communication between different cell types (directly or indirectly) of cytokines is to amplify
i n v o l v e d i n i m m u n i ty .T h e y are l ow mol ecul ar immune responsesand inflammatory responses
 Ch a pt er68 : I M M UN O L OC Y 421

Therapeutic uses of cytokines
lt is now possibleto produce cytokines in vitro.
Some of the cytokines have potential applications
in t he p ractice o f me dic ine. For ins t anc e, lL- 2 is
used in can ce r im m unot her apy , and in t he
t reat m e nt o f immu nodef ic ienc y dis eas es . lL- 2
induces th e p rolife r at ion and dif f er ent iat ion of
T -and B-cells, b esides inc r eas ing t he c y t ot ox ic
caoacitv of na tura l killer c ells .
A group of cytokines namely interferons can
comba t viral infe ctio nby inhibit ingt heir r eplic at ion.
viral or any foreign antigen. Some of the epitopes
of foreign atrtigens are similar (homoiogous) to
epitopes present on certain host proteins. This
results in cross reaction of antigens and antibodies
w h i c h m a y l e a d t o a u t o i m m L r n ed i s e a s e s .
ORGAN TRANSPLANTATION
T h e p h e n o m e n o n o f t r a n s f e r o f c e l l s , t i s s u e so r
organs from one site to another (in the sarne
organism, autograft or from another organism
allograft) is regarded as organ transplantation.In
case of humans, majority of organ
t r a n s p i a n t a t i o n s a r e a l l o g ra f t s ( b e t w e e n t w o
i n d i v i d u a l s ) .T h e t e r m x e n o g r a f t i s u s e d i f t i s s u e s /
organs are transferred from one species to
another e.B. from pig to man (For more details on
xenotransplantation Refer Chapter 41).

The prime function of imune system is to protect Organ transplantation is associated with
t he ho st a ga instth e inv ading pat hogens .The body i m m u n o l o g i c a l c o m p l i c a t i c n s ,a n d t i s s u e r e j e c t i o n .
tries its best to overcome various strategies of This is because the host body responds to the
infec tiou s a ge nts (ba c t er ia, v ir us es ) ,and pr ov ides t r a n s p l a n t e dt i s s u e i n a s i m i l a r w a y a s i f i t w e r e a n
immunity. i n v a d i n g f o r e i g n o r g a n i s m .M a i o r h i s t o c o m p a t i b i l i t y
complex (MHC) is primarily involved in allograft
S o me of th e impo rt ant im m unologic al as pec t sr n rejection. This is due to the fact that MHC proteins
human h ea lth an d d is eas ear e br ief lv des c r ibed are unique to each individual, and the immune
system responds promptly to foreign MHCs.
A UT OIMMUNE DISEASES
Organ transplantation between closely related
In general, the immune system is self-tolerant family members is preferred,since their MHCs are
r e not responsive to cells or proteins of self. also likely to be closely related. And major
Sometimes, for various reasons, the immune system immunological complicationscan be averted.
fails to discriminate between self and non-self. As
a consequence,the cells or tissuesof the body are CANCERS
attacked. This phenomenon is referred to as
autoimmunity and the diseases are regarded as Crowth of tumors is often associatedwith the
autoimmu ne disea se s .The ant ibodies or oduc ed t o formation novel antigens. These tumor antigens
(also referred to as oncofetal antigens e.g.
self molecules are regarded as autoantibodies:
S ome examp les of aut oim m une dis eas es ar e a-fetoprotein) are recognized as non-self by the
I isted. immune systems However, tumors have developed
several mechanisnrsto evade immurre responses.
. I nsulin -de pe nd en t d iabet es ( panc r eat ic p- c ell
autoreactive T-cells and arrtibodies). AIDS
. Rheuma toid arth ritis ( ant ibodiesagains t pr ot eins Acquired immunodeficiency syndrome (AIDS),
pres en t in jo ints). caused by human immunodeficiency virus, is
r Mya sth en ia gra vis ( ac et y lc holine r ec ept or c h a r a c t e r i z e d b y i m m u n o s u p p r e s s i o n ,s e c o n d a r y
auto an tibo dies). neoplasma and neurological manifestationsA . lDS
p r i m a r i l y a f f e c t st h e c e l l - m e d i a t e d i m n r u n e s y s t e m
o A utoirn mun e he moly t ic anem ia ( er y t hr oc y t e
which protectsthe body from intracellularparasites
autoantibodies).
s u c h a s v i r u s e s ,a n d b a c t e r i a .M o s t o f t h e i m m u n o -
Mechanism of autoimmunity : lt is widely d e f i c i e n c y s y m p t o m so f A I D S a r e a s s o c i a t e dw i t h a
accepted that autoimnrunity generally occurs as a reduction in CDo (cluster determinant antigen 4)
consequence of body's responseagainst bacterial, c e l l s .
 enetics is the study of heredity. lt is
appropriately regarded as the science that
ex plains t he s im ilar it iesand dif f e r e n c e sa m o n g t h e
related orqanisms.
The blood theory of inheritance
in humans
For many centuries, it was customary to explain
inheritance in humans through blood theory.
People used to believe that the children received
blood from their parents, and it was the union of
blood that led to the blending of characteristics.
That is how the terms 'blood relations', 'blood will
tell', and 'blood is thicker than water' came into
existence.They are still used, despite the fact that
blood is no more involved in inheritance.With tne
advances in genetics, the more appropriate terms
should be as follows
. Gene relations in place of blood relations.
. Genes will tell instead of blood will tell.
BRI EF HI STO RY AND DEV E L O P M E N T
O F G ENETI CS
Cenet ic s is r elat iv elyy oung, n o t e v e n 1 5 0 y e a r s .
The blood theory of inheritancewas questioned in
1850s, based on the fact that the senren contained
no blood. Thus, blood was not being transferredto
the offspring. Then the big question was what was
the hereditary substance.
Mendel's experiments : lt was in 1866, an
Austrian monk named Gregor Johann Mendel, for
422
the first time reported the fundamental laws of
inheritance. He conducted several experinrentson
the breeding patterns of pea plants. Mendel put
forth the theory of transmissible facfors which
states that inheritance is controlled by certain
factors passedfrom parentsto offsprings.His results
were published in 1866 in an obscure journal
Proceedingsof the Society of Natural Sciences.
For about 35 years, the observations made by
Mendel went unnoticed, and were almost forgotten.
Two European botanists (Correns and Hugo de
V r i e s ) i n 1 9 0 0 , i n d e p e n d e n t l y a n d s i m u l ta n e o u sl y
rediscoveredthe theoriesof Mendel. The year 1900
is important as it marks the beginning the modern
era of genetics.
The origin of the word gene : In the early years
of twentieth centurv. it was believed that the
Mendel's inheritancefactorsare very closely related
to chromosomes (literally coloured bodies) of tne
cells. lt was in 1920s, the term g'ene (derived from
a Creek word gennan meaning to produce) was
introduced by Willard Johannsen. Thus, gene
replaced the earlier terms inheritance factor or
inheritance unit.
Chemical basis of heredity : There was a
controversy for quite sometime on the chemical
basis of inheritance. There were two groups-the
protein supportersand DNA supporters.lt was in
1944, Averyand his associatespresentedconvincing
evidence that the chemical basis of heredity lies in
DNA, and not in protein. Thus, DNA was finally
Chapter69 : CENETICS 823

identifiedas the genetic material. lts structurewas femaleshaveXX.The sexof the child is determined
elucidatedin 1952 by Watsonand Crick. by the father.

lmportance of genes in inheritance- Monogenic and polygenic traits

studies on twins The genetictraitsor characters arecontrolledby
Monozygoticor identicaltwinscontainthe same si ngl egenesor mul ti pl e genes.The changesi n
genetic material- DNA or genes. Studies genesare associated with geneticdiseases.
conducted on identical twins make starting Monogenicdisorders: Thesearethe singlegene
revelationswith regardto inheritance.One such disease in the corresponding
traitsdue to alterations
study is describedhere. gene e.g. S i ckl e-cel lanemi a, phenyl ketonuri a.
Inheritance of monogenicdisordersusuallyfollows
OskarStohrand JackYufewere identicaltwins
the Mendelianpatternof inheritance.
separatedat birth. Oskar was taken to Germany
where he was broughtup by his grandmother as a Polygenicdisorders: The genetictraitsconferred
Chr is t ian.
J ac k wa s ra i s e db y h i s fa th e ri n l s ra e as
l by morethan on gene(i .e mul ti pl egenes),
and the
a Jew.The two brotherswere reunitedat the ageof disordersassociated with them are verv important
47. Despitethe differentenvironmental influences, e.g. hei ght, w ei ght, ski n col ours, academi c
their behaviouralpatternsand personalities were performance,blood pressure, aggressiveness,length
rem ar k ably s im il a r of l i fe.
. Both men had moustaches,wore two pocket PATTEBNS OF INHER'TANCE
shirts,and wire-rimmedglasses. The heredity is transmittedfrom parent to
o Both loved spicyfoodsand tendedto fall asleep offspri ngas i ndi vi dualcharacterscontrol l edby
in f r ont of t ele v i s i o n . genes. The genes are linearly distributed on
. Both flushedthe toilet beforeusing. chromosomes at fixed oositionscalled loci.
o Both read maganizes from back to front. A gene may have differentforms referredto as
r Both storedrubberbandson their wrists. alleles.Usuallyone allele is transferredfrom the
o Both liked to sneezein a room of strangers. father,and the otherfrom the mother.The allele is
regardedas dominantif the trait is exhibiteddue to
BesidesOskar and Jack, many other studies its presence. On the otherhand,the alleleis saidto
conducted on identical twins point out the be recessiveif its effect is maskedby a dominant
importanceof geneson the inheritedcharacters
allele.The individualsare said to be homozygous
relatedto personalityand mannerisms. if both the allelesare the same.When the alleles
are differentthey are said to be heterozygous.
BA S I C P RI NCI PL E S OF H E R E D IT Y
IN HUM A NS The patternof inheritanceof monogenictraits
may occur in the followingways (Fig.69.1).
The understandingof how geneticcharacteristics
1. Autosomaldominant
are passedon from one generationto the next is
basedon the principlesdevelopedby Mendel. 2. Autosomalrecessive
As we know now, the human genome is 3. S ex-l i nked.
organized into a diploid (2n) set of 46 1. Autosomaldominantinheritance: A normal
chromosomes.They exist as 22 pairsof autosomes allele may be designated ar a *hile an autosomal
and one pair of sexchromosomes(XX/XY) During domi nantdi sease al l el eas A (Fi g.69.1A ).The mal e
the course of meiosis,the chromosomenumber with Aa genotypeis an affectedone while the
becomes haploid (n). Thus, haploid male and femalewith aa is normal.Half of the genesfrom
female gametes - sperm and oocyte respectively, the affectedmale will carry the diseaseallele.On
are formed.On fertilizationof the oocyte by the mating,the male and femalegametesare mixed in
sperm,the diploid statusis restored.This becomes differentcombinations. The resultis that half of fhe
possible as the zygote receivesone member of children will be heterozygous(Aa) and have the
each chromosomepair from the father,and the dis,ease. Exampleof autosomaldominantinherited
other from the mother. As regards the sex diseases are familial hypercholesterolemia,
chromosomes, the maleshave X and Y while the p-thalassemia, breastcancergenes.
824 B IOTE CHNO LO CY

(A) Autosomal dominant

PARENTS v CHILDREN
Male (d\ {). Genotyperatio-1: 1 Aa to aa
Genotype-Aa Phenotype- 50% affected
Phenotype-Affectedmale -50% normal
>A
Femate (Q)
Genotype- aa >a
Phenotype- Normalfemale

(B) Autosomal recessive

PARENTS CHILDREN
Mate(d) ,/ )Y
Genotyperatio-1:2: 1 BB/Bb/Bblcb
Genotype- Bb Phenotype-25% affected
Phenotype - Carriermale -25"k normal
/' -507ocarriers
Femate (Q) d'
Genotype-Bb t*o
- Carrierfemale
Phenotype

(C) X-chromosorne(sex chromosome)-linked inheritance

PARENTS CHILDREN
Mate (d) Genotyperatio-1 : 1 : 1 : 1 WXY/X'XA"Y
Genotype- XY Phenotype-50% of malesaffected
Phenotype-Normal
male
y'x
Female (Q) d'
Genotype-X"X \"
Phenotype- Carrierfemale

Fig. 69.1 : Patterns of inheritance-autosomaldominant, autosomal recessive and

X-linked (Note : Genotype refers to the description of genetic composition, while phenotype
represents the observable character displayed by an organism).

2. Ar r t os om al r ec es s iv e in h e r i t a n c e : I n t h i s possessonly one X chromosome, and there is no

c as e,t he nor m al allele is des ig n a t e da s I w h i l e t h e
disease-causingone is a (Fig. 69.1B). The gametes
of c ar r ier m ale and c ar r ier f e m a l e ( b o t h w i t h
genotype Bb) get mixed. For these heterozygous
carrier parents,there is one fourth chance of having
an af f ec t edc hild. Cy s t ic f ibr os i s ,s i c k l e - c e l la n e m t a
and phenylketonuria are some good examples of
autosomal recessivedisorders.
3. Sex (X)-linked inheritance : In the Fig. 69.1C,
s ex - link ed pat t er n of inher it a n c e i s d e p i c t e d . A
nor m al m ale ( XY) and a c ar r i e r f e m a l e ( X C Y )w i l l
pr oduc e c hildr en wher ein, half o f t h e m a l e c h i l d r e n
are affected while no female children is affectet-r.
This is due t o t he f ac t t hat t h e m a l e c h i l d r e n
dominant allele to mark its effects (as is the case
w i t h f e m a l e s ) .C o l o u r b l i n d n e s sa n d h e m op h i l i a a r e
good examplesof X-linked diseases.
A selected list of genetic disorders (monogenrc
traits) due to autosomal and sex-linked inheritance
i n h u m a n s i s g i v e n i n T a h l e 6 9 . 1.
GENETIC DISEASES IN HUMANS
The pattern of inheritanceand monogenic traits
along with some of the associateddisorders are
described above (Table 69.1). Besides gene
mutations, chromosomal abnormalities (aberrations)
a l s o r e s u l t i n e e n e t i cd i s e a s e s .
Chapter69 : CENETICS 425

Tlrrr 69.1 Selected eranples of genetlc dlsorders (monogenlctraltsf In humans

I n he r it ed patter n/cli sease Estitnated incidence Salient features

Autosomaldominant
Familial
hypercholesterolemia 1 500 Highriskfor heartdiseases
Huntington
disease 1 5000 Nervousdisorders,dementia
Familial
retinoblastoma 'I 12000 Tumcrsof retlna
Breastcancergenes 1 800 Highriskforbreast
andovarian
cancers
(BRAC1 and2)
B-Thalassemia 1 : 2500(inpeople
of A blooddisorder;
thebloodappears
to
Meditenaneandescent) be blueinstead
of red

Autosomalrecessive
Sickle-cell
anemia 1 : 100(inAfricans) Severe lifethreatening
anemia;
confers
resislance to malaria
Cysticfibrosis 1 : 2500(inCaucasians) Defective severelung
iontransport;
infectionsandearlydeath(beforethey
reach30 years)
Phenylketonuria 1 : 2000 Mental
retardation
dueto braindamage
deficiency
cr,-Antitrypsin 1 : 5000 Damage to lungsandliver
Tay-Sachs
disease 1 : 3000 Nervous
disorder; and
blindness
(inAshkenazi
Jews) pararysrs
immunodeficiency
Severecombined Rare(only100cases Highly immune
defective system;
early
(SCID)
disease reported
worldwide) death

Sex-linked
Colour
blindness 1 : 50 males Unable
to distinguish
colours
(li/B)
Hemophilia 1 : 10,000
males Defective
bloodclotting
Duchenne dystrophy
muscular 1 : 7000males Muscle
wastage

Mitochondrial
opticneuropathy
Leberhereditary Not known Damage mayleadto
to opticnerves,
blindness

Aneuploidy : The presence of abnormal number Selected examoles of chromosomal disorders

of chromosomes within the cells is referred to as
:ne up loid y. Th e m os t c om m on aneuploid c ondit i o n
, trisomy in which three copies of a particular
:r'omo so me are pr es ent in a c ell ir r s t eadof th e
- rr.nal two e.g. trisomy-21 causing Down's
stndrome; trisomy-1B that results in Edward's
*ndrome. These are the examples of autosomal
.-e up loid y.
in case of s ex - link ed aneuploidy , t he s e x
--'Dmo so mes oc c ur as t hr ee c opies . e. g. phen o -
-.::ca lly male c aus ing Klinef elt er ' ss y ndr om e h a s
t r\: triso my-X i s phenot ic ally a f em ale wit h XX X .
along the with the syndromes and their
characteristicfeatures are siven in Table 69.2.
EUGENICS
Eugenics is a sclence of improving human race
based on genetics. lmproving the traits of plants
and animals through breeding programmes has
been in practice for centuries.
E u g e n i c si s a h i g h l y c o n t r o v e r s i a ls u b j e c t d u e t o
social, ethical, and political reasons. The
proponents of eugenics argue that people with
826 B IOTE C H NO LO CY

Tlru 69.2 Scte€fedexanples of chromosonal dlsorders in humans

Condition Chromosome Syndrome Salient features

Normal
Females 2n (46,XX)
Males 2n (46,XY)

Autosomalaneuploidy 2n + specific chromosome

Trisomy-21 4 7 ,X Y+, 21 Down'ssyndrome lQ<50;
Congenitalhearl
lowlife
disease,
expecrancy
Trisomy-18 4 7 ,X Y+, 21 Edward's
syndrome Malformation
of heart,
kidneyandotherorgans
Trisomy-
13 47,XY,+13 Patau's
syndrome Smallhead,polydactyly,
heart
congenital
diseases
Sex-linkedaneuploidy
Phenotypically
male 47,XXY Klinefelter's
syndrome Smalltestes,infeltility
47,XYY Jacob's
syndrome Usuallyasymptomatic,
oftentall
Phenotically
female 47,XXX (trisomy-X)
Triplo-X Usually
asymptomatic,
about20%mentaly
handicapped
47,XO(missing
Y) Turner's
syndrome Shortstature.

desirable and good traits (good blood) should d u r i n g 1 9 3 0 s . A l a w o n e u g e n i c s t e r i l i z a t i onw a s

reproduce while those with undersirablecharacters p a s s e d i n 19 3 3 . I n a s p a n o f t h r e e ye a r s,
(bad blood) should not. The advocatesof eugenics, c o m p u l s o r y s t e r i l i z a t i o n w a s d o n e o n a b o u t
however, do not force any policy, but they try to 250,000 people, who allegedly suffered from
convince the people to perform their duty hereditarydisabilities,feeble mindedness,epilepsy,
vo iunt ar ily . The objec t of eugenic s i s t o l i m i t t n e s c h i z o p h r e n i a ,b l i n d n e s s ,p h y s i c a l d e f o r m a t i e s,a n d
p roduc t ion of people who ar e unf i t t o l i v e i n t h e d r u g o r a l c o h o l a d d i t i o n .
society.
The Cerman Covernment committed many
atrocities in the name of racial purity. Other
Eugenics in Nazi Germany
countries, however do not support this kind of
Cermany developed its own eugenic programme e u S e n r c s .
 icrnformaiics is the combination (or marriage!)
Q
L) 6! biology and information technolo7y.
Ba sically, b ioin f or m at ic s is a r ec ent ly dev elop e o
scien ce using in f or m at ion t o under s t andbiologi c a l
ph en ome no n.lt br oadly inv olv est he c om put at io n a r
to ols an d meth ods us ed t o m anage, analy s e a n d
man ipu late volu m es and v olum es of biologi c a l
data.
Bioinformaticsmay also be regardedas a part of
the computational biology. The latter is concerned
rvith the a pp lic at ion c f quant it at iv e analy t i c a l
:e ch niq ue s in m odeling and s olv ing pr oblem s i n
th e b iolo gical s y s t em s . Bioinf or m at ic s is a n
inte rdiscip lina ry appr oac h r equir ing adv anc e d
kno wled ge o f co m put er s c ienc e, nr at hem at ic sa n d
sta tistical me thods f or t he under s t anding o f
bio log ica l p he no m eha at t he m olec ular lev el.
History and relevance of bioinformatics
The term bioinformatics was first introduced in
1 99 0s Orig ina lly , it dealt wit h t he m anagem e n t
and an alysis o f t he dat a per t aining I o DNA, RN A
and protein sequences.As the biological data rs
being produced at an unprecedented rate. its
m an ag eme nta nd int er pr et at ioninv ar iably r equir e s f u n c t i o n s o f p r o t e i n s
bio info rmatics.Thus , bioinf or m at ic s now inc lud e s
many other types of biological data. Some of the
mo st imp orta nt o nes ar e lis t ed below
. Cer,e expression profiles
o Protein structure
r Pro tcin in tera c t ions
. Microarrays(DNA chips)
o F r - r n c t i o n aal n a l y s i so f b i o m o l e c u l e s
. D r u F ,d e s i g n i n g .
Bioinformatics is largely (not exclusively) a
c o m p u t e r - b a s e dd i s c i p l i n e . C o m p u t e r s a r e i n f a c t
v e r y e s s e n t i a lt o h a n d l e l a r g e v o l u m e s o f b i o l o g i c a l
data, their storage and retrieval.
We have to accept the fact that there is no
computer on earth (however advanced) which can
s t o r e i n f o r m a t i o n ,a n d p e r f o r m t h e f u n c t i o n s l i k e a
living cell. Thus a highly complex information
technology lies right within the cells of an
organism. This primarily includes the organism,s
g e n e sa n d t h e i r d i c t a t e sf o r t h e o r g a n i s m sb i o l o g i c a l
processesand behaviour.
BROAD COVERAGE OF BIOINFORMATICS
Bioinformatics covers many specialized and
advanced areas of biology.
Functional genomics : ldentification of genes
a n d t h e i r r e s p e c t i v ef u n c t i o n s .
Structural genomics: Predictions related to
Comparative genomics : For understandingthe
genomes of different species of organisms.
DNA microarrays : These are designed to
measure the levels of gene expression in different
tissues, various stages of development and in
different diseases.
827
a28
Medical informatics : This involves tne
m anagem ent of biom edic al d a t a w i t h s p e c i a l
referece to biomolecules. in vitro assavs ano
c linic al t r ials .
CO M PO NENTS O F BI O I NF O R M A T I C S
Bioinformatics comprises three components
1. Creation of databases : This involves tne
organizing,'storageand managementthe biological
data sets. The databases are accessible ro
researchersto know the existing information and
submit new entries. e.g. protein sequence data
bank f or m olec ular s t r uc t ur e.D a t a b a s e sw i l l b e o f
no us e unt il analy s ed.
2 Development of algorithms and statistics :
This inv olv es t he dev elopm e n t o f t o o l s a n d
r es our c est o det er m ine t he r ela t i o n s h i pa m o n g t h e
members of large data sets e.g. comparison of
pr ot ein s equenc e dat a wit h t h e a l r e a d y e x i s t i n g
protern sequences.
3. Analysis of data and interpretation : The
appr opr iat e us e of c om ponen t s 1 a n d 2 ( g i v e n
above) to analyse the data and interpret the results
in a biologic ally m eaningf ul m a n n e r . T h i s i n c l u d e s
DNA, RNA and pr ot ein s e q u e n c e s , p r o t e i n
structure,gene expressionprofiles and biochemical
pathways.
BI O I NFO RM ATI CS AND T H E INTERNET
The internet is an infernational cemputer
network. A computer network involves a group of
c om put er s t hat c an c om m unica t e ( u s u a l l y o v e r a
telephone system) and exchange data between
u sers.
It is the internet protocol (lP) that determines
how the packets of information are addressedano
routed over the network. To access the internet, a
computer must have the correct hardware (modem/
network card), appropriatesoftware and permission
for accessto network. For this purpose, one has to
subscribe Io an internet service provider (ISP)
World wide web (www) : www involves the
exchange of information over the internet using a
programme called browser. The most widely used
browsers are Internet explorer and Netscape
navrgator.
www works on the basis of Uniform resource
loc at or ( URL) whic h is a doc u m e n t w i t h a u n i q u e
address. URLs takes the format hftp.// (hypertext
B IOTE CHNO LO CY
fransfer protocol) that can identify the protocol for
communication over www.
BIOLOGICAL DATABASES
The collection of the biological data on a
computer which can be manipulatedto appear in
varying arrangements and subsets is regarded as a
database.The biological information can be stored
in different databases.Each database has its own
w e b s i t e w i t h u n i q u e n a v i g a t i o nt o o l s .
The biological databases are, in general,
p u b l i c l y a c c e s s i b l eS
. e l e c t e de x a m p l e so f bi o l o g i ca l
databasesare briefly described (Table 70.1).
Nucleotide sequence databases
T h e n u c l e o t i d e s e q u e n c ed a t a s u b m i t te d b y th e
s c i e n t i s t sa n d g e n o m e s e q u e n c i n gg r o u p s i s a t th e
d a t a b a s e s n a m e l y C e n B a n k , E M B L (Eu r o p e a n
M o l e c u l a r B i o l o g y L a b o r a t o r y ) a n d D DBJ ( D N A
Data Bank of Japan).There is a good coordination
between these three databases as they are
s y n c h r o n i z e do n d a i l y b a s i s .
Besides the primary nucleotide databases
(referred above), there are some other databases
also to provide information on Benes,genomes and
ongoing research projects.
Protein sequence databases
Protein sequencedatabasesare usually prepared
from the existing literature and/or in consultation
with the experts. In fact, these databasesrepresent
the translated DNA databases.
Molecular structure of databases
T h e t h r e e d i m e n s i o n a l ( 3 - D ) s t r uctu r e s o f
macromolecules are determined by X- r a y
c r y s t a l l o g r a p h y a n d n u c l e a r m a g n e t i c re so n a n ce
( N M R ) . P D B a n d S C O P a r e t h e p r i m a r y d a ta b a se s
o f 3 - D s t r u c t u r e so f b i o l o e i c a l m o l e c u l e s.
Other databases
K E C C d a t a b a s e i s a n i m p o r t a n t o n e th a t
provides information on the current knowledge of
m o l e c u l a r b i o l o g y a n d c e l l b i o l o g y w i th sp e ci a l
reference to information on metabolic pathways.
i n t e r a c t i n gm o l e c u l e s a n d g e n e s .
 Chapt er70 : BIOIN F O R M AT IC S 8.29

Tlsrs 70.1 selected examplesof biological databasesin bioinfornatics

Database Salient features

Primarynucleotidesequencedatabases
GenBank Provides
nucleotide
sequence
databases
maintained
by the
(www.
ncbi.
nih.gov/GeneBank/) National
Center
for Biotechnology
Information
(NCBI),
USA.
EMBL European Molecular
Biology
Laboratory(EMBL)maintains
nucleotide
(wwwebl.ac.uUembl/) sequence databasesunderthe aegisof European
Bioinformatics
(EBl),UK.
Institue

Other nucleotidesequencedatabases
UniGene Thenucleotide
sequences
of GenBank
in theformof ciusters,
(www.
ncbi.
nih.goviUniGene/) genesareavailable.
representing
GenomeBiology Theinformation
aboutthecompleted
genomes
is available.
(www.
ncbi.nlm.
nih.gov/Genomes/)
EBIGenomes Provides
dataandstatistics
for thecompleted
genomes,
besides
(wwwebi.
uk./genomesi) theinlormation
on theongoing projects.

Protein sequencedatabase
SWISS-PROT Provides thedescrlption of a protein,
of thestructure its domains
(www.
expasy.ch/sport) structure,post-translational
modifications,
variants
etc.lt hashigh
levelof integration
withotherdatabasesandminimal levelof
reoun0ancy
PIR Protein
information
resource
(PlR)is a database
by the provided
(pir.georgetown.edu) *'l'::1':"::::'1 us'r
"::':'l:::T:":l-aRD
:n
Protein sequencemotif databases
PROSITE Provides
information
on proteinfamilies
anddomains.lt alsohas
(www.expasy.ch/prosite/) patterns
andprofilesfor sequences
andbiological
functions.
Pfam A database
of protein
families
defined
intheformof domalns.lt has
(www.cgr.ki.se/Pf
am/) multiple
alignmentof a setof sequences.

Macromolecular databases
P DB Thisis theprimary
database (3-D)slructures
for 3-dimensional
(www.rcsb.org/pCb) ol biological
macromolecules
(deterrnined
by X-rayandNMR
studies).
SCOP Provides
informalion
on thestructural of proteins
classification
(www.
mrc.
lmb.cam.
ac.uUscop/) The
er:l'::' "l ln'o"''l '_l"u'olll'
lscoP] ::':n'"]:o
Other databases
KEGG TheKyotoEncyclopedia of GenesandGenomes (KEGG)is a
genome.ad.jp/kegg/)
www. database withlatestcomputerised
informationon biomolecules
ano
cellbiology.
KEGGprovides detailson information
pathways,
interacting
molecules andtheconnecting linkswithgenes.
830 B IOTE CHNO LO CY

APPLI CATI O NS O F BI O I N F O B M A T I G S o Prediction of functional gene products.

The advent of bioinformatics has revolutionized . To trace the evolutionary trees of genes.
t he adv anc em ent s in biolog i c a l s c i e n c e . A n d
biot ec hnology is lar gel y benefited by . F o r t h e p r e d i c t i o n o f 3 - d i m e n s i o n a l str u ctu r e o f
bioinf or m at ic s The bes t ex am p l e i s t h e s e q u e n c i n g prorerns.
of hum an genom e in a r ec or d t i m e w h i c h w o u l d
o M o l e c u l a r m o d e l l i n g o f b i o m o l e c u l e s.
not hav e been oos s ible wit ho u t b i o i n f o r m a t i c s .A
s elec t ed lis t of applic at ions o f b i o i n f o r m a t i c s r s o D e s i g n i n g o f d r u g s f o r m e d i c a l t r e a t me n t
given below.
. H a n d l i n g o f v a s t b i o l o g i c a l d a t a w h i c h o th e r w i se
. Sequenc em apping of biom o l e c u l e s( D N A , R N A ,
is not possible.
oroteins).
o ldent if ic at ion of nuc leoti d e seeuences of . D e v e l o p m e n t o f m o d e l s f o r t h e f un cti o n i n g
f unc t ional genes . v a r i o u s c e l l s , t i s s u e sa n d o r g a n s .
. Finding of s it es t hat c an b e c u t b y r e s t r i c t i o n The above list of applications however, may be
enz y m es . treated as incomplete, since at present there is no
. Des igning of pr im er s eque n c e f o r p o l y m e r a s e f i e l d i n b i o l o g i c a l s c i e n c e st h a t d o e s n o t i n vo l ve
c hain r eac t ion. bioinformatics
 :,1

t.t:tltttll
:l :i , I I I , I I l

,i,.1'':,:'l'
'l ',,,, r
Clossary 83
,t"l' .' , ,
,.,, ,,.
Abbreviations

tt,
-lr , I ' , I i I I I l
,1,,,1

831
 Agarose gel electrophoresis Electrophoresis carried
out on agarosegel to separate DNA fragments.
{ bz y m es Th e c at aly t ic ant ibodies or ant ibo d y Agrobacterium tumefaciens A rod shaoeo
zymes. bacterium that causes crown gall disease by
-^
{biotic stress The stress caused to plants clue to inserting its DNA into plant cells.
-:'bicides, water deficiency ozone, intense light
AfDS Acquired immunodeficiency syndrome, a
disease caused by a retrovirus resulting In
{bscisic acid An important plant growth regulator diminished immune response, and increased
' - th e ind uction of em br y ogenes is . s u s c e p t i b i l i t yt o v a r i o u s i n f e c t i o n s a n d c a n c e r s .
{ cid ra in Wh en t he pH of t he r ain wat er is less Aleurone The outmost layer of the endosperm in a
' - :n 5 .5, it is con s ider ed as ac id r ain.
seed.
\ctive site Th e sit e on t he enz y m e at whic h t h e
Algalization The process of cultivation of brue-
.
-:rstra teb ind s. green algae (cynobacteria)in the fields as a source
{ dap tor A synt het ic , double- s t r anded oligo - of biofertilizer.
' -.:leo tide used t o at t ac h s t ic k y ends t o
a blun t
Allele Alternative forms (one or mor:e)of a gene
--::e d mo lecule .
occupying a given locus on the chromosome.
{erated lagoons (aerated ponds) Facultatrve
.- ponds with surface aerators to Aflogeneic cells The cells taken from a person
-otlization
.eTco meba d od our s ( due t o ov er load of or ganr c o t h e r t h a n t h e p a t i e n t f o r u s e i n c u l t u r e , t i s s u e
^^a terials). e n g i n e e r i n ge t c .

{erated ponds See aerated lagoons. Allosteric regulation An enzyme regulating

process in which binding of a small effector
\ero be A microo r ganis mdependent on O , f or it s
molecule at one site (allosteric site) affects of the
: 't\\'In .
activity at other site (active site) of an enzyme.
{ninity chromatography A type of chromato-
Ames test A test used for the detection of
--.:-,.h ),in which t he m at r ix c ont ains c hem ic a l
chemical mutagens and their carcinogenecity.
-' --cs th at ca n s elec t iv ely bind ( ligands ) t o t he
- , ecule s be ing pur if ieo. Amino acid The buildine blocks or monomeric
fn inity ta g Th e t agged am ino ac id s equence u n i t s o f p r o t e i n s.
- :h forms a part of the recombinant protein and Aminoglycosides Oligosaccharide (carbohydrate)
, -:s a s an ide ntifi c at iont ag. antibiotics.

833
834
Amplified fragment length polymorphism (AFLP)
A sensitive method for the detection of
p oly m or phis m in t he genom e. lt i s b a s e d o n t h e
p r inc iple of RFLP and RAPD.
A m phipat hic St r uc t ur esor m ol e c u l e s p o s s e s s i n g m a n r p u l a t e d ,
two s ur f ac es , hy dr ophilic ( wa t e r l o v i n g ) a n c l
hydrophobic (water hating).
Anaerobe A microorganism that can grow in the
a bs enc e of O r .
Androgenesis Development of plants from male
Bametophytes.
A neuploidy An abnor m a l c o n d i t i o n o f
c hr om os om es , dif f er ing f r om t h e u s u a l d i p l o i d
c ons t it ut ion.This m ay be due t o a l o s s o r g a i n c f
c hr om os om es .
A nnealing The pair ing of c om p l e m e n t a r y s i n g l e
s t r andsof DNA t o f or m a doubl e h e l i x .
A nt ibiot ic A biologic al m olec ul e t h a t i s p r o d u c e d
by one or ganis m whic h c an k ill/i n h i b i t t h e g r o w t h
of anot her or ganis m .
Ant ibody A pr ot ein ( im m unoglo b u l i n ) s y n t h e s i z e d
by B-lymphocytesthat recognizes ar.rtigen.
Ant ic odon A s et of t hr ee nuc l e o t i d e s i n I R N A
molecule that are complementary to a set of three
nuc leot ides ( c odon) in m RNA.
Ant igen A s ubs t anc et hat elic it s i m m u n e r e s p o n s e
a nd induc es t he pr oduc t ion of a n t i b o d i e s .
Antioxidants A group of substances that can
pr ev ent ox idat ion e. g. v it am in E
Antisense therapy The in vlvo treatment of a
genet ic dis eas e by bloc k ing t r a n s l a t i o n ( p r o t e i n
s y nt hes is )wit h a DNA or RNA s e q u e n c e t h a t i s
c om plem ent ar y t o s pec if ic m RN A .
Apoptosis Programmed cell death.
Aptamers A special type of oligonucleotides that
can specifically bind to target proteins and not to
n uc leic ac ids .
Aspartame A low-calorie artificial sweetener used
i n s of t - dr ink indus t r y . Chem ic a i l y , i t i s a s p a r t y l -
p heny lalanine m et hy l es t er
Assisted reproductive technology (ART) The
n r anipulat ions of r epr oduc t ion i n a n i m a l s a n d
h u m ans .
Attenuation A regulatory process used by some
b ac t er ia t o c ont r ol t he ex pr es s io no f c e r t a i n a m r n o
a c id ooer ons .
B i OTE C HNO LO CY
Autoimmunity A n a b n o r m a l i m m u n e r e sp o n se
against his/her body's own components
Autologous cells Cells taken from an individual,
cultured/stored, and (sometimes) genetically
a n d i n f u s e d back into the original
Autoradiography The process of deiection of
r a d i o a c t i v e l yl a b e l e d m o l e c u l e s b y e x p o s ur e o f a n
X - r a y s e n s i t i v ef i l m .
Autosomes Chromosomesthat are not involved tn
sex determination
Autotroph An organism that is capable of
s y r r t h e s i z i n g i t s o w n f o o d e . g . p l a n t s th r o u g h
phctosynthesis.
A u x i n s A g r o u p o f p l a n t g r o w t h r e g u l a t or sw h i ch
a r e i n v o l v e d i n c e l l e l o n g a t i o n , r o o t i n i t i a ti o n e tc.
e.g indole aceiic acid.
Auxotroph A mutant microorganismthat can grow
w h e n a n u t r i e n t i s s u p p l i e d ( t h i s n u t r i e nt i s n o t
needed by the wild type).
B
Bacillus thuringiensis A rod shaped bacterium
w h o s e t o x i c c r y s t a l s a c t a s a n i n s e c t i c i d e a g a i n st
certain species of arthropods
Bacterial artificial chromosome (BAC) : A vector
system based on the F-factor plasmid of E. coli.
BAC is used for cloning large (100-300 kb) DNA
segments.
Bacteriophage A virus that infects a bacterium
Also called phage.
B a k e r 'sy e a s t T h e l i v i n g c e l l s o f a e r o b i c a ll y g r o w n
yeast, Saccharomyces cerevisiae, used in bread
making.
Base pair (bp) Ihe hydrogen bonded structure
formed between two complementary nucleotides
( i . e . p a r t n e r s h i po f A w i t h T o r o f C w i t h C ) i n D N A
structure.
Base ratio The ratio of A to I or C to C in a
d o u b l e - s t r a n d e dD N A
Batch culture (batch fermentation) A cell or
m i c r o b i a l c u l t u r e t h a t i s g r o w n f o r a l i m i t e d ti m e . l t
follows a sigmoid pattern of growth
Bergmann's plating technique The most widely
used method for culture of isolated single plant
cells.
 CLOSSARY
Binary vector system A two plasmid vector system
for transferring T-DNA into plant cells. l-he
vir ule nce g en es ar e on one plas m id while t he
engineered T-DNA region is on the other plasmid.
Bioaccumulation (biomagnification) Increasingthe
concentration of chemical agents by way of
increasingthe quantities in the organismsof a food
cha in.
BiodegradationBiological transformationof organic
comp ou nd s by living or ganis m s , par t ic ular ly t he
mrcro org an rSms
Biofertilizers The nutrient inputs of biological
origin that support piant growth.
Bioaugmentation The addition of microorganisms Bioprosthesismaterials The natural materials
to waste sites so that the hazardous wastes are modi fi edto becomebi ol ogi cal liynerte.g.col l agen-
rendered harmless. based connective tissue forming porcine heart
Biofiltration The process of removing complex
rvastes (from domestic or industrial sources) by
usr n Smrcro orSa nts m s .
Biogas A mixture of gases mainly composed of
rrethane, COr, nitrogen and hydrogen sulfide. lt is
used for cooking purposes,generationof electricity
etc.
Biohazards The accidents or risks associatedwith
biolo gical ma teria ls .
Bioinformatics A science of using information
technology to understandbiological phenomena. lt
may be regarded as a marriage between biology
and information technology.
Bioleaching The use of bacteria to recover valuable
metals {rom ores.
Biolistics The process of introducing DNA into
plan t an d a nim al c ells , and or ganelles by
bombardment of DNA-coated pellets under
pressure a t h igh s peed. This is als o c alled as
m i c rop roj e ctil e bombard ntent.
Biochemical oxygen demand (BOD) The oxygen
requil'ed to meet the metabolic needs of aerobic
orga nismsin wate r c ont aining or ganic c om pounds'
Biological databases The collection of biological
data on a cofitputer in different arrangementsand
sub)sets.
Bionrass The organic rnassthat can be used as a
source of energy. Biomass also refers to the cell
ma ss pro du ce d b y a populat ion of liv ing or ganis m s. the i nternalmassof yol k.
835
Bionretry Application of statisticalmethodsto
studybiologicalproblems.
BiopesticidesThe toxic compoundsproducedby
l i vi ng organi smsthat can speci fi cai l yki l l a
particularpestspecies.
BioplasticsThe biodegradable
plastics,chemically
composedof polyhydroxyalkanoates (PHAs).
Biopol A biodegradableplastic composed of
pol yhydroxybutyriaci
c d and val eri caci d.
Bioprocesstechnology A more recent usage to
replacefermentation technologythat involveslarge-
scale cultivationof microorganisms for industrial
purposes.
valves.
Bioreactor A growth chamber or a vessel
(i'ermenter)
for cellsor microorganisms.The cellsor
cell extractscarry out biological reactionsirr a
bioreactor.
BioremediationThe biologicalprocessinvolving
Iiving organismsto remove unwantedsubstances
(contaminants or pollutants)from soil or water.
Biosensor An analyticaldevice containing an
bi ol ogi calmateri al(enzyme,
i mmobi l i zed anti body,
organel l eor w hol e cel l ) w hi ch reactsw i th an
analyteto producesignalsthat can be measured.
Biosorption The processof microbialcell surface
adsorptionof metals.
BiosphereAll the zoneson the earthin which liie
is present.
Biostimulation Addition of soecific nutrientsto
enhance. the growth of naturally occurring
microorganismsthat converttoxic compoundsto
non-toxi ccompounds.
B i otechnol ogy The appl i cati onsof bi ol ogi cal
principles,organismsand products to practical
purposes.
BioticstressThe stresscausedto plantsby insects,
pathogens(viruses,fungi, bacteria),woundsetc.
BiotransformationThe use of biologicalsystems
of bi omol ecul es.
for the conversi on
BlastodermA stagein embryogenesis in which a
layerof nucleior cellsaroundthe embryosurround
836 BIOTECHNOLOCY

Blotting The transfer of macromoleculesby Cell cloning The productionof a populationof

capillaryactionfrom a gel to a membrane. cel l sderi vedfrom a si ngl ecel l .
B-lymphocytesAlso called B-cellg maturein bone Cell culture The culture of dispersed (or
marrow and are the precursorsfor the antibody disaggregated) cells obtained from the original
p ro d u c i n gc e l l s(p l a s m ac e l l s ). tissue,or from a cell line.
BOD Seebiochemicaloxygendemand. Cell cycle The seriesof eventsthat occur in a cell
betweenone divisionand the next.
Brewer'syeast A strainof yeastusuallybelonging
to Saccharomycescerevisiae that is used for the Cell immortalization The acquisitionof an infinite
productionof beer. l i fe spanby a cel l .

Broth Any fluid mediumsupportingthe growthof C el l l i nes A ni mal or pl ant cel l s that can be
microorganisms. cultivatedunder laboratoryconditions.

Bt plants The plant carryingthe toxin producing Cell-mediatedimmune response The activationof
gene from Bacillus thuringiensis,and capable of the T-lymphocytesof the immune system in
protectingthemselvesfrom insectattack. responseto a foreignantigen.

Bystandereffect The phenomenonof the death of Cell synchronization Manipulationof cells at

unmodifiedcells by a cytotoxicmetabolitethat is differentstagesof cell cycle in a cultureso that the
producedby geneticallymodifiedcells. cel l sw i l l be at the samephase.
Cell transformation A changein the phenotypeof
c a cell due to a new geneticmaterial.
Cell viability The capabilityof cellularexistence,
CAAT box A promoter sequenceof DNA for survivaland development.
eukaryotictranscriptionunits.
CellularsenescenceSeesenescence.
Callus A mass of undifferentiated olant tissues
regarding the
formed from plant cells or tissuecuttingswhen Central dogma The statement
grown in culture. unidirectionaltransfer
of informationfrom DNA to
RNA to protein.
Cancer Uncontrolledgrowthof cellsof a tissueor
an organin higherorganisms. Chaperone A protein required for the proper
assembly or folding of other proteins or
Cap The chemicalmodificationat the 5'- end of polypeptidesin vivo.
eukaryoticmRNA molecules.
Chaperonin A multisubunitproteinwhich formsa
Capsases A group of proteaseenzymesthat are structuralconformationto facilitatethe folding of
associatedwith apoptosis(programmed cell death). other proteinsin vivo.
Capsid The externalproteincoatof a virusparticle. Chemical oxygen demand (COD) Oxygen
equivalents of organicmatterthat can be oxidized
CasetteemutagenesisReplacementof a wild type
by usingstrongchemicaloxidizingagents.
DNA by a synthetic double-strandedoligo-
(a
nucleotide small DNA fragment). Chemiluminescence The phenomenon of emission
of light from a chemicalreaction.
Cataboliterepression The phenomenonof the
decreased expression of bacterialoperonsby extra- Chimeric antibodies Antibodies in which the
c e l l u l a rg l u c o s e . individual polypeptidechains are composedof
segments from two differentspecies(usuallyman
cDNA (complementaryDNA) A double-stranded
and mouse).
DNA synthesizedin vitro from an mRNA by using
reversetranscriptase and DNA polymerase. Chimera A recombinant DNA molecule that
containssequences from differentorganisms.
cDNA library A collection of cDNA clones,
generated in vitro from mRNA sequencesof a Chloroplast The photosyntheticorganelle of a
singlecell (or tissue)population. pl antcel l .
 GLOSSARY
Chromatin The complex of DNA and protein rn a
cel l.
Chromatography An analytical technique dealing Concatemer A DNA molecule composed of a
with the separation of closely related compouncts n u m b e r o f i n d i v i d u a l p i e c e s ( o r s e q u e n c e s )j o i n e d
from a mixture. together via cohesive enos.
Ch romo so m e A phy s ic ally dis t inc t unit o f t h e
Senome/ carrytng many genes.
Chromosome jumping A technique used for the
identification of two segments of DNA in a
chromosome separatedby thousandsof base pairs.
Chromosome walking lt is a technique used to
identify the overlapping sequences of DNA in a
chromosome in order to identify a particular locus
of interest.
Cistron The smallestfunctional unit (sequence)of
DNA) that encodes a polypeptide chain.
Clean gene technology The processof developing
transgenicplants without the presenceof selectable
marker genes or by use of more acceptable genes.
Clonal propagation See micropropagation.
Clon e A p opulat ion of c ells or , or ganis m s , o r
molecules that are identical to the oarent cell
(org an ism)or pr ec ur s or m olec ule.
Cloning vector (vehicle) A plasmid or a phage that
carries an inserted foreign DNA to be introduceo
into a ho st c ell.
Codon A triplet nucleotide sequence of mRNA
co din g for a n am ino ac id in a poly pept ide.
Coenzyme See cofactor.
Cofactor A low-rnolecular r,veight non-protein
compound required for an enzymatic reaction.
Cofermentation Fermentation carried out by
simultaneously growing two microorganisms in a
bioreactor.
Cointegrate vector The modified Ti plasmid rn
combination with an intermediate cloning vector.
Co lch icin e A n alk aloid us ed t o bloc k m it os i s o y
a rrestingsp indle f or m at ion.
Cofony hybridization A technique that employs
'ucleic acid probe to identify a bacterial colony
*',,itha vector carrying specific gene(s).
Complement system A group of serum proteins
:rat help in the formation of membrane attacr
complex to destroy invading pathogens.
837
Composting The processof biological degrao'ation
of solid organic wastes to stable end products.
Congenital The term used to describe genetic
abnormalities present at birth.
Conjugation The transfer of DNA from one
bacterium to another through cell-to-cell contact.
Constitutive genes Also called as house-keeping
genes. They are expressed as a function of
interaction between RNA polymerase and the
promoter without any additional regulation.
Contaminant A substancenot present in nature but
released due to human activities e.g. methyl-
rsocyanate.
Contig map A chromosomemap in which various
DNA segmentshave been joined together to form
a continuous DNA molecule.
Continuous fermentation Fermentationcarried out
by continuous addition of fresh medium that
balances with the removal of cell suspension(from
the bioreactor)
Copy number The number of plasmids in a
bacterial cell. lt also refersto the number of copies
of a gene in a genome of an organism.
Cosmids A high capacitycloning vector consisting
of the )" cos site inserted into a olasmid
Crossing-over The reciprocal exchange of genetic
material between chromosomes during meiosis.
Crown gall disease A plant disease caused by Ti
plasmid of Agrobacterium tumefaciens.
Cryopreservation Storage and preservation at very
low temperatures(-1 96"C).
Cryoprotectant A chemical agent or a cornpound
that can prevent damage to cells while they are
frozen or defrosted
Cultivar A term used to refer to the plants founo
only under cultivation.
Cufture A population of microorganismsor cells
(from plants/animals) that are srown unoer
controlled conditions.
Culture medium The nutrients prepared in tne
form of a fluid (broth) or solid for the growth of
cells/tissuesin the laboratory.
838 B IOTE C HNO LO CY

C'ybrid(cytoplasmichybrid) A viable cell formed Differentiation The development of cells or tissues

by the {usionof a cytoplastwith a whole cell. or orSans.

CybridizationThe processof formationof cybrids. D i p l o i d A c e l l o r a n o r g a n i s m t h a t h a s a s e t o f a l l

pairs (i.e. two sets)of chromosomes.
Cyclins The proteinsthat accumulateduring the
cell cycle and regulatethe biochemicaleventsin a Diploidization The process of doubling
cell cycle specificmanner. chromosomes of a cell.
C y n o p l ra g e sT h e v i ru s e sth a t c an ki l l al galcel ts. DNA chip A DNA microarray that consists of
Cysticfibrosis A diseaseaffectinglungsand other o l i g o n u c l e o t i d es e q u e n c e si m m o b i l i z e d o n a ch i p .
tissuesdue to defectsin ion transoort-lt is caused It is used for the analysis of gene structure and
by the deficiencyof CFfR gene. fu nciion.

Cytoplasmichybrid Seecybrid. DNA fingerprinting A technique for the

i d e n t i f i c a t i o n o f i n d i v i d u a l s b a s e d o n t h e sm a l i
Cytoplasnric
inheritance Inheritance
as a property differences in DNA sequences.
of geneslocatedin the cytosol(in rnitochondria
or
c h l o ro p l a s ts ). DNA hybridization The pairing of two DNA
molecules used to detect the specific sequence in
Cytoplasmic transfer The transier of cytoplasm
the samole DNA.
from a donor (w.ithaciive mitochondria)into the
oocytes. DNA marker A DNA secuence that exists in two
o r n r o r e r e a d i l y i d e n t i f i a b l e f o r m s ( p o l yr n o r p h r c
Cytoprotoplasts The sub-protoplasts containing
f o r m s ) w h i c h c a n b e u s e d t o m a r k a m a p p o si ti o n
th e o ri g i n acl y to p l a s m incra te ri a(il n partor ful l )but
on a genome rnap.
lack nucleus.
DNA microarray See DNA chip.
Cy.toskeleton The network of fibres in the
c y to p l a s mo f a e u k a ry o ti c e l l . DNA probe (gene probe) A segment of DNA that
CytotoxicityThe toxic effectson cellsthat resultrn is tagged with a label (i.e. isotope) so as to detect
m e ta b o l i ca l te ra ti o nisn c l u d i n gthe deathof cel l s. a complementary base sequence in the DNA
sample after a hybridization reaction.
Cytotoxic T-cells (T.) The lymphocytesthat
mediatethe lysisof targetcells. DNA profiling The term used to describe different
methods for the analysis of DNA to establish the
identity of an individua,.
D DNA repair The biochemical processes that
Dedifferentiation The process of dirferentiated c o r r e c t m u t a i i o n s o c c u r r i n g d u e t o r e p l i ca ti o n
cells getting reverted to non-differentiatedcells. crrors or as a consequcnce of mutagenic agents.

Degeneracy This term is used in relation to genetic DNA sequencers The machines that are used for
code representingthe fact that there may be more the determination of specific sequences of
than one codor-rfor most amino acids. n u c l e o t i d e si n a g i v e n D N A m o l e c u l e .

Denaturation The process of unfoldrng ot a D N A v a c c i n e A g e n e o r D N A s e q u e n c e ,g n co d i n g

protein, or separation of the two complementary a n a n t i g e n i c p r o t e i n , i n c o r p o r a t e d i n t o t h e ce l l s o f
s t r andsof DNA. a target animal

Diabetes mellitus A disease characterized by D o l l y T h e f i r s i m a m m a i ( s h e e p )c l o n e d b y Wi l m u t

increased blood glucose concentration (hyper- and Campbell in 1997.
gly c em ia) .
Domain A segmentof a pOlypeptideor protein that
Diazotrophs The microorganisms involved in has soecific conformation and function.
diazotrophv.
D o t - b l o t A t e c h n i q u e i n w h i c h s m a l l do ts ( o r
Diazotrophy The process of fixation of soots) of nucleic acid are immobilized on a
at m os pher ic ni' , r ogenby m ic r oor g a n i s m s n i i r o c e l l u l o s eo r n y l o n m e m b r a n ef o r h y b r i d i za ti o n .
CLOSSARY 839

Double helix The double-stranded DNA structure EndornitosisThe processof doublingchromosornes

in the nativeforrn in a cell, 'without division of the nucieus.This resultsin
polyploidy.
Doublingtime The time requiredto double the
num berof c ellsi n a ti s s u ec u l tu re . EndospermThe nutritivetissuethatdevelopsin the
embryosac of most angiospermplants.
DownstreamTowardsthe 3'-endof oolvnucleotide
or DNA s equen c e . Energy-richcrops The plants which are very
efficientin convertingCO, into biomass.
Downstreamprocessing The methods/procedures
used to isolate and purify the products (e.g. Enhancer A regulatorysequenceof DNA that
proteins)of fermentationprocesses. enhancesthe rate of transcriptionof a gene or
genesthat are locatedat distantplaces.
Dry anaerobic composting (DRANCO) An
anaerobicdigestingprocessfor the compostingof Enzyme A biocatalystsynthesized by living cells.
solid wastes. Enzyme-linkedimmunosorbentassay (ELISA) A
techniquefor the detectionof small qLrantities of
proteinsby utilizingantibodieslinkedto enzymesr
E which in turn catalysethe formationof coloured
oroducts.
Ediblevaccines The vaccinesproducedin plants
which can enterthe body on eatingthem. Enzyme technology Involves the production,
isolation,purificationand use of enzymes.
Electrophoresis An analytical technique that
separates chargedmoleculesin an electricalfield. EpitopesThe specificantigendeterminants located
on the antigens.
Electroporation Thetechniqueof introducingDNA
into cells by inducingtransientpores by electric Eutrophication Excessgrowth of algae(in sewage/
DUtSe. \/aste waters)which leadsto oxygendepletion.
Elicitors The compounds of biological origin that Exon A region of the eukaryoticgene that is
stimulate the production of secondary metabolites. expressedas mRNA which is translatedinto a
prorern.
ELISA See enzyme-linked immunosorbent assa;,.
ExosomeA multiproteincomplexthat is involved
El-Sl The ethical, /egal, and social implicaiions that
in the degradation
of mRNA in eukaryotes.
arise as a result of developments in genetic
engineering or biotechnology. Explant A piece of tissueisolatedfrom the intact
plantthat is usedto initiatecultureor development
Embryo An organism in the early stages of
of plant.
development. In humans, the first two months in
the uterus. Exponential phase This refersto a phasein cuiture
i n w hi ch cel l sdi vl de at a maxi mumrate.
Embryogenesis The process by which an embryo
develops from a fertilized egg cell, or asexually Expressedsequencetag (EST) A cDNA that is
from a group of cells. sequencedin order to gain rapid accessto the
genesIn a Senome.
Embryonic stem cells (ES cells) The cells of an
early embryo that can give rise to all differentiated Extein The functional comoonent of a
ce lls, includ ing ger m c ells . protei n.
di sconti nuous
Embryo rescue The culture of immature embryos Ex vivo Outsidethe body.This term is commonly
to rescue them from unripe or hvbrid seeds which usedto describethe manipulations of genesoutside
fail to germinate. the body for genetherapyi.e. ex vivo genetherapy.
Embryo transfer The process of irnplantation of Eugenics The scienceof improvinghuman stock
embryos from a donor animal, or developed by in by selectivebreeding. lt involves giving better
vitro {ertilization into the uterus of a recipient chancesfor more suitablepeoplein the societyto
a nima l. reproducethan the lesssuitablepeople.
840 B IOTE C H NO LO CY

EukaryotesThe organisms with a well definedand Fusionprotein A proteinthat is formedby fusion

m e m b ra n eb o u n d n u c l e u s . of two polypeptides,normallycoded by separate

F FusogenAn agentthat inducesfusionof protoplasts

i n somati chybri di zati on.
Ft Also called first filial generation,refersto the
first generationof offspringsfrom a cross.
G
t2 The secondgenerationof offspringsfrom a
crosswith at leastone F, parent.F, is also called Gamete The haploidmale (sperm)or female(egg)
s e c o n dfi l i a l g e n e ra ti o n . cel l sthat fuseto producea di pl oi dzygotedur ing
sexualreproducti on.
Familialtraits The traitssharedby membersof a
family.Theseincludethe hereditaryas well as the Gameteintrafallopiantransfer(GtFT) The transfer
environmentallyinfluencedtraits e.g. social and of both spermand oocyteinto the fallopiantube so
behaviouraltraits. as to allow the fertilization'tooccur naturallyln
vtvo.
Fed-batch fermentation The process of
f e rm e n ta ti o b n y g ro w i n gc e l l s o r mi croorgani smsGametoclonalvariations The variationsobserved
d u ri n gw h i c h n u tri e n tsa re a d d e dperi odi cal l y(to in the regenerated plantsfrom gameticcells (e.9.
the bioreactor). antherculture).
Fermentation The growth of cells or GametophyteThe haploidstageof the life cycleof
microorganismsin bioreactors (fermenters)to pl ants.
synthesize special products. Fermentation in GE Ms S eegeneti cal lengi
y mi croorganism s.
neered
biochemistryrefersto the degradationof carbon
Gene A segmentof DNA that encodesa functional
compoundsby cellsor organismsunderanaerobic
(lack of oxygen)conditions. protein.lt is the basicunit of inheritancelocatedon
a chromosome.
Fermenter A containment system for the
of
Gene amplification Increasein the expression
cultivationof prokaryoticcells.
a gene severalfold.
Fertilization The fusion of male gamete(sperm)
Genebank A libraryof genesor clonesof an entire
with female gamete(egg)to produce a zygote.
genomeof a species.
Flavr Savr Transgenic tomatodevelopedby using
Gene cloning lt basicallyinvolvesthe insertionof
antisensetechnology. (Note : lt was not a
a gene (a fragmentof DNA) or recombinantDNA
commercialsuccess).
into a cloningvector,and propagationof the DNA
Flow cytometry A method used to sort out cells, moleculein a hostorganism.
o rg a n e l l e so r b i o l o g i c a l m a te ri al sby passi ng
Gene knockout The processof destroyinga
throughaperturesof definedsizes.
specificgene by blockingits function.
Fluidizedbed reactors See packed bed reactors. Gene map The linear array of genes of a
Ffuorescentin situ hybridization (FISH) The chromosome.
methodof employingfluorescent labelsfor locating Gene probe See DNA probe.
markers on chromosomes by detecting the
h y b ri di z a ti o np o s i ti o n s . Generati onti me S eedoubl i ngti me.
The instabilityof geneexpression
F-plasmid lt is a fertilityplasmidthat directsthe Gene silencing
in transgenic plants.
conjugaltransferof DNA betweenbacteria.
Gene therapy Treatmentof diseasesby use of
Frameshiftmutation A mutationthat occursas a
genesor DNA sequences.
result of insertion or deletion of a group of
s a t a re n o t m u l ti p l esof three.Thi s Genetically engineeredmicroorganisms(GEMs)
n u c l e o ti d e th
resultsin the alterationof the frame in which the The microorganisms with geneticmodificationsare
codonsare translated into proteins. collectivelvreferredto as CEMs.
CLOSSARY 8,41

Genetically modified (GM) foods The entry of Golden rice The geneticallyengineeredrice with
transgenicplantsarld animalsinto the food chain provitaminA (p-carotene)
enrichment.
representsGM foods.
GRAS This is a shortform for generallyrcgarded
Geneticallymodified organisms(GMOs) A term as safe,and is in usein somecountriesto represent
usedto represent an organisms that are genetically the safety(no historyof causingillnessto humans)
engineer ed l.t u s u a l l y d e s c ri b e sth e tra n sgeni c of foods,drugsand other materials.CRAS is also
plant sand tra n s g e n iac n i m a l s . usedto representthe hostorganismsemployedin
Genetic code The total set of 64 codonsthat code geneticengineeringexperiments.
for 20 amino acids,and threeterminationcodons. Gratuitousinducer A substancethat can induce
Genetic disease A diseasecaused by defective transcription of gene(s),
but is not a substrate
for the
gene(s) from one generatron
that can be transmitted inducedenzyme(s).
to the next. Greenhouseeffect The ohenomenonof retention
Genetic engineering Broadly involvesall the in of earth'sheat by the atmosphere.
vitro geneticmanipulations. Greenhousetechnology The growingof plantsin
Genetic portfolios The identificationof all the greenhouses
beforethe plantculturesaretransfered
geneticportfolios. to fields.
genesin individualsrepresents
Genetic immunization The immune responseof Creen revolution The improvement in crop
the body stimulatedby DNA vaccines. varietiesand their manaqementfor increasein
world'sfood supply.
Genetics A branchof biology involvingthe study
of genes. CynogenesisOvary or ovule culturethat resultsin
the productionof haploids(gynogenichaploids).
Genome The total contentof DNA represented by
t he genesc o n ta i n e di n a c e l l .
Genomic library A collection of clones H
the entiregenomeof an organism.
representing
Hairy root diseasesA plantdiseasecausedby the
Genomics The studyof the structureand function infection ol Agrobacterium
rihizogens.
of genomes.
Haploid Containing one set of chromosomes
Genotype The geneticconstitutionof an organism. (opposite
to diploid).
(oppositeof phenotype).
Hel-acells A pure cell line of humancancercells
Germ cell A reproductive cell which on maturation
usedfor the cultivationof viruses.
can be fertilizedto producethe organism.
Helix-loop-helix motif A domaincommonlyfound
Germ line Reproductive cellsthat producegametes
i n D N A -bi ndi ngprotei ns.
which in turn give riseto spermsand eggs.
Helix-turn-helixmotif The structuralmotif for the
GermplasmThe hereditarymaterialtransmitted to
attachment of a proteinto DNA molecule.
the offspringthroughgerm cells.
Gibberellins A group of plant hormonesthat Herbicides The weedkillers that destrov the
induc ec ell e l o n g a ti o na n d c e l l d i v i s i o n . unwantedand uselessplants.

Global warming The retentionof eafth's heat by Heritabletraits The characteristics that are unoer
atmosphere(greenhouseeffect) resultsin global the control of genes, and transmitted from one
war m r nS . generation to another.

Glufosinate Seephosphinothricin HeterochromatinThe relativelymore condensed

part of chromatinwhich is believedto contain
Glycinin A lysine-richproteinof soybean. DNA that is not being transcribed.
GlyphosateAn herbicideusedto destroyunwanted Heteroduplex A DNA moleculeformed by base
olants. pairing between two. strandsthat do not have
GMOs See genetically
modified organisms. complete complementary sequences.
442 B IOTE C H NO LO G Y

Heterogeneousnuclear RNA (hnRNA) The Hr.rmoralimmune response The productionof

unprocessed nuclearRNAproducedin transcription. antibodiesby B-cells of the immune system in
This is also regardedas primary transcript. responseto antigens.
HeterokaryonA cell in which two or more nuclei Human mouse The transqenicmousewith human
of differentgeneticmake-upare present. i mmunesvstem.
Heterologous Refersto gene sequencesthat are DNA.
Hybrid DNA See heteroduplex
not identical, but show variable degrees of Hybridization At the molecularlevel, it refersto
s i m i l a ri ty . joining togetherof artificiallyseparatednucleic
Heterozygous Having two differentallelesfor a acid strands via hydrogen bonding between
gi v e ntra i t i n a d i p l o i d o rg a n i s m( cel l /nucl eus). complementary bases. lt also refers to the
productionof hybrids or hybrid offspringsfrom
High fructosecorn syrup(HFCS)A goodsubstitute
y ssi mi l arparents.
geneti cal ldi
for sugarin the preparation of softdrinks,processed
f o o d sa n d b a k i n g .H F C Sc o n ti i n sequalamountsof Hybridoma A clone of hybrid cells producedby
glucoseand fructose. fusion of a myeloma celJ' with an antibody
producingcell. Eachhybridomaproducesonly one
Hirudin A naturalanticoagulant proteinproduced
type of monoclonalantibody.
by salivaryglandsof leeches.
Hydrosphere The,water on the earths surface
Histotypiccultures The growthand propagation of which includesoceans,seas,rivers,polar ice caps
c e l l s i n th re e -d i me n s i o n ama l tri x to hi eh cel l etc.
density.
Hypervariableregion A regionof the genomethat
Hognessbox (TATAbox) A DNA sequencefound possesses variablenumberof repeatedsequences
in eukaryotic promoters.This has a sequence w hi ch are usefulfor the di agnosi sof i ndividuals
TATAAAT, and is comparableto Pribnowbox found (throughDNA fingerprinting).
in eukaryotes.
Homologous,chromosomes Membersof the paired
c h ro mo s o m e si n d i p l o i d o rg ani smsthat are I
id e n ti c a li n D N A s e q u e n c e(g s e nes)and i n thei r
lce-minus bacteria The bacteria that do not
visiblestrutture.
synthesizeice nucleationproteins.
HomozygousPossessing two identicalallelesfor a
lCl protein The singlecell proteinproducedby the
gi v e ntra i t i n a d i p l o i do rg a n i s m(cel l /nucl eus). (l Cl) f r om
companyl mperi alC hemi calIndustri es
Host A cell or an organismused to propagate methanoland ammoni a.
recombinantDNA rnolecules.
lmhoff tanks An improved septic tank w'ith two
H o s t c e l l s T h e l i v i n g c e l l s i n w hi ch the carri er compartments-onefor the sedimentation and the
of recombinantDNA (or vector)can be propagated. other for the digestion.
Holliday structure An intermediatestructure lmmobilizedcells The cellsthat are entrappedin
formed ciuringrecombinationbetweentwo DNA the matrices(of alginate,agarose,polyacrylamide)
molecules(or chromosomes). and used in bioreactors.
Homokaryon A cell with two or more identical lmmobilizedenzyme An enzvmethat is physically
nuclei as a resultof fusrr-rn. localized in a defined region so that it can be
reusedin a continuousprocess.
Humufin Human insulin used for the treatmentof
diabetic patients.lt was developedbv Eli LiPly lmmune system The networkof cells,tissuesand
companv and was approvedfor human use in organs that defends the body against foreign
19 8 2 . i nvaders.
Human Genome Proiect(HGP) An international lmmunoglobulins The specialgroup of proteins,
megaproject for the identification of huma'n commonly referredto as antibodies,producedby
genomesequences, and involvedin humoralimmunity.
the genesand their functions. B-lymphocytes,
CLOSSARY 843

lndigo A blue pigmentextensivelyused to dye

cotton and wool. K
Inducer A small molecule that triggersgene Karyotyping The method
of photographing the
transcriptionby bindingto a regulatoryprotein. complete set of chromosomes for a particular cell
Induciblegenes The genesthat can be stimulated type and organizing them into pairs based on size
by induc e rs . and shape.
Innate immunity The inherentcapabilityof an Kilobase (kb)
A unit consisting of one thousand
organismto offer resistance againstdiseases. n i t r o g e n o u sb a s e s i n a D N A o r R N A m o l e c u l e .
Insulin A hormonesynthesized by B-cellsof the
pancreas.lt facilitatesthe uptakeand metabolism Klenow fragment A fragment of DNA polymerase
I that lacks 5'-exonucleaseactivity.
of glucose.
Insertion sequence(lS) A small transposable Knockout mouse A genetically altered mouse
elementfound in bacteria.lt carriesgenesneeded lacking the genes for an entire organ or organ
for its own transposition. system.
Intein The part of a polypeptide removed by Kozak consensus The nucleotide seouence
splicingprocessaftertranslation. surrounding the initiation codon of eukaryotic
Interferons A group of glycoproteinsthat resist m R N A .
viral infectionsand regulateimmune responses.
Intergeneric A crossbetweentwo genera. L
InterleukinsA groupof lymphokinesimportantfor
the functionof immunesystem. Lac operon See lactoseoperon.
Interspecific A crossbetweentwo differentspecies Lactoseoperon (lac operon) The clusterof genes
bf a genus . responsible for the coding of enzymesinvolvedrn
Intron A segmentof the DNA that is transcribed, utilizationof lactoseby E. coli.
but removed from within the transcriot.Thus laggingstrand The DNA strandof a double helix
intronsrepresentthe non-codingregionswithin a that i s copi ed i n a di sconti nuous fashi onduri ng
dis c ont inu o ugse n e .(l n tro ni s a l s oth e tra d ename repl i cati on.
for imerteron-a\.
LandfarmingA techniquefor the bioremediation of
In vitro Literally means /in glass', refers to
hydrocarboncontaminated soils.
biologicalactivities/reactions carriedout in the test
t ube r at he rth a n th e l i v i n gc e l l o r o rg a n i sm. Landfilling A method for the final disposalof
In vitro fertilization (lVF) The fertilizationof eggs sl udge.
of a female in the laboratorvconditions.and Leadingstrand The DNA strandof a double helix
transferringthe fertilizedeggs (zygotes)into the that i s copi ed i n a conti nuousfashi on duri ng
uterusa few days later. reol i cati on.
In vitro mutagenesis The techniques used to LeghemoglobinA proteinpresentin the nodulesof
producea specifiedmutationat a predetermined in l egumi nous pl ants, that i s comparabl e to
a DNA . hemogl obi ni n ani mal s.
In vivo Literallymeans'in lifd, refersto the natural
Leucinezipper motif A domaincommonlyfound
s it uat ionw i th i n a c e l l o r o rg a n i s m.
i n D N A -bi ndi ngprotei ns.
In vivo genetherapy The directdeliveryof gene(s)
to a tissueor an organto alleviategeneticdisorders. linkers The short known sequences of DNAs (or
lsotopes The elementswith the same atomic oligonucieotides) that are joined to the ends of
numberbut differentatomic weiehts. duri ngthe process
D N A mol ecul es of genecl oni ng.
[ipoplexesThe lipid-DNAcomplexes,
alsoreferred
to as lrposomes.
I LithosphereThe outer boundarylayerof the solid
funk DNA The intergeniccontent of DNA is also earthon which the continentsand the oceanbasrns
r ef er r edt o a s i u n k D N A. rest.
8,44 B IOTE C H N O LO CY

Locus The site on the chromosomewhere a Michaelis-Menten constant The substrare

specificgene is located. concentration iter)to producehalf-maxi
(moles/l mal
Longterminalrepeats([TRs) Similarsequences of velocityin an enzyme-catalysedreaction.
geneticinformationthat are locatedat the endsof Seehumanartificial
Microchromosome chromosome.
the genomesof retroviruses. Microinjection The delivery of DNA or other
lymphoma Cancerof the lymph tissue. compoundsi nto eukaryoti ccel l s usi ng a f ine
Lytic cycle The replicationcycle of bacteriathat mi croscopi needl
c e.
ulti m a te l yre s u l tsi n th e l y s i so f h o stcel l s. Microprojectilebombardment Seebiolistics.
MicropropagationThe propagation of a plant in a
M controlledand artificialenvironmentunderaseptic
conditions,usinga definedgrowth medium.
Macrolides Antibiotics with large lactone rings.
Microsatellites A type of sequence length
Major histocompatibility complex (MHC) The polymorphismthat has tandemcopiesof di-tri-or
special group of proteins present on the cell tetranucleotidereoeatunits.Microsatellites
are also
surfacesof T-lymphocytes.MHC is involved in the called as shorttandemrepeats.
recognition of antigens on T-cells.
Miniprotoplasts The sub-protoplasts
containing
Magnetic resonance imaging (MRl) A medical nucleus, and are capable of regenerationinto
technology designed to observe the internal organs p Iants.
and structureswithout invasive procedures.
Mismatch The absenceof base pairing between
Marker genes The set of genes used for selecting
of two hybridizednucleic
one or more nucleotides
the transformed plant materials (cells/tissues).
acid strands.
Megabase (Mb) Indicates 106 bp of DNA.
Mi tosi s N ucl eardi vi si on i n somati ccel l s . Tne
Me ios is Nuc lear div is ion of ger m c e l l s t o p r o d u c e parent and the daughtercells contain the same
e gg c ells or s per m s c ont aining h a p l o i d ( h a l f ) numberof chromosomes.
number of chromosomes.
MolassesA byproductof sugarindustry,usedas a
Melting temperature (Tn') The midpoint of cheap source of carbohydratesin fermentation
temperature range at which DNA gets denatured. i ndustri es.
Meristem A localized region of actively dividing
Molecularbreeding The plant breedingmethods
cells in plants i.e. tips of stems and roots.
that are coupled with genetic engineering
Mesophile A microorganism that can grow at an techni oues.
optimum temperaturearound 37"C, and is capable
Moleculamarker A DNA sequencein the genome
of surviving within the temperature range of
which can be locatedand identified.
20 -5 0 ' c .
Monellin A proteinfound in the fruitsof an African
MessengerRNA (mRNA) A single-stranded RNA
plant Discorephyllumcumminsii which is about
that is capable of synthesizingone or more
100,000times sweeterthan sucrose.
po l y p e p ti d e
chains.
Metabolic load lt representsthe changes in Monoclonalantibody(MAb) A specificand single
metabolicand cellular functionsof the host cell type of antibodythat is producedby hybridoma
due to the presenceof cloned DNA. cel l s.MA b i s di rectedagai nsta speci fi cantigenic
determinant(epitope).
MetabolismThe entirerangeof chemicalreactions
Monocotyledons (monocots)A classof plantsthat
occ u rri n gi n a l i v i n gc e l l o r a n o rgani sm.
oossess one seedleaf.
M e ta b o l i teA l o w mo l e c u l abr i o l o gi calcompound
essentialfor cell'sor body'smetabolism. Monoploid(monohaploid)A cell or an organism
(usual l y a pl ant) w i th the l ow est number of
Metabolomics The use of genome sequence
chromosomes, denotedby x e.g. barleyn = x - 7.
an a l y s i sfo r d e te rm i n i n gth e c a p abi l i tyof a cel l /
tissue/organisms to synthesize metabolites. Morphogenesis The growthand development of an
undifferentiated structure to a form.
differentiated
Metallothioneins A group of small peptides
pr o d u c e di n a n i ma l sd u e to m e ta lpol l uti on. MRI Seemagneticresonanceimaging.
CLO S S A R Y
Motif The part of a protein that mediates the
binding of regulatoryproteins (transcriptionfactors)
to DNA.
Multicellular tumor spheroids (MCTS) ln vitro
cellu lar th ree- dim ens ionalpr olif er at ingm o d e l s f o r
the stu dy of t um or c ells .
Multiple alleles The alternative forms of a gene
tha t h as mor e t han t wo alleles .
Multiple myeloma A cancer of B-lymphocytes.
Multiple ovulation See superovulation.
Mu sh roo ms The f ungi belonging t o t h e c l a s s
b asido myc et es . Som e of t hem ar e edib l e e . g .
Agaricus bisporus (button mushroom).
Mutagenesis The changes in the nucleotides of
DNA o f an or ganis m by phy s ic al or c h e m i c a l
treatments.
Mutagens The agents that increase the rate of
mu tatio n by induc ing c hanges in DNA.
Mutation The changes in the base sequencesof
Oleosin partition technology The purificationof
DNA that are heritable
forei gn protei nsthrough thei r producti oni n oi l
Mu tein s The s ec ond gener at ion r ec om b i n a n t bodi es.
therapeutic proteins are collectively referred to as
mu tein s.
Myasthenia gravis An autoimmune diseasecausing
neuromusculardisorders.The diseaseoccurs due to
the production of antibodies againstacetyl choline
receptors.
Myelo ma A t um or c ell line der iv ed f r o m a
lymp ho cyte whic h us ually pr oduc es a s ing l e t y p e
o f immu no globulin.
Mycelium A mass of interwoven thread-like
filaments of a fungus or bacteria.
N cancer.lt was grantedU.S.patentin 1988,the first
Natural selection The preservationof favourable
a llele s whi le r ejec t ing t he injur ious one s i n a
natural way.
Nick Th e oos it ion in a double- s t r ande d D N A
where one of the polynucleotides is broken due to
lack of phosphodiesterbond.
Nitrogen fixation The process of conversion of
atmo sp he r ic nit r ogen t o am m onia. Bio l o g i c a l
nitrogen fixation occurs in prokaryotes and is
catalysed by the enzyme nitrogenase.
Nodulation The formation of nodules on the roots
of plants by symbiotic bacteria.
845
Nodule A tumor like growth on the roots of
legumes(beans,peanuts)that containssymbiotic
nitrogenfixing bacteria.
Northern blotting The transferof RNA from an
electrophoresisgel to a membraneto perform
N orthernhybri di zati on.
NorthernhybridizationThe techniqueusedfor the
detecti on of speciifc R N A mol ecul e through
Northernblotting.
Nucleoid A term used to reoresentthe DNA-
contai ni ngregi onof a prokaryoti cel
c l.
o
Okazaki fragment A short segment of DNA
synthesi zed
duri ngrepl i cati on
on the l aggi ngstrand
of doubl ehel i x.
Old biotechnologySeetraditionalbiotechnology.
Oleosins The oil body proteins that are
hydrophobicin nature, and are associatedwith
ol antseeds.
OligonucleotideA smallpieceof syntheticsingle-
strandedD N A mol ecul e.
Oligonucleotide-directed mutagenesis A technique
to al terone or morespeci fi cnucl eoti desi n a gene
(DNA sequence)so that a protein with specific
ami no aci d changei s produced.
Oncogene A genethat promotescell proliferation
to resultin uncontrolledgrowth.
Oncomouse The animal model of mouse for
animal to be patented.
Oocyte lt is a stagein the developmentof female
gameteor ovum (egg).The wordsoocyteand ovum
are used interchangeably.
Operon A clusterof bacterialgenes under the
control of a sigle regulatorysequence.
Opine An amino acid derivativeformed in some
plantsby the condensation
of an amino acid with
a sugaror a keto acid.
Organ culture The in vitro cultureof an organso
as to achievethe developmentand/orpreservation
of the ori gi nalorgan.
846 B IOTE C HNO LO CY

Organogenesis The processof morphogenesis that Phosphinothricin(glufosinate) A broad spectrum

finally resultsin the formationof organse.g.shoots, herbi ci de.'
roots.
PhotosynthesisThe synthesisof carbohydratesin
Osmolyte A compound that is involved in the greenplantsby assimilation of carbondioxide.
regulationof osmoticpressurewithin a cell. produced
PhytoalexinsThe secondarymetabolites
Osmoticum The agent or a compound that in plantsin responseto infection.
s e o s m o ti cp re s s u re
i n c re a s eth of a l i qui d.
Phytochelatins A groupof smallpeptidesproduced
Ovum The fully maturefemalegameteor eggcell. i n pl antsas a resul tof metalpol l uti on.
Ozone hole Depletion of ozone layer in the PhytohormoneThe generalname usedfor all the
atmospheremay result in ozone hole that may hormonesproducedby plants.
causeharmfuleffectsto living organisms.
PhytoplanktonsThe aquaticplantsthat freelyfloat
on water surface.
P PlantibodiesThe antibodiesproducedin plants.
Packed bed reactors (fluidized bed reactors) Plantlet Smallrootedshootor germinated embryo.
Devices used for the removal of BOD, and Plasmid An autonomous,circular,self-replicating
n i tri fi c a ti o n . extrachromosomal DNA, found in bacteria ano
Packedcell volume (PCV) lt is a test for the someothercel l s.
determination of viabilityof cells.PCVis expressed Platingefficiency The percentage of cells plated
as percentage of volurneof cellsaftersedimentation w hi ch oroducecel l col oni es.
in a low speedcentrifuge.
Pointmutation A mutationcausedby an alteration
ParthenogenesisProductionof an embryo from a
of a si ngl enucl eoti dei n a D N A mol ecu le.
female gametewithout the participationof male
Samete. Pollutant A substance which increases in quantity
in the air and adversely affectsthe environment e.g.
Patent A government-issueddocument that
CO, lead.
provides the holder the exclusive rights to
manufacture, useor sell an inventionfor a defined Poly(A)tail A seriesof A-nucleotides attachedto
period (usually20 years). the 3'-end of eukaryotic mRNA.
Pathogenesis-related (PR) proteins The low- Polyclonalantibodies Differentantibodieswhich
molecularweight proteinsproducedby plantsto can reactwith the sameantigen.
protect themselvesagainst invading pathogensPolyhydroxyalkanoates(PHA) Intracellular
carbon
(fungi,bacteria). and energy storage compounds. They are
Pedigree A chart form depicting the genetic biodegradablepolymers.
relationshipsbetweenthe membersof a human Polymerase chain reaction(PCR) A techniqueof
familv. amplifyingDNA ln vitro.
Phage A virus infectingbacterium. P ol ymorphi sm The al l el i c vari ati on sin t he
Phage display A technique involving the genomesthat resultin differentphenotypes.
identificationof proteinsthat interactwith one Positioneffect The phenomenonof differentlevels
another. of gene expressionthat is observedafter insertion
PhagemidA cloningvectorcreatedby a mixtureof of a new gene at different positions in the
p l a s mi da n d p h a g eD N A . eukaryoticgenome.
Pharmanimal A geneticallyengineeredanimalto Post-transcriptionalmodifications The changes
producepharmaceutical
products. that occur in the primary transcript(RNAs)after
they are synthesized.
PhenotypeThe appearance or othercharacteristics
of an organismthat resultfrom an interactionof its Post-translationalmodifications The changesthat
geneticconstitutionwith the environment. take place afterthe proteinis synthesized.
CLOSSARY
Pribnow box A DNA sequence found in the
prokaryotes that is required for transcription
in itiatio n.
Primary cells The eukaryotic cells taken directly
{rom an a nim al f or c ult ur e pur pos e.
Primary culture The culture produced by the
fre sh ly is olat ed c ells or t is s ues t ak en f r o m a n
orSa nrsm .
Primary transcript The initial or primary product
of tran scr ipt ion of a gene( s ) . M at ur e m R N A t s
produced after prccessing.
Primer A s hor t s equenc e of oligonuc leo t i d e st h a t
hybridizes with template strand and provides
in itiatio n f or t he nuc leic ac id s y nt hes is .
Primer extension Synthesisof a copy o{ a nucleic
acid from a prirner.
Primer walk ing A t ec hnique f or s equen c i n g l o n g
(>1 kb) p iec es of DNA.
Primordium The earliest detectable stage of an
org an in plant s .
Prion A proteinaceous infectious agent which
be ha ve sl ik e an inher it ablet r ait ( alt hough n o D N A /
RNA is present).
Prob e A labeled m olec ule us ed in hy b r i d i z a t i o n
te ch niq ue.
Prokaryote An organism whose cells lack a
memb ran e bound or dis t inc t nuc leus .
Protein engineering Ceneration of proteins with
subtly modified structures conferring improved
pro pe rties e. g. higher c at aly t ic f u n c t i o n ,
thermostability.
Protein targeting The process of transport of
proteins.from one compartment to other within a
cell. Also called as protein sorting.
Proteome The total population of protetns
p rod uced by a c ell.
Proteomics The study of proteome.
Protoplast A cell from which the cell wall is
completely removed.
Psuedogene An inactivated and non-functional
copy of a gene.
Pyrosequencing A DNA sequencing method
in vo lvin g t he det ec t ion of nuc leot ide add i t i o n f r o m
the re leas e of py r ophos phat e whic h e m i t s
che milum ines c enc e.
8'47
Quantitative PCR A polymerase chain reaction
that is used to estimate the number of DNA
molecules in a samole.
RAC Recombinant DNA Advisory Committee, a
s u p e r v i s o r y g r o u p t h a t o v e r s e e st h e r e c o m b i n a n t
DNA exoeriments
RACE See rapid amplification of cDNA ends.
R a d i o i m m u n o a s s a y( R l A ) A n a n a l y t i c a lt e c h n i q u e s
b a s e d o n t h e p r i n c i p l e s o f r a d i o a c t i v i t yo f i s o t o p e s
and immunological reactions of antigen and
antibody. RIA is a sensitive technique for the
estimationof severalbiological compounds.
Random amplified polymorphic DNA (RAPD) A
P C R - b a s e dm e t h o d o f D N A p r o f i l i n g . l t b a s i c a l l y
i n v o l v e st h e a m p l i f i c a t i o n o f D N A s e q u e n c e su s i n g
r a n d o m p r i m e r s , a n d u s e o f g e n e t i c f i n g e r p r i n t st o
i d e n t i f y i n d i v i d u a l o r g a n i s m s( m o s t l y p l a n t s ) .
RAPD See random amplified polymorphic DNA.
Rapid amplification of cDNA ends (RACE) A
technique that employs reversetranscription and
PCR for the rapid amplification of cDNA ends.
Recalcitrant xenobiotics The xenobiotics that ocr
not easily undergo biodegradation, and therefore
p e r s i s ti n t h e e n v i r o n m e n t f o r a l o n g p e r i o d .
Recombinant vaccines The new generation of
vaccines produced by employing recombinant
DNA technology.
Recombination Exchange of genetic information
between two different DNA molecules
Regeneration Development and formation of new
orSans.
Renaturation The reassociation of two nucleic
acid strands after denaturation.
Replication The synthesiscf DNA from a parent
DNA molecuie.
Readingframe A seriesof triplet codons in a DNA
sequence.
Recombinant DNA (rDNA) A DNA molecule
created in the laboratory by ligating different pieces
of DNA which are not normally joined together.
848 B IOTECHNO LO CY

Recombinant DNA (rDNA) technology The Satellite DNA ReoetitiveDNA that forms a satellite
techniquesinvolvedin the construction, and useof band in a density gradient.
re c o m b i n a nDt N A m o l e c u l es.
Scale-up The expansion of laboratory experiments
Recombinantprotein A proteinthat is produced to full-sized industrial processes.
by the expression of a cloned gene of a
S C P S e e s i n g l e - c e l lp r o t e i n .
re c o m b i n a ncte l l .
Secondary metabolite A metabolite that is not
Reportergene A gene whose phenotypecan be
required for the growth and maintenanceof cellular
assayedwhich in turn can be usedto determinethe
functions.
functionof regulatoryDNA sequence.
Senescence The biological aging of ce l l s,
R e p re s s i o nIn h i b i ti o no f transcri pti on
by bl ocki ng
characterizedby loss of functions and degradatron
the binding of RNA polymeraseto transcription
of biological molecules.
i n i ti a ti o ns i te .
Septic tanks Anaerobic digestersof solids of the
Restriction endonuclease An enzyme that
sewage settled at the bottom of tanks.
s p e c i fi c a l l y c u ts D N A mol ecul e at speci fi c
n u c l e o ti d es e o u e n c e s . S e w a g e T h e l i q u i d w a s t e a r i s i n g ma i n l y fr o m
domestic and industrial sources.
Restrictionfragmeni length potymorphism(RFLP)
A restrictionfragmentwith variablelengthsdue to Sex chromosomes The non-autosomal X and Y
the presenceof polymorphicrestrictionsitesat one c h r o m o s o m e s i n h u m a n s t h a t d e t e r m i n e th e se t o f
or both ends. i n d i v i d u a l s . M a l e s a r e X Y , f e m a l e s X X.

Restrictionmapping The processof determining Short tandem repeats See microsatellites.

th e re s tri c ti osni te si n a D N A mol ecul eby anal ysi ng Shot gun approach A technique for sequencingof
the sizesof the restrictionfragments. g e n o m e i n w h i c h t h e m o l e c u l e s t o b e se q u e n ce d
Retrovirus A virus with RNA as geneticmaterial. are randomly broken down into fragrnents,which
are then individually squenced.
Reversetranscription The processof synthesis
of
'
D N A fro m R N A. Shoot tip The terminal portion of a shoot
comprising the meristemdome and adjoining leaf
fragmentlengthpolymorphism
RFIP Seerestriction
and stem tissues.
Rheology The ability of a solutionto modify its
Shuttle vectors' The plasmid vectors that are
flow characteristics.
designed to replicate in two different hosts e.g. F.
Ribosome-inactivatingproteins (RlPs) The coli and Streptomyces sp.
antifungal/antimicrobial proteinssecretedby plants
Siderophore A low molecular weight compound
th a t i n h i b i tp ro te i nb i o s y n thesi s.
that can tightly bind to iron. They are produced by
Ribosomes The centres(or factories)for protein c e r t a i n s o i l m i c r o o r g a n i s m sa n d p l a nts to o b ta i n
biosynthesis. iron from the surroundings.
Ribozyme An RNA moleculethat has catalytic Signal peptide A short sequenceof amino acids at
activity. the N-terminal end of some proteins that facilitates
R N A v a c c i n e s R N A m ol ecul es w hi ch can the orotein to cross membrane.
synthesize antigenicproteinsand offer immunity. Single-cell protein (SCP) The cellular mass or
Rotatingbiologicalcontactor(RBC) A devicefor protein extracts of microorganismsgrown in large
the biologicaltreatmentof sewage. q u a n t i t i e s .S C P i s u s e d a s h u m a n o r a n i m a l p r o te i n
supplement, or food substitute.

Single copy sequence A sequence of nucleotides

S that occur only once in a genome.

Salvagepathway The directconversionof purines Single-locus probe Probe used in DNA finger-
and pyrimidines into the corresponding printing that identifies a single sequence (locus) in
n u c l e o ti d e s . the qenome.
 CLOSSARY 849

Single nucleotide polymorphisms (SNPs) The Sub-protoplasts The fragments derived from
po sitio ns on t he genom e wher e s om e i n d i v i d u a l s protoplaststhat do not contain all the contents of
ha ve one nuc leot ide ( e. g. C) while ot h e r s h a v e a the plant cells.
d iffere nt nuc leot ide ( e. g. C) .
Subunit vaccine An immunogenic protein
Site-directed mutagenesis The techniques used to produced from a cloned gene or purified from a
produce a specified mutation at a predetermined d i s e a s e - c a u s i n go r g a n i s m .
po sitio n in a DNA m olec ule.
Suicide gene A gene that producesa protein which
Slud ge The s em i- s olid m as s pr oduc ed d u r i n g t h e i s c a p a b l e o f d e s t r o y i n gt h e c e l l b y d i r e c t o r i n d i r e ct
course of sewage/wastewater treatment processes. m e a n s .
SNPs S ee s ingle nuc leot ide poly m or p h i s m s .
S u p e r b u g T h e f i r s t g e n e t i c a l l ye n g i n e e r e do r g a n i sm
Solid substrate (state) fermentation Fermentatron (bacterialstrain of Pseudomonadthat was oatented.
processwherein the growth of the microorgantsms It carries different hydrocarbon-degradinggenes on
is carri ed out on s olid s ubs t r at es . p l a s mi d s .

Somaclonalvariations The genetic variationsfound Superovulation The process of inducing more

in the c ult ur ed plant c ells when c om p a r e d t o a o v a r i a n f o l l i c l e s t o r i p e n a n d p r o d u c e m o r e e g g s.
pu re b r eeding s t r ain. S y m b i o t i c T h e e c o l o g i c a l r e l a t i o n s h i pi n w h i c h t w o
Somaclones The plants derived from somaclonal different species or organisms live together for
va riatio ns . mutual benefit.

Somatic cell Any body cell as opposed to germ
ce ll. Som at ic c ell is non- r epr oduc t iv e,a n d d i v i d e s
by mito s is .
Somatic cell gene therapy The delivery of gene(s)
to somatic cells to correct genetic defects.
Somatic embryogenesis Formation of embryos
from a s ex ual c ells .
Southern hybridization A technique used for the
detection of specific DNA sequences (restriction T-cells See T-lymphocytes.
fragments). T-DNA The part of the Ti plasmid that is transferred
Sparger A device that introduces air into a t o t h e p l a n t D N A .
bioreactor in the form of a fine stream.
Splicing A term used to describe the removal of
intro ns and joining of ex ons in RNA. Th u s , i n t r o n s
are spliced out wh'ile exons are spliced together
Stem cell A progenitor cell that is capable of
dividin g c ont inuous ly t hr oughout t he l i f e o f a n
org anrs m .
Synchronization A term used to representthe cells
when they are at the same stageof cell division.
T
I
Tandem repeat A repeat consisting of an array of
DNA sequencethat is repeatedcontinuously in tne
same direction.
Telomere The natural end DNA sequence in a
cnromosome.
Template The polynucleotide strand (of nucleic
acid) that determines the sequence of nucleotides
i n a n e w l y s y n t h e s i z e dn u c l e i c a c i d . T e m p l a t e i s
used by polymerases(DNA or RNA) for new strano
synthesis.

Stirred tank fermenter A fermentation vessel in
which t he c ells or m ic r oor ganis m sar e m i x e d b y
mecha nic ally dr iv en im peller s .
Stop codons The three triplets (UAA, UAC, UCA)
wh ich ter m inat e pr ot ein bios y nt hes is( tr a n s l a u o n l .
Stuffer DNA The non-essentialDNA of a vector
that can be replaced by a foreign DNA.
Subculture The transfer of cells from one culture
vessel to another culture vessel.
Termination codons See stop codons.
Thaumatin A protein extractedfrom berries whicn
is about 3000 times sweeter than sucrose.
Ti plasmid The large-sizedtumor inducing plasmid
found in Agrobacterium tumefaciens. lt directs
crown gall formation in certain plant species.
Tissue culture A process where individual celrs,
or tissues of plants or animals are grown
artificially.

Biotechnology [54]
850 B IOTE CHNO LO CY

Tissue engineering The application o{ the Tumor An enlargement or swellingof tissuedue to

p ri n c i p l e so f e n g i n e e ri n g
to cel l cul turefor the pathologicalovergrowth.
constructionof functionalanatomicalunits.
Turbidostat An open continuousculture system
T-lymphocytes(T-cells) The lymphocytesthat are into which freshmedium flows in responseto an
dependenton the thymusfor their differentiation, increasein the turbiditvof culture.
andareinvolvedin cell-mediated immuneresponses.
Totipotent A term usedto describea cell that is
not committedto a singledevelopmental pathway,
U
and thus it is capable of forming all types of Upstream Refersto the 5rend of a polynucleotide.
differentiated cells.
Traditional (old) biotechnology The age old
practicesfor the preparationof foodsand beverages,
V
basedon the naturalcapabilities of microorganisms.Vaccine A preparationintroducedinto the body to
Transcript A gene productin the form of RNA. sti mul atei mmuni tyagai nsta pathoge n.
of RNA from DNA (or
TranscriptionThe synthesis Variable number tandem repeats (VNTRs)
gene). Repetitive DNA composedof a numberof copiesof
Transcriptome The entire content of RNA a short sequence.VNTRs are involved in the
m o l e c u l e si n a c e l l . generati onof pol ymorphi cl oci w hi ch ar e usef ulr n
D N A fi ngerpr
i nti ng.
TranscriptomicsThe studyof transcriptome.
TransductionTransferof bacterialgenesfrom one Vector Seecloning vector.
cell to anotherby packagingin a phageparticle. Vegetative cell A non-reproductive
cell thatdivides
Transfection The insertionof a purified phage by mi tosi s.
D N A m o l e c u l ei n to a b a c te ri alcel l . Vegetativepropagation The asexualpropagation
TransferRNA (tRNA) The RNA involved in tne of plantsfrom the detatchedpartsof plants.
transferof aminoacidsto ribosomes duringprotern Veggie(vegetable)
vaccines Seeedible vaccines.
biosvnthesis. IRNA. also called as soluble RNA
contains7.1-80 nucleotides. Vermicomposting The process of compost
formationby earthworms.
TransformationThe acquisitionof new genesby a
cell throughthe uptakeof nakedDNA. Vinegar An aqueoussolutioncontainingabour4"h
acetic acid, and is widely used as a flavouring
Transgene The target gene responsiblefor the
agent.
developmentof transgenig
organisms.
Virus An infectiousagent that cannot replicate
TransgenicAn organismthat carriesa foreignDNA
(transgene). w i thouta hostcel l .

of a polypeptide,the
TranslationThe biosynthesis Vitrification An undesirable
conditionin in vitro
sequencebeing determinedby codonsof mRNA. tissues,characterizedby brittlenessand glassy
appearance.
TransposonA geneticelementthat is capableof
moving from one positionto another in a DNA VNTRs Seevariablenumbertandemrepeats.
m o l e c u l el.t i s a l s oc a l l e da s transposableleement.
Trickling filters Oxidation units used for the W
biological treatment of sewage in small
c o mmu n i ti e s . Westernblotting The transferof proteinfrom a gel
Triplex A type of DNA structurewith three to a membrane.
p o l y n u c l e o ti d e s . Wild type The term usedin geneticsto describea
Trisomy The occurrenceof three copies of a commonly observedphenotype(in the normall
h o rn o l o g o ucsh l o m o s o mei n a nucl eus. nativestate)of an organism.
Trypsinization Disaggregation
of tissuesby the Wobble hypothesisThe processin which a single
e n z y m etry p s i n . tRNA can decodemorethanone codon(of mRNA).
CLO S S A R Y
".t
Xe no bio tic s The unnat r : r al,f or eign anc l s y n t h e t i c
che mica ls . s uc h as pes t ic ides , her bic ic l e s a n d
vario us org anic c om pounds
Xe no ge ne ic c ells The c ells t ak en ir om d i f f e r e n t
spe cie s (e g pig s our c e f or hum ans ) f or u s e i n
e xp erime nis s uc h as t r s s ueengineer ing
Xe no tran s plant at ion The r eplac em ent o f f a i l c c l
hr.rma no rgans by t he [ r , r nc t ionalanim al or g a n s .
X-ra y crys t allogr aphy A t ec hnique r r s e c l t o
s lr Lr c t ur co f l . r n , e
d ete rmirret he t hr ee- c linr ens ional
moie cules
q
Ye ast arti f ic ial c hr om os om e ( YAC) A c l o n i n g
vector constructeclfrom the conroonentsof a veast
chro mosom e
851
Yeasttwo-hybrid system A technique to detect the
proteins that interact with eacfr other.
Z
Z-DNA A type of DNA conformationin which two
p o l v n r - r c l e o t i d eas r e w o u n d i n t h e f o r m o f a i e f t -
h . r n d e c lh e l i x
Z i n c f i n g e r m o t i t A c o n r r r r o ns l r u c t u r a l m o i i i t h a t
a t t a c h e sa p r o t e i n t o . r D N A n r o l c c u l e .
Zoo blotting A biotting techniquc used to
rleternrine the gerres oi relatecl organisms by
h v l t r i ciiz . t t i o n
Zygote The fertilized egg formed by the fusion of
t w o g a n r e t e sd u r i n g m e i o s i s
Zygote intrafallopian transfer (ZIFT) The transfer
o f t h e f e r t i l i z e d e g g s ( z y g o t e s )w i t h i n 2 4 h o u r s i n t o
the fallopian tube by using laparoscopy
 carboxylic acid
ACC 1- Am inoc y c lopr opane- .1 CFCs Chlorofluorocarbons
Ac M NPV Aut ogr aphac alif or ni c a m u l t i p l e n u c l e a r
regulator'
CFTR Cyslicfibrosistransmembrane
poly hedr os isv ir us . homogeneou elect
C H E F C ontour-cl amped s r ic-
ACP Ac y l c ar r ier Pr ot ein field electrophoresis.
ADEPT Antibody-directedenzyme prodrug therapy. c e steri l i tY
C MS C vtoP l asmimal
ADA Adenos ine deam inas e. C MV C ucumbermosai cvi rus
AFLP Am plif ied f r agr lent leng t h p o l y m o r p h i s m C OD C hemi caloxY gendemand
AH As s is t edhat c hing. C OH C ontrol l edovari anhypersti mul a t ion
AI DS Ac quir ed im m unodef ic i e n c y s y n d r o m e . CPPP Cyclopentenoperhydrophenanthrene
AK Aspartokinase CpTi Cowpeatrypsininhibitor
A[S Acetolactate sYnthase CSTR Continuousstirredtank reactor
AP-PCR Arbitrarily Primed PCR. CT Cytoplasmictransfer
ART Assisted reproductive technology' ammoni
C TA B C etyl tri methY l um
ATPS Aqueous two-Phase systems. C S C al f serum
BAC Bac t er ialar t if ic ial c hr om o s o m e . D E A E D i ethyl ami noethY l
BCG Bac illus Calm et t e- Cuer i n . DHFR Dihydrofolatereductase
BNR Biologic al nit r ogen r em o v a l ' synthase
DHPDS Dihydropicolinate
BO D Bioc hem ic al ox y gen de m a n d . DMD Duchenne'smuscular dYstroPhY
bp Base pair. modification
DMEM Dulbecco's medium'
of Eagle's
BPTI Bov ine panc r eat ict r y psi n i n h i b i t o r . DMSO Dimethylsulfoxide
Bt Baccillus thuringiensis D N A D eoxyri bonucl eiaci
c d
CAM s Cell adhes ion m olec ul e s DSBs Double-stranded breaks
CaM V Caulif lower m os aic v ir u s DVT Deerrvein thrombosis
c DNA Com plem ent ar YDNA. ' E A A s E ssenti al
ami no aci ds
CDK Cy c lin- dependentk inase . virus
EBV Epstein-Bar
CDRs Com plem ent det er m in i n g r e g i o n s ' E.C. E nzymecomml ssron
CF Cystic fibrosis EGF Epidermalgrowthfactor

852
A B B RE V IAT ION S 853

EtISA En z y m e- link edim m unos or bant ass a y H N P C C ' H e r e d i t a r y n o n p o l y p o s i sc o l o n c a n c e r

EMEM Eagle' sm inim al es s ent ialm edium hnRNA Heterogeneousnuclear RNA
EMS Eo s inophilia- m y algias y ndr om e HPIC High performanceliquid chromatography
EPAs Environmental protection agencies HPV Human papilloma virus
EPSPS 5-Enoyl-pyruvylshikimate
3-phosphatesynthase H S V H e r p e s s i m p l e x v i r u s
ES Embryonic stem lC Inhibitory concentration
ESC Emb r y onic s t em c ell lcl lmperial Chemical Industries
ET Embryo transfer ICM Inner cell mass
FACS Fluorescentactivated cell sorter I C P I n s e c t i c i d a lc r y s t a l l i n e p r o t e i n
FDA Fluor es eindiac et at e lCSl intracytoplasmicsperm injection
FENI Flap endonuc leas eI lg lmmunoglobulin
FCF Fibroblastgrow'th factor I G F I n s u l i n - l i k eg r o w t h f a c t o r
FMD Foot and mouth disease I H G S C I n t e r n a t i o n a lH u m a n C e n o m e S e q u e n c i n g
GAG Cly c os am inogly c ans Consortium

Gb Cig abas e pair lL I n t e r l e uk i n s

CBSS Cr anule- bound s t ar c h s y nt has e lmviC Indole, methyl red, voges-Proskaverand

Citrate tests (i used for phonetic purpose).
GCV Ca nc ic lov ir
lPRs Intellectual property rights
GDH Clutamate dehydrogenase
IRES Internal ribosomal entry site
GEMs Ce net ic ally engineer ed m ic r oor ga n i s m s
ISFET lon-selectivefield effect transistors
CEOs Cenet ic ally engineer edor ganis m s'
lUl I n t r a u t e r i n ei n s e m i n a t i o n
GFAP Cl ial f ibr illar y ac idic pr ot ein
I V C I n t r a v a g i n a lc u l t u r e
CH Crowth hormone
M In vitro fertilization
GIFT Camete intrafallopian transfer
fVM ln vitro maturation
GLUT Clucose transporter
kb Kilobase
GM Cen et ic ally m odif r ed
kDa Kilodaltons
GMEM Clasgow's modification of Eagle'smedium
Kn,, Michaelis-Menten constant
gp Clycoprotein
tDt Low density lipoprotein
HAC Hu m an ar t if ic ial c nr om os om e
LEAR Low-erucic acid rape
HAT Hypox ant hine am inopt er in and t hym i d i n e
l-Hb Leghemoglobin
HBsAg Hepatitis B surface antigen
LINEs Long interspersednuclear elenrents
HDGS Hom ology - dependentgene s ilenc i n g
tOS Low oxygen storage
HEAR Hi gh er uc ic ac id r ape
tPS Low pressurestorage
HFCS High fructose corn syrup
LTR Long ternrinal repeats
HFS High fructose syrup
MAb Monoclorral antibody
hGH Hum an gr owt h hor m one
Mb Megabase pair
HCP Hum an genom e pr oiec t
MCTS Multicellular tumor spheroids
HGPRT Hy pox ant hine- guanine phos p h o r i b o s y l
transferase. MEOR Microbial enhanced oil recovery

HIC Hydrophobic interaction chromatography MFT Membrane filtration technique

HIV Hum an im r nunodef ic ienc yv ir us MCT Mean generation time

Hl-A Histocompatability locus antigen MODY Maturity onset diabetes of the young

HMP Hexose monophosphate MOET Muitiple ovulation with embryo transfer

 854 BIOTECHNOLOCY

mRNA Messenger
RNA rRNA RibosomalRNA
MSG Monosodiumglutamate RT-PCR Reversetranscriptionpolymerasechain
mtDNA MitochondrialDNA reaction.
MTF Multiple-tubefermentation SARs Scaffoldattachmentregions
NASA NationalAeronautics SpaceAdministration SCF Siliconcarbidefibres
NHGRI National Human Genome Research SCF Sisterchromatidexchange
. Institute fluid
SCF Suoercritical
NI I D M N o n -i n s u l i nd e p e n d e ndt i abetesmel l i rus SCID Severecombinedimmunodeficiency
OHSS Ovarianhyperstimulation syndrome SCS Soecializedchromatinstructure
p Promotersquence SCP Single-cellprotein
pa Polyadenylation
sequence slgA SecretoryimmunoglobinA
PAGE Polyacrylamidegel electrophoresis SINEs Shortinterspersed nuclearelements
PBR Plantbreeders'rights S IP C S urfacei mmobi l i zedpl ant cel l
PCBs Polychlorinated
biphenyls S N P s S i ngl enucl eoti depol ymorphi sms
PCNA Proliferating
cell nuclearantigen SPECT Single photon emission computed
PCR Polymerase
chain reaction tomography
PCV Packedcell volume bi ndi ng
S S B S i ngl e-stranded
PDGF. Platelet-derived,growth
factor ssDNA Single-stranded DNA
P D T Po p u l a ti o n
d o u b l i n gti me SSF Solid substrate(solidstate)fermentation
PEC Polyethylene glycol STRs Shorttandem repeats;stirredtank reactors
gel electrophoresis
PFGE Pulsed-field SUZI Subzonalinsertion
PC Prostaglandins;
Polygalacturonase T-DNA TransferredDNA
PGD Preimplantationgeneticdiagnosis TE Tissueengineering
PHA Polyhydroxyalkanoates TEUSA ThermometricELISA
PHB Polyhydroxybutyrate ThOD Theoreticaloxygendemand
PPi Pyrophosphate TCF-p Transforminggrowth factor p
P Q Q Py rro l o q u i n o l i nqeu i n o n e TGS Transcriptionalgenesilencing
PR Pathogen-related TlLs Tumorinfiltratinglymphocytes
PTGS Posttranscriptional
gene silencing TK Thymi di neki nase
PUFA Polyunsaturated
fatty acids Tm Meltingtemperature
RAC RecombinantDNA AdvisoryCommittee TMV Tobaccomosaicvirus
RACE Rapidamplificationof cDNA ends TNF Tumornecrosisfactor
RAIT Radioimmunotherapy TOC Totalorganiccarbon
RAPD RadomamplifiedpolymorphicDNA tPA Tissueplasminogenactivator
rDNA RecombinantDNA intellectualpropertyrights
TRIPS Trade-related
RFC ReplicationfactorC IRNA TransferRNA
RFLP Restriction
fragmentlengthpolymorphism TTC Triphenyltetrazoliumchloride
RI A R a d i o i m m u n o a s s a y TUNET ldt-mediated dUTP nick end-labeling
RlPs Ribose-inactivating proteins assay
RN A R i b o n u c l e i ac c i d VEGF Vascularendothelialgrowth factor
ROSNI Roundspermidnucleusinjection VNTRs Variablenumbertandemrepeats
RPA ReplicationproteinA XP Xerodermapigmentosum
RPMI RoswellParkMemorialInstitute YAC Yeastartificialchromosome
 Index

Ae r atcrl l ag,oon-.,(r()'1 A i r pol l utron control , 6;:l

Ae r : rted ponds, (r91 A i r-l i ft fcrmenter,.l 5.l
Ae r ated sl ,rti c-prl C of A i r-l i ft i crmentcr r Lrl ture..15-1
cor.r'rl x)strng, 7l 2 A i rl i i t l ri oreactors,2.1I
\ a k p l a n t, - 3 9 9
Ac.r obi c ,rttachei l grow th A l cohol , 311, 395
, { b a s i c s i tcs, 3 ,1
treatnrent prOr:c'sses, 692
\ b i o t i c e r licito r s,5 1 4 A l cohol i c beveragc:, 367
Ae r obi c l racteri r, 7i -1
(r6t)
A l gal bi oassa.vs,
A b i o t i c str e ssr e sista n ce ,6 0 7
Ae r obi c: l ri oremedi afi on, 72 I
A b i o t i c str e r sse s,
596 A l gal bl ooms, 66/
Ae r obi c r omposti ng, 713
\ b s c i s i c acid ,5 2 0 A l gal i zati on,6,l (r
r \cr obi c cl i gesters,(t92, 711
Abz,ymcs,226 A l gi nate.,.3B 5
Ae r obi c: rl rgesti on,692
A C C d c a r r in a se , 6 1 .5 A l gi nate l vase, l 9B
Ae r obi c ponds, 6()6
{ C C s y n th a se ,6 1 5 A Ikal i es, 762
Ae r ohi c si udge di gcsti on, Zl l
, A c e t r ca cid , 3 2 6 A l kal i nc phospl ral ase,79
Ae r obi c suspendedgrow th
A l kanes to producc. S C P ,375
\cetone, 3 I 5 treal nl ent processes,690
A Il el es, 823
\ c e t y l c h o lin e e ste r a se ,6 6 1 Affini ty r hromatography,27!)
A l l ogenei c cel l s,,173
\ c i d f o r m in g b a cte r ia , 6 9 6 Affini ty tag,gi ng,14tr
'I A l l ograft, 821
\ c i d r a i n ,2 3 4 AF L P, I, , 651
A l opeci a ri ni versal i s,'l B 3
\ c r d o g e n e sis, 6 9 5 Ag a rose g,el el ectrophoresi s,cl 2
A l tepl ase, 196
{ c i d s , 7 62 Ag ly conc, 723
A l u secl uerrces,3.i
\ c o u s t i c b io se n so r s,3 0 2 Ag r ob ac l t'r i u m-deri ved
promoter, 592 A l zhei mer's di sease, 1B 1
\ c t i n o myr in D, 4 5
Agrobar tcrium rhizoS4enes,577 A i zhei nrer's mouse, 485
\ctivase, I 95
Agrobac teriun tLrneiaciens, 57 3 A mes test, 662
\ c t i v a t e d slir d g e p r o ce ss, 6 9 0
Ag roLtac'tc r i u m-rnediated gene L-A mi no aci d acyl ase, 290
\ c t i v e s i te , 7 8 9
t ransfcr,573 A mi no aci d metabol i sm-i nuorn
l c v l c a r rie r p r o te in , 8 0 6
Ag r ob;tck r i u n rne,di .i tedp1.rrrt errc>rs,81 4
\ c l a p t o r s, 7 B t ransk)rrrratron,580 A mi no aci d moti l s, 65
\ c l e n o - . i s so cia te dvir u s ve cto r Ag r opi nt,s,5/'1 A mi no aci ds, 19,778
s v s t e m , I6 5 AIDS , 82I
A nri no aci ds mi <robi al
\ J e n o > i nrd, e a m in ,t' e , 1 6 2 anti scnsetherapy, 1 71
pro(l ucti on, 144
\ c l e n o s i n e m o n o p h o sp h a te , I - 3 cl i agnosi s,1 7/
gcnl ' fherap1,,I69 7 A mi nocephal osporani c ac:i cl ,335
\ c l e n o V i raI ve cto r syste m , I 6 5 nronoc.l onalantrbodi es,222 1-A mi rror,yci oprop:rne1-carboxl ,l i c
\ D P - g l u c o se p h o sp h o r yla se ,6 2 2 vac:ri nes, 204 aclo, lr l5

i r l s o r p t i on , 2 7 8 ,7 6 5 Air pol l utants,66,.) 6 A mi no peni ci l l ani c aci ci , .13,1

855
856 BIOTECHNOLOCY

Am in o su g a r s,7 7 3 A ni mal cel l -geneti c Assistedhatching,235

Am in o a cyla se , 2 9 6 engi neeri ng, 41 3 Assistedreproductive
A ni mal cel l structure, 750 technology,227
Am in o a cyl IRNA, 5 3
7 - Am in o ce p h a lo sp o r a n ica cid , 3 3 5 A nther cul ture, 539 Asymbioticnitrogenfixation,640

1- Am in o cyclo p r o p a n e - 1 ca r b o xvlic A nther cul ture crops, 545 Asynrbioticnitrogenlixers,646

a cid , 6 1 5 A ntl -rocyani di n,785 Asymmetriccarbon,761
Am in o g lyco sid e a d e n yltr a n sfe r a se , A nthocyani n pathw ay, 61 6 Asymmetrichybrids,534
5 BB
A nthocyani ns, TB 5 A s y m m e t r i cP C R ,1 1 6
Am in o g lyco sid e s,3 3 6 Atherosclerosis,22.l
A nti bi oti c resi stancegenes, 5B B
Am m o n ifyin g b a cle ( ia , 7 5 6
A nti bi oti cs-mi crobi al Attenuatorgene,62
Am p e r o n r e tr ic b io se n so r s,2 9 !) producti on,329 657
Atmosphere,
Am p h o lyte s, 7 6 2 A nti body-directed enzyme Attenuatedrecombinant
Am p lifie d fr a g m e n t le n g th prodrug IheraP Y ,224 vaccines,208
p o lym o r p h ism , 1 1 7 , 6 5 1
A nti codon, 48 226
Autoantibcdyfingerprinting,
cr - Am yla sein h ib ito r s, 6 0 1
Antigene therapy, 169 Autograft,821
Am ylo id p r e cu r so r p r o te in , 4 8 6
A nti metabol i te marker genes, 589 A u t o i m m u n ed i s e a s e s8,21
Am yo tr o p h ic la te r a l scle r o sis,18 .1
A nti mei abol i tes, 790 32l
Autoimmunity,
An a b o lism ,7 9 7
A nti mi crobi al protei ns. 605 Autologouscells,164, 473
Anaerobic attached-growth
A nti sense ethyl ene technol ogy, 61 5 106
AutomatedDNA sequencer,
treatment processes, 696
A nti sensegene, 604, 61 5 100
Autoradiography,
Anaerobic bacreria, 7 54
.l
An a e r o b ic b io r e m e d ia tio n , 7 2 1 A nti sense ol i gonucl eoti des, 71 823
Autosomes,
An a e r o b ic co n ta ct p r o ce ss, 6 9 6 A nti sense R N A s, 603, 614 Autotrophicbacteria,755
.l
An a e r o b ic d ig e ste r ,3 9 7 , 6 9 5 , 7 0 6 Antisense therapy, 69 A u x i l l a r yb u d c u l t u r e ,5 53
An a e r o b ic d ig e stio n , 6 9 5 , 7 0 6 crt-A nti trypsi n,134 A u x i n s ,5 1 3 , 5 2 0
An a e r o b ic filte r p r o ce ss, 6 9 6 Apligrafru, 476 Auxotrophicbacteria,755
An a e r o b ic p o n d s, 6 9 7 A poptosi s, 29, 435 Auxotrophicmutants,53.1
An a e r o b ic slu d g e d ig e stio n , 7 1 0 Aptamers, 172 Avidin,633
Anaerobic suspended-grorvth A puri ni c si tes, 34 Axillarybud culture,555
treatment processes, 694
Aqueous two-phase systems, 277 Azolla, 396, 646
An ch o r e d PCR,1 1 5
A rabi dopsi s,612 Azoospermia,23l
An d r o g e n e sis,5 3 8
A rachi doni c aci d, 310
An d r o g e n ic h a p lo id s, 5 4 0
A rbi trari l y pri med P C R , 115
An e u p lo iciy,8 2 5
A ri d pl ant bi otechnol ogy, 653
An im a l b io r e a cto !' s,4 9 0
A romati c anti bi oti cs, 34 |
An im a l ce ll cu ltu r e
A rti fi ci al chromosome vectors, 86
B
a d va n ta g e s,4 1 .1
a p p lica tio n s, 4 1 1 A rti fi ci al cul ture medi a, 415
a se p tic co fr d itio n s, 4 0 9 A rti fi ci al i nsemi nati on, 227 B aci l l us subti ti s,317
b io h a za r d s,4 l4 Bacillus thuringiensis, 597
A rti fi ci al l i ver, 477
ce ll q u a n tita tio n , 4 5 0
Artificial pancrcas, 477 Bacmid, 142
co n ta m in a tio n , 4 0 9
fa cilitie s,4 0 T A rti fi ci al seeds, 559 B acteri a-characteri sti cs754
,
g e n e r a l co n sid e r a tio n s,4 5 0 Ascorbic acid, 327 B acteri a-i mportance,755
g r o wth kin e tics, 4 5 1
A si l omar recommendati ons,90 B acteri al arti fi ci al chromos ome, 85
h isto r ica l t' a ckg r o u n d , 4 0 7
lir n ita tio n s,4 1 1 Asparaginase, 13 5 B acteri al di seases(pl ants ),604
p r o d u cts o f im p o r ia n ce , 4 1 3 A spartame, 344, 367 B acteri al i uci ferase,591
r isks in la b o r a to r y, 4 1 3 B acteri ophage1,, B 3
A sparti c aci d, 354
safety regulations, 414 .103
Aspect ratio, 240 B acteri ophageM13,
sle r iliza tio n , 4 1 0
typ e s o f cu ltu r e s, 4 5 2 Aspergillus niger, 283, 318 B acteri ophages,83
 INDEX

Ba cu lovirus,
14 1 B io in fo r m ati cs-appl i cati ons,830 B i osphere,658
Baker'syeast,365 Bio le a ch ing,400 Biostat, 454
Ba lan ce dsa lts olut ion,417 Bio fe a ch ing o( copper, 402 B i osti mul ati on, 719, 727
g en e,183
Ba ldn ess Bio le a ch ing of urani um, 402 B i otechnol ogi cal rnethods for
pol l uti on management, 663
Baseexcisionrepair,35 Bio listics,584
Bases,762 B i otechnol ogy ar-rddevel opi ng
Bio lo g ica l cal ci fi cati on, 664
countri es,746
BastaAventis,609 Bio lo g ica l databases,828
B i otechnol ogy and soci ety, 739
Batchculture/fermentation,
259 Bio lo g ica l fi l m, 692
B i otechnol ogy tree, 6
BCC vaccine,2O3 Biological nitrogen fixation, 639
B i otechnol ogy-deti ni ti ons, 3
Beer,369 Bio fo g ica f ni trogen removal , 700
B iotechnoiogy-history, 4
Beer-Lambert
law, 767 Bio lo g ica l phosphorus
I i otechnol ogy-publ i c
Beerwort, 369 r e m o val , 731 percepti on, 7
Be ntDNA, 17 Bio lo g ica l treatment of B i oti c el i ci tors, 5i 4
sewage, 689
Be nth ics,66 6 B i oti c stresses,596
Bio lo g ica l w arfare, 743
platingtechnique,506
Bergmann's B i otransformati onof anti bi oti cs,
Bio m a g n ificati on,666 309
Bia lph os,60 9
Bio m a r ke r sof pol l uti on, 660 B i otransformati onof steroi ds,308
Binaryvector,579
Bio m a ss,3 93 B i otransforrnati ons,306
Bio accu mula t ion,
666, 720
Bio m e th ylati on, 666 B i otri ckl e fi l ters, 678
Bioassays
in environmental
monitoring,660 t' | o m o te cu tes, ././| Biovenling, 727
Bioaugmentation,
7 19, 727 Bio m o n ito ri ng of ai r pol l uti on, 672 B i rnboi m and D ol y method, 95
Bioblaster,
584 Bio m o n ito ri ng of pol l uti on, 659 B i speci fi c monocl onal anti bodi es,
Biochemicaloxygendemand,683 Bio p e sticide,59B 218
B l ood gas moni tori ng, 304
Biocon ve rsio ns , 256 Bio p h o to ly si s,398
B l ood gl ucose bi osensor,299
plastics,386, 390,
Biodegradable Bio p la stics,626
626 B l ue-green al gae, 646
Bio p o l, 3 9 0
polymers,386
Biodegradable B l unt ends, 78
Bio p r o ce sstechnol ogy, 239
Bio de gra da tion,
718 B -Lymphocytes,21 7
Bio p r o sth esi smateri al s,475
Biodegradationof contaminated B OD bi osensor,663
Bioreactor-operalion, 245
soils,72 7 Bovine growth hormone, 741
Bio r e a cto r s,239
Biodegradation
oi herbicides,722 Bovine somatotropin story, 24l
Bio r e cla m ati on, 718
Biodegradation
of B oyer and C ohen experi ments, 75
hydrocarbons,722 Bio r e m e ciiati on,7.18
Brassica napas, 631
Biodegradation Bio r e m e d iati on of contami nated
of organic B read, 365
matter,695 so|s, / l/
B reast cancer, 181
Bio r e m e d iati on of ground
of pesticides,
Biodegradation 722
waIer, 728 B rew i ng, 369
Bioethanol,
399
Bio r e m e d iati on of w aste B romoxyni l ni tri l ase, 59C
Biofencingtechnique,728 ta n o s, / z/ B t resi stanti nsects,599
Biofe rtilize rs,645 Bio r e so r b abl epol ymers, 475 B t toxi n, 597
Biofilte rs,6 TS Bio sca ve n gersof metal s, 666 B t toxi n genes, 598
Biofuels,395, 398 Bio scr u b b ers,678 B ubbl e col umn bi oreactors,241
Biogasplant,397 Bio se n so r s ,29T B ud cul tures, 554
Biogas,395 Buffer action, 762
Bio se n so r s -appl i cati ons,
304
Bioh yd rog en ,39S Buffers, 762
Bio se n so r si n evi ronmental
Biohydrometall
ugy, 4OO m o n ito ri ng, 663 B utanol , 315
b to tn Io rma U CS , ull .l
Bio so r p tio n, 404 Bystander effect, 68
854 BIOTECHNOLOCY

Cell viability,459 C hromopl asts,785

G Cell viabilityassays,
460 C hymosi n, 364,741
Cell wall, 753 C i stron, 59

CAAf box, 42 Cell-mediated

immunity,Bl 7 C i tri c aci d, 3.18

Ca llu s, 4 9 8 Cellularsenescence,
435 Citric acid cycle, 799

Ca llu s cu ltu r e , 5 0 0 Cellulase,533 C l ean gene technol ogy, 59 0

Ca lo r im e tr ic b io se n so r s,3 0 0 C e l l u l o s e3, 9 4 , 7 7 4 C l onal propagati on, 552

Ca lvin cycle , 8 0 4 C e n t r ad
l o g m a ,1 1, 2 3 Clone, 82

Ca n ce r , 8 2 1 Centrifugal
elutriator,
466 Cfoning efficiency, 466
.l
antisense therapy, 70 Cephalosporins,
334 C l oni ng of fetal cel l s, 491
d ia g n o sis, 1 8 1
Cerezyme,666 C foni ng of humans, 744
gene therapy strategies, 16/
m o n o clo n a l a n tib o d ie s, 2 2 1
Chaga'sdisease-diagnosis,
177 C l oni ng of pet ani mal s, 47 1
.l
Ca n ce r a n d ce ll cycle , 2 9
Chainterminationmethod, 07 C l onogeni c assays,461

Carbohydrates, 772
Chaperones,
55 Clostr id i u m acetobutyl i cu m, 3 16

Ca r d io va scu la rd ise a se s,2 2 0

Chaperonin,
56 C l otti ng factor V l l l , 194
Chargaff's
rule, 14 C odons, 46
B- Ca r o te n e ,3 5 9 , 6 1 8
Ca r o te n o id s. 7 8 5
Cheddar,364 Coenzymes, 792
C h e e s e3, 6 2 , 7 4 1 Cointegrate vectors, 577
Ca se tte em u ta g e n e sis,13 1
Caspgses,436
Chemicaloxygendemand,683 C ol chi ci ne, 5A 4, 543
Chemical-aided 689
sedimentation, C ol d trypsi ni zati on, 439
Ca ta b o lism , T 9 6
Chemostat
bioreactors,
262 C ol d-tol erant genes, 61 2
Catabolite gene activator
p r o te in , 6 0 Chillproofing,3T0 C ol i form organi sms, 684
Catalase, 752 Chimeric anribody,217 C ol i l ert techni que, 685

Ca t- ta ils,6 6 7 ChimericDNA, 76 C ol l agenase,439

Ca ta lytic M Ab s, 2 2 6 Chimericoligonucleotides,
171 C ol l oi dal state, 763
Ca ta lytic RNA, 2 2 Chiral, 761 C ol l oi ds, /63

Catechol, 722 Chitinase,605 C ol on cancer, 182

Ca u liflo we r m o sa ic vir u s, 5 8 1,5 9 2 C h l o r a m p h e n i c o3l 4, 1 C ol ony and pl aque bl otti n g, 100

Ca u liflo we r m o sa ic vir u s Chloramphenicol acetyl C ol ony hybri di zati on, 126

p r o m o te r , 5 9 2 transferase,
592 Lol onmeter, ./b/
Ca u lim o vir u se s,5 8 1 Chlorofluorocarbons,
730 C ompeti ti ve i nhi bi ti on, 790
Ce le r a Ce n o m ics, ' l4 8 Chlorophyll,753, 8O3 C ompl ementary D N A , 123
Ce ll clo n in g , 4 6 3 , 4 6 5 Chloroplast
engineering,
.594 C ompl ementary D N A l i brari es , 123
Ce ll cycle , 2 9 genome,594
Chloroplast C ompl ementary D N A
C;ll d isr u p te t 2 2 4 Chloroplast
transformation,
594 vacci nes, 205

Ce ll g r o wth m o n ito r in g in Chloroplasts,

594, 753 C ompl ete cul ture medi a, 417
sca le - u p , 4 5 8 Chlortetracycline,
339 C omposti ng, 711
Ce ll im m o r ta liza tio n , 4 5 3 Choler,a,
208 Lomposi l ng ptanl 6, /t/
Ce ll lin e s, 4 2 7 , 4 3 O, 4 4 2 Cholesterol,
806 C omputati onal bi ol ogy, 82 7
Ce ll lin e s- m a in te n a n ce ,4 4 4 Cholesterol
biosynthesis,
806 C ondi ti oni ng of sl udge, 71 4
Ce ll lin e s- n o m e n cla tu r e ,4 4 3 Chromatin,19, 750 C onducti metri c bi osensors ,300
C e ll lin e s- se le clio n ,4 4 3 Chromatography,
766 C onl ugati on, 87
Cell support materials, 474 Chromosome jumping,105 C onsti tuti ve genes, 59
C e ll su r viva l a ssa ys,4 6 1 Chromosome
karyotyping,
430 Contact condenser, 677
C e ll syn ch r o n iza tio n ,4 3 3 C h r o m o s o mw
e a l k i n g ,1 0 5 C ontami nant,.669
Cell transformation, 463 Chromosomes
isolation,96 C onti nLous cel l l i nes, 443
INDEX 859

C o n t i n u o u s cu ltu r e / Cu l tured stem cel l s- D extran, 385

f e r me n ta tio n , 2 6 2 appl i cati ons, 449 D ext!'orotator, 761, 773
C o n t o u r- cla m p e d h o m o g e n o u s Cu rdl an,386 D i abetes mel l i tus, 190
c l e c tr ica l- iie ld cle ctr o p h o r e sis. Cyanophages,665 D i abetes-D N A probes, 182
93
Cybri di zati on,535 D i ami nopi mel ate, 350
C o n t i n u o u s flo w cu ltu r e , 4 5 4
Cybri ds,535 D i auxy,260
C o n t r o l l e d o va r ia n
Cycl i ns,29 D i azotrophrc mrcroorgani sms,646
h y p e r stim u la tio n ,2 3 2
Cycl odextri n gl ucosl ,l D i azotrophs,639
C o n v e n tio n a l p la n t b r e e d in g , 498,
transferase,623
572 D i azotrophy,639
Cycl odextri ns,623
C o o r d i n a te d g e n e e xp r e ssio n ,143 D i ci stroni c expressi on vector, 144
Cycl one col l ectors, 673 D i cots,574
Copy nature strateBy,601
Cynobacteri a, 646 D i cotyl edonous pl ants, 57+
Cory n ebacter i u m g I uta m i c u m, 347,
350 Cysti c fi brosi s, 56, 106, 166, 179 D i ctyosomes, 751
C o s m i d s, 8 4 Cysti c fi brosi s transmembrane .l
D i deoxynucl eoti de method, 02
'l
regulator, 66
C o s u b s tr a te s,7 9 2 D i esel oi l ,375
Cytogeneti c bi oassays,662
C o w p e a tr yp sin in h ib ito r D i ffusi on, 263
g e n e,6 0 1 Cytogeneti c map, 149
D i goxi n, 516
Cristae,783 Cytoki nes, & 20
D i hydrofol ate reductase,135, 589
C r o p s a s b io r e a cto r s,6 2 8 Cytoki ni ns, 513, 52O
D i l uti on cel l cl oni ng, 465
C r o p y i eld im p r o ve m e n t, 6 1 2 Cytopl asmi c hybri ds, 535
D i pl oi di zati on of hapl oi d
C r o w n ga ll d ise a se ,5 7 4 Cytopl asmi c mal e pl ants, 543
steri l i t, 536, 617
L r y o p r e se r va Uo n ,) b c) D i rect D N A transfer
Cyt opl asmi c Iransfer,234 (i n pl ants), 583
C r y o p r ese r va tio n - ART2, 3 5
Cyt opl asts,528 Disaccharides, 77 4
C r y o p r ese r va tio nb y co ld
s t o r ag e , 5 6 9 Cyt oskel eton, 252 D i sc centri fuge, 272
C r y o f r e se r va tio n b y fr e e zin g , 5 6 8 Cytosol ,752 D i sease-freepl ants-producti on,55 9
c r y o p r o te cta n ts,5 6 8 Cyt otoxi ci ry 459 D i si nfecti on of sew age, 703
C r y s t a l pr o te in s, 5 9 8 Cytotoxi ci ty assays,460 D i si nfecti on of sl udge, 714
C r y s t a l l o id s,7 6 3 D i ssol ved oxygen, 679, 683

C u l t u r e m e d ia - a n im a l ce lls, 4 .15 D i stiI l ery-w astew ater

a r tificia l m e d ia , 4 1 5 treatment, 704
c o mp le te m e d ia , 4 1 7 DNA
c o mp o sitio n , 4 1 B E' chemi cal synthesi s,109
n a tu r a l m e d ia , 4 1 5 damage and repai r, 33
p h ysico - ch e mica l p r o p e !' tie s, denaturati on, 18
415 Da i ri es-w astew ater treatment, 703 doubl e hel i x,.l 4
serum-free media, 421 De afnessgenes, 183 fi nger pri nti ng (profi l i ng), 184
w i th se r u m , 4 1 9 methyl ati on, 64
DEA E dextran-medi ated
C u l t u r e d a n im a l ce lls transfer, 5BB organi zati on i n cel l , 19
c h a r a cte r istics,4 2 3 puri fi cati on, 94
De ami nati on, 809 repl i cati on, 23
c e l l a d h e sio n , 4 2 3
c e l l d iffe r e n tia tio n ,4 2 4 De di fferenti ati on(pl ant cel l s), 598 sequencrng,I 01
c e l l lin e d e ve lo p m e n t, 4 2 7 De ep vei n thrombosi s, 221 structure, l 4
c e l l p r o life r a tio n , 4 2 4 topoi somerases,26
De fensi ns,606
c e l ! syn ch r o n iza tio n , 4 3 3 transposi ti on,33
c h a r a cte r iza tio n ,2 2 8 De ni tri fi cati on, 700 vacci nes,205
.l
g r owth cycle , 4 3 2 De oxyri bonucl easel , 9B D N A chi ps, 108, 176
rneasurement o{ growth, 432 De pth fi l ters, 252, 271 D N A hel i cases,25
m eta b o lism ,4 2 5
De rmagraftrM,476 D N A i mbi bi ti on, 588
p l atin g e fficie n cy, 4 3 3
De wateri ng of sl udge, 714 'l
t r a n sfo r m e d ce lls, 4 3 0 D N A i n drseasedi agnosi s, l l
8eo BIOTECHNOLOCY

DNA ladderingtest,435 Ediblevaccines,2O7,636 Enzymesin beverage

DNA library-screening,
125 Electrochemical
biosensors,
299 induskies,371
DNA ligases,78 Electrofusion,
530 791
Enzymespecificity,
DN A m ar k er s185
, Electrophoresis,
766 791
Enzymestereospecificity,
DNA microarrays,
827 Electroporation,
87 Fnzymetechnology,
281
DNA modifyingenzymes,79 (plants),583
Electroporation Enzymesin diseasediagnosis,
795
DNA-molecular 167
conjugates, precipitators,675
Electrostatic Enzymesin fruit juice
production,371
DNA polymerase,
25, 27, 114 Elicitors,5 14
nature,787
Enzymes-chemical
DNA probes,1O8,126, 174, 178, ELSIof biotechnology,739
662 786
Enzymes-classification,
Embryobiopsy,229
DN A pr of iiing,184 297
Enzymethermistor,
Embryocloning,231
DNA repairand cancer,37 Eosinophilia-myalgia
Embryoculture,230
DN A s c r eening,
I 25 syndrome,241
Embryoculture(plarrt),562
DN A s equenc ing,
101 Epithelialstemcells,448
Embryoculture-applications,
564
DNA s huf f ling,136 Epitopes,213
Ernbryo-endosperm
transplant,
563 .l
DNA topoisomerases,
26 Epogen, 98
Embryorescue/562
DNA vaccines,205 Epstein-Ba
v irr u s , 2 1 7
Embryosexing,229
Dolly, 490 Erythromycin,
340
Embryosplitting,229
Dot-blotting,100 Erythropoietin,
l93
Embryotransfer,
228, 232, 233
Double-strand
breakmodel,31 EScell cultures,477
Embryo-endosperm
transplant,
563
Double-strand
breakrepair,37 EScells and tissuerepai, 449
Embryonicstellcell
Dow'nstreamprocessing,270 Essential
amino acids,778
engineering,4TT
Down'ssyndrome,825 Ethanolproduction,312
Embryonicstem cell rcsearch,744
DRANCO process,717 production,377
Ethanol-SCP
Embryonicstemcells,447, 477,
Drug deliveryand MAbs,225 744, 482 Ethicsof biotechnology,740
Dry aneaerobiccomposting Endogenous
elicitors,5 14 Ethylenebiosynthesis,
615
process,717
Endomitosis,
544 Eugenics,825
Duchenne'smuscular
Endonucleases,
79 749
Eukaryotes,
dystrophy,179
Endoplasmic
reticulum,75-l cell, 750
Eukaryotic
Dye exclusionassayfor cell
viability,460 &Endotoxins,
597 hosts,82, I37
Eukaryotic
Dye uptakeassayfor cell Energymetabolism,797 Eukaryoticinitiation factors,5l
viability,460 Energy-rich
crops,399 Eutrophication,
665
Dynamiccircularreactors,713 Engerix-8,
202 Evan'sblue staining,505
Dynamic in-vesselcomposting Enhancers,
65 Exogenous
elicitors,514
r eac t or s , 713
phosphate
Enolpyruvylshikimate Expandedbed process,696
Dynamic precipitators,673 synthase,589, 608
Explant,498
Dynamicrectangular
reactors,713 pollution,657
Environmental
Exons,44
polIution-sources,
Environmental
Exonucleases,
79
U
Exopolysaccharides,
382
Environmentalprotection
agencies,
659 Ex situ bioremediation,721
E Environmental
sustainability,
735 Ex-situconservationof
germprasm, 5o5
Enzymeaction-mechanism,793
Earthworms,
714 Ex vivo gene therapy,158
E n z y m ei n h i b i t i o n 7
, 90
Eco--lech,735 Exosome,79
Enzyme-l
inked immunosorbant
Ediblemushrooms,
380 assay,77O Exteins,56
 INDEX
a6t

F r a m e sh iftmutati ons, 34, 47 C ene expressi on,6O, 68, 137

F Freund's adjuvant, 214 C ene gun, 584
F r ie d r e ich 'sataxi a, 180 C ene i nhi bi ti on therapy, 152
Fabric filters, 625 F r o st to le r arrce,612 C ene knockout, 484
Facultative bacteria, 7 54 F r o stb a n ,61 2 C ene l i brari es, 120
F a c u l t a t i v e p o n d s, 6 9 7 F r u cta n s,623 .l
C ene l i brari es-screeni ng, 25
F a m i l i a l h y p e r ch o le ste r o le m ia ,16 4 F r u it d isco lorati on-geneti c C ene l i nkage map, 149
e n g in e eri ng, 61 6
F a t t y a c i d b i o s yn th e sis,8 0 6 C ene rearrangement,67
F r u it r ip e n i ng-geneti c
F a t t y a c i d o x i d atio n , 8 0 4 C ene regul ati on, 59, 67
m a n ip ul ati ons, 614
Fotty acids, 624, 776 Lenes, 59
F u n g a l d iseases(pl ants),604
F e c a t o a c t e i l a , b tJ5 C ene si l enci ng, 593
F u n g i- ch a r acteri sti cs,
756
Fed-batchculture/ C ene synthesi s,111
F u n g i- im p ortance,756
fermentation,261
F u sa r iu m mycoprotei n, 378 C ene taggi ng, 70
F e m a l e i n f e r t i l i t y, 2 3 .1
F u sio n p r o tei ns, 138, 146 C ene therapy, 157
F e r m e n t a t i o n - c la ssifica tio n2,6 4
F u so g e n s,529 C ene therapy,
Fermented foods, 362 .l
A ID S vi rus, 61
F e r m e n t e r ,2 3 9 bone marrow cel l s, 162
F e r m e n t e r p r e c u ltu r e , 2 6 6 for ADA deficiency, 162
for A ID S , 169
F i b r e o p t i c l a c t a te b io se n so r ,3 0 .1
ror cancer, tt,./
F i l t e r p a p e r r a f t -n u r setissu e for hemophi l i a, 164
technique,505 G for hyperchol esterol emi a,164
Finite cell lines, 442 for Lesch-N yhan
F i r e f l y l u c i f e r a s e,5 9 .1 p - Ca la cto sidaseand syndrome, 164
se n e scence,435 vectors, 159
Fischer's template theroy, 794
vi ruses, 159, 165
F i s h e s i n e n v i r o n m e n ta l b io a ssa ys, Ca m e te in trafal l opi an transfer,233
Geneti cal l y engi neered
661 Ca m e to clo nal vari ati ons, 546, 551
mi croorgani sms,668, 724, 726
Fixed bar screen, 687 Ca m e to clo nes, 551
C eneti cal l y engi neered
Fixed-bed rcacIots, 457 Ca n ciclo vir , 168 organi sms, 91 , 480,742
Flag peptide, 147 Ca se o u s a ir pol l utants, 671 C eneti cal l y mani pul ated
F l a v e r S a v r t o m ato , 6 - 1 4 Ca se o u s p o l l utants, 675 oryanisms, 742
F l a v o u r e n h a n c er s,3 6 7 Ca s b io se n sors,663 C eneti cal l y modi fi ed foods, 742
F l o w e r p i g m e n t atio n - g e n e tic Ca se o u s fu el , 395 C eneti cal l y modi fi ed
e n g i n e e r i n g ,6 l6 Ca s o il, 3 7 5 organi sms,480
F l u i d i z e d b e d b i or e a cto r s,2 4 2 Ca so h o l, 3 1 2 Cenetic code, 46
.l
F l u i d i z e d - b e d r e acto r s, 4 5 7 , 6 9 4 Ca u ch e r ' s di sease,634 Cenetic detective, 84
F l u o r e s c e i nd i a c eta te , 5 0 5 Ce l- filtr a tio n chromatography,278 C eneti c di seases,824
F l u o r e s c e n c e - a c tiva te ce
d lI Ce lla n , 3 8 6 C eneti c engi neeri ng gui del i nes, 90
sorler, 228, 434
Ce m in ivir u s es,5S 2 C eneti c engi neer's tool ki t, 76
F lu o r e s c e n c e - a ctiva te dce l I
CEM s in b ioremedi ati on, 226 C eneti c heterogenei ty,546
sorting, 96, 434
Ce n e a m p lifi cati on, 67 Cene transfer methods
Food preservation, 269
Ce n e a u g m entati on therapy, 157 i n pl ants, 573
Food technology, 268
Ce n e b a n ks, 120, 184 Cene transfer-methods, 86
F o o t a n d m o u t h d ise a se ,2 0 2
Ce n e ch ip , 1 76 C ene-transgeni cbul l cal f, 491
F o r t i f i e d w i n e s , 37 0
Ce n e clo n ing strategi es,89 C eneti c i mmuni zati on, 205
Fossilfuels, 393
Ce n e d e livery C eneti c i mprovement of mi crobi al
F o u r - s t r a n d e dD NA, 1 7 stratns, 257
b y n o n - vi ral systems,166
F r a g i l e X s y n d r o me , 1 8 0 b y vir u s es, 165 Cenetic portfolios, 743
862 BIOTECHNOLOCY

Ceneticrecombination,
259 C o l d e n R i c e2 , 6 1 9 -l84
Hemochromatosis,
Cenetics,822 751
Colgi apparatus, H e m o p h i l i a1, 6 4 , 1 9 4
Cenom e,38, 59 hormone,228
Conadotrophic HepatitisB, 201
149
Cenomesequencing, Cram-negativebacteria,754 HepatitisB vaccine,2O2
Cenom iclibr ar ies120
, Cram-positivebacteria,754 HepatitisB
Cenomesof organisms,I53 Cranularactivatedcarbon surfaceantigen,201

Cenomics,38, 827 contactors,699 Herbicideresistance,

607
CRASorganism,140 genes,589
Herbicideresistance
Cenotoxicityrating,659
Cratuitousinducers,60 Herbicideresistancemarkers,589
Ceometricalisomerism,
761
Cravitysettlingchambers,673 plants,610
Herbicide-resistant
Cerm cell genetherapy,I57
protein,591
Creenfluorescent 607
Herbicides,
565
Cermplasmconservation,
Creen houseeftect,664,73O Hereditarynonpolyposis
colon
Cermplasmstorage-
cancer,37,182
applic at ions , 5Tl Creen houseeffect-measures
to control,732 Heredity,822
Cibberellins,361, 521
Creen housegases,730 Herpessimplexvirus,166, 203
Clauc om a,183
Creen manuring,646 Heterocyclicrings,758
Clobal warming,664, 730
Creen petrol,3l l DNA, 32
Heteroduplex
Clucanase,
605
612, 652, 722
Creen Revolution, bacteria,325
Heterofermentative
630, 634
Clucocerebrosidase,
653
Creenhometechnology, nuclearRNA,21, 43
Heterogeneous
Cluconeogenesis,
800
Creenhouse 653
technology, 531
Heterokaryons,
Cluconic acid,324
Criseofulvin,34l 775
Heteropolysaccharides,
Clucosebiosensors,
299, 304
Crit chambers,687 Heterotrophicbacteria,755
Clucoseisomerase, 296
Cround water pollution,680 alleles,823
Heterozygous
p- Cluc ur onidas 59l,
e, 631, 633
Growth kineticsof lag, 146
Hexahistidine
Clufosinate,
609
microorganisms,263 Hexopofysaccharide, 382
Clutamate,B.l1
Cynogenesis,
542 Hexosemonophosphateshunt, 800
Clutamic acid,346
Cynogenichaploids,542 High-erucicacid rape,625
Clutamine,81.1
corn syrup/296, 367
High-fructose
Clut ein,365
High-fructosesyrup,296
Clyceraldehyde, 761, 773
Iiquid
High-performance
Cly c er ol,316
chromatography,767
Clycine,809 H
High-amylose starch,623
Clycine betaine,611 H i r u d i n ,'1 3 5 ,6 3 1
G I y c inin,618 Hairy root culture,512
Histoneacetylation,64
Clycogen,774,8OO Hairy root disease,577
Histotypic cultures, 47O
Clycogenesis,
800 Haploid plants,538
HMP shunt,800
Clycogenmetabolism,
800 Haploid plants-applications,
544
Hognessbox, 42
Clycogenolysis,
800 Haploidplants-identification,
543
Hollidaymodel,31
Clycolysis,798 Haploidplants-limitations,
545 Homocyclicrings,758
Clycosides,773 of microbialcells,271
Harvesting bacteria,325
Homofermentative
Clyoxylatecycle,803 HAT medium,214, 483 Homokaryons,
529
Clyoxysomes,
752 Heap leaching,402 Homologousrecombination,
31
Clyphosate,
345 Heat shockproteins,55 Homologydependentgene
Clyphosateresistance,
607 a-Helix,782 silencing,593
Cobar gas,396 moril,67
Helix-foop-helix Homopolymertailing,78
Cobar gas plant,397 motif, 65
Helix-turn-helix Homopofysaccharides,
774
Colden Ric e,618 Hematopoietic 221
malignancies, Homozygousalleles,83
 INDEX
863

H o m o zyg o u s lin e s, 5 3 8 l ndustri al fermentdti on-

Hops, 369 substrates,249
H o s t ce lls fo r clo n in g , 8 1 Industri al pol l utants, 670
H o s t c e lls fo r g e n e Inferti l i ty,23l
lce-mi nus bacteri a, 6i 2
e x p r e ssio n , 13 7 l ni ti ati ng codons,46
lce-nucl eati ng bacteri a, 61 j
H u m a n a r tificia l ch r o m o so m e , 8 6 , Innate i mmuni ty, 826
lC l pressurecycl e fermenter, 3Zl
1 6 2 ,1 6 7 Inorgani c ai r pol l utants, 670
lC l pruteen,3/7
H u m a n clo n in g , 7 4 4
l norgani c w ater pol l utants, 6B O
r oropnase /55
H u m a n ES ce ll r e se a r ch ,4 7 7 , 7 4 4 Insect cel l expressi onsystems,.1 41
L -l duroni dase,634
H u m a n g e n e th e r a p y,7 4 3 Insect resi stance,-597
lmhoff tanks, Z0Z
H u m a n g e n e th e r a p y tr ia ls, 1 - 5 9 Insect resi stanceto B t crops, 599
lmrdazol i nones,610
H u m a n g e n o m e a n d e th ics, 1 5 4 Insecti ci dal crystal l i ne protei n, 597
lmmobi l i zati on of arti fi ci al
H u m a n g e n o m e - h ig h lig h ts,l+ 9 /n sl fu bi ol eachi ng, 4O2
cel l s,305
H u m a n g e n o m e - m a p p in g , 1 4 9 /n si tu bi oremedi al i on, 720
lmmobi l i zati on of
H u m a n g e n o m e - o r g a n iza tio n ,15 1 enzymes and cel l s, 288 /n si tu conservati on of
.l4 8 germptasm, 5b5
H u m a n g e n o m e p r o je ct, lmmobi l i zati on-methods,288
H u m a n g e n o m e - se q u e n ce ,1 4 9 Irt situ sterilization, 245
lmmobi l i zed cel l cui tures
.l
(pl ants),51 1 Insul i n, 34, 189
H u m a n g e n o m e - sid e lig h ts,i 5 1
lm mobi l i zed cel l s and Insul i n l i spro, 'l 92
H u m a n g r o wth h o r m o n e , 1 9 2
appl i cati ons, 295 lntegrarv, 476
H u m a n im m u n o d e ficie n cy
v f f u s, | / / lm mobi l i zed cel l s i n pol l utron Intei ns,56
management, 667 Intei n spl i ci ng, 56
H u m a n le u ko cyte a n tig e n , 8 2 0
lm mobi l i zed enzyme reactors,294 Intel l ectual property ri ghts, 744
H u m a n m o n o clo n a l
a n t ib o d ie s, 2 1 6 Im mobi l i zed enzymes- l nterferon, 134, 196
appl i cati ons,295
H u m a n m o u se , 4 8 5 Intergeni c D N A , I52
lm mortal i zati on of cel l s, 463
H u m a n p a p illo m a vir u s, 1 2 8 Interl euki n, S 20
lm mune response,820
H u m a n p r o te in r e p la ce m e n ts,1 8 9 Intermediary metabolism, 797
lm mune system, 816
H u m a n ize d a n tib o d y, 2 1 7 Internal ri bosomal entry si te, 144
lm munoaffi ni ty puri fi cati on, 14Z
H u m a t r o p e , I9 3 Internati onal human genome
Im munobi osensors,303 sequenctng consortrum, I48
H u m o r a l im m u n ity, 8 1 7
lm munogl obul i ns, 817 Intracytopl asmi csperm
H u m u l i n, 1 9 1
lm munol ogy, 816 injection, 234
H u n t i n g to n ' s d ise a se , 1 8 0 Intrauteri ne i nsemi nati on, 232
lm munosci nti graphy,220
hup genes, 644 Intravagi nalcul ture, 234
lm munosuppressi on,222
H y b r i d o m a ce lls, 2 .13
lm munotoxi ns, 223 - l ntron, 'l 97
H y b r i d p la n t ce lls, 5 3 1 , 5 3 1 Introns,44
lm munoel ectrophoresi s,762
H y b r i d pr o te in , 7 .1 Introns and gene expressi on,593
IM Vi C test, 685
H y d r o g en io n ,7 6 2 Inverse P C R ,-l 15
In dex organi sms of sew age, 684
H y d r o g e n a se ,6 4 ' l In vivo ferilization, 229, 23O
In di go, 310, 392
H y d r o g e n a se g e n e , 6 4 4 In duced embryogeni c ln vivo gene therapy, 158, 164
H y d r o p h o b ic in te r a ctio n determi ned cel l s,558 l on-exchange chromatography,229
c h r om a to g r a p h y,2 T g f nduced fit theory, 794 l on-sel ecti vefi el d effect
nyorospnere/ b5l In d uci bl e genes, 59 transi stors,300
H y g r o m ycin p h o sp h o - In d uci bl e promoters, 624 Iron bi ndi ng pepti des, 644
t r a n s fe r a se ,5 8 8 l rreversi bl ei nhi bi ti on, 791
In d ustri al enzymes-producti oni n
H y p o a l bu m in e m ia , 7 6 4 pl ants, 631 Isoel ectri cfocussi ng, Z6Z
H y p o x a n th in e g u a n in e In d ustri al enzymes-sources l soenzymes,795
p h o sp h o r ib o r yltr a n sfe r a se2,' l 4 and appl i cati ons, 284 tsomefl sm, /56
 BIOTECHNOLOCY
864

Leachate,715 L y o p h i l i cc o l l o i d s ,7 6 3
lsomers,758
786 Leafprimordia,498 Lyophobiccolloids,763
lsoprenoids,
95 Leber'shereditaryoptic Lysine,350
lsopycniccentrifugation,
neuropathy/ 5B Lysine-export,351
lsotopes,765
Lectins,60.1 Lysosomalenzymes,633
Itaconic acid, 328
641
Leghemoglobin, 751
Lysosomes,
cr-L-lduronidase,634
L e i s h m a n i a s i2s1, 0 L y s o z y m e2,7 5 , 6 0 5
Lesch-NyhansYndrome, 164 B-Lymphocytes, 817
Leucinezipper motif,66 817
T-Lymphocytes,
L e u k e m i a1, 3 5
J 753
Leucoplasts,
761, 773
Levorotatory,

.lunkDNA, 152 Lewy bodies,183

Libefty,609
M
Lignocellulose,393
223
MAbs as imn-runotoxins,
Plot, 788
Lineweaver-Burk
225
MAbs in proteinPurification,
Linkageanalysis,652
K Linkers,TB Macrolides,340

Lipids, 775 Major histocomPatibility

c o m p l e x ,8 1 9
528
Karyoplasts, 88
Lipofection,
.105 Malaria-diagnosis, 177
Klenowsuburrit, Lipofuscin,752
Male infertilitY, 231
K, value,788 Lipolase,287
.l Male sterility (plants)-genetic
Knockoutmouse,486 Lipoplexes,67 e n g i n e e r i n g6,17
Knockoutpigs,492 Lipoproteins,777 Mammaliancell exPression
Kogenate,194 L i p o s o m e s1,6 7 , 2 2 5 vectors,143
model,794
Koshland's Liposomefusion,586 Maniatistechnique,12.1
Kozakconsensus 51
sequence, Liposome-mediatedgene Manipulationof gene
transfer,B8 expression, l3B
Liposome-mediated Markergenesfor Plant
586
transformation, 588
transformation,
in drug deliverY,225
Liposomes transgenic
Marker-free plints, SOO
L 658
Lithosphere, MatureembrYoculture,562
l-ockand key model,794 Maxam and Cilberttechnique,101
elements,33
Long intersPersed Media for industrial
Lac operon, 60
derepression, 60 L o n gP C R ,1 2 3 fermentation, 248
repression, 60 367
Low caloriesweeteners, Medicalethicsand culture
B-Lactamtechnology,336 techniques,44l
Low-erucicacid raPe,625
371
Lactasein dairYindustrY, Low-oxygenstorage,570 Membranefillers,272
Lacticacid, 325 storage,570
Low-pressure Membranefiltration
technique,685
Lactobacillus,325 Luciferase,177, 3O2
Membranereactors, 295
Lactoseintolerance,371 Luminescence testfor cell
822
Mendel'sexPeriments,
Lagooning, 716 viability,461
Luminescent 302
biosensors, Meningitis,203
715, 717
Landf illing,
Lux reportergenes,662 Menkesdisease,184
Landfillsites,397
Meristemculture,553
728
Landfarming, Lymedisease,178
culture,560
Meristem-tip
Latticeentrapment,2BB Lymphoidorgans,8.17
.l Meselson-Raddingmodel,3i
Leachliquor,402 Lynch syndrome, 82
 INDEX 865

Messenger
RNA, 21, 44, 96 Microbialenzymeproduction- Mitochondria,650
Metabolic assaysfor cel regulation,286 MitochondrialDNA, 58
viab ility,4 62 Microbialleaching,400 Molality,763
Metabolicdrain/burden,
139 M ic r obia ll i p i d s ,3 9 1 Molarity,763
of
Metabolicengineering Microbialmetabolicproducts,254 Mofasses,
249, 314
622
carbohydrates, M ic r obia lm i n i n g ,4 0 0 Molds,756
Metabolicengineering
of Microbialproductionof amino Molecularbreeding,652
lipids, 624 acids, 344
Molecular farming,622
Metabolicengineering
of Microbialproductionof
plant
Molecularmarker-aided
plants,622 antibiotics,329
breeding,648
Metabolicengineering
of Microbialproductionof foodsand
Molecularmarkerassisted
proteins,628 beverages,362
selection,652
lvletabolicload, 139 Microbialproductionof
Molecularmarkers,648
Metabolicpathways,796 indigo ,3 9 2
Molecularpharming,622
Metabolicpathways- Microbialproductionof organic
ac ids ,3 18 M o n e l l i n ,3 6 7 , 6 1 8
integration,
813
Microbialproductionof Monocistronic
mRNA,50
Metabolicpathways-
v it am i n s , 3 5 5 214
Monoclonalantibodies,
re gu latio n,
815
Microbialrecoveryof Monoclonalantibodies-
Metabolismof amino acids,808
petroleum,403 a p p l i c a t i o n s2,1 9
798
Metabolismof carbohydrates,
Microbialrubber,391 Monogenicdisorders,
823
806
Metabolismof cholesterol,
slime,692
Microbiological Monolayercultures,455
Metabolismof lipids,804 pollutants,680
Microbiological Monopartiteviruses,682
Metabolisms,
796 Microcarrierculture,456 Monoploids,538
Metabolite.796 technique,505
Microchamber Monosaccharid
es, 773
Metabolomics,
38 Microdrop method, 506 Monosodiumglutamate,367
Metacleavagepathway,722 289
Microencapsulation, Moondust,617
Metal biotechnology,400 Microfluidizer, 274 MTT-basedcytotoxic assays,462
Metallothioneins,
667 89, 48"1,586
Microinjection, arides,775
Mucopolysacch
Metal pollution-managment,
566 Microinjectionof DNA, 586 Multicellulartumor spheroids,
471
Metal scavenging,66T 234
Micromanipulation, Multichambercentrifuge,273
Methane,396 test,662
Micronucleus Multipartiteviruses,682
production,375
Methane-SCP Microorganisms as oil Multiplefixed bed adsorber675
Methanogenesis,
496, 695 f ac t o r i e s , 3 9 l M u l t i p l em y e l o m a 2
, 13
production.377
Methanol-SCP Microorganisms-growth Multipleovulation,228
kinetics,263
Methanogenic
bacteria,696 Multipleovulationwith
Microorgan 252
isms-isolation, embryo transfer,229
Methylationof DNA, 64
Micioprojectile bombardment,584 Multiple-tubefermentation
constant,7BB
Michaelis-Menten
584
Microprojectiles, technique,684
Microalgalphotosynthesis,
664
Micropropagation,
552 Multisurface
culture,456
Microarrays,
1O8,176
559
Micropropagation-applications, Murine leukemiaviruses,161
Microbialadhesive
Microprotoplasts,
528 Mushrooms,380
biopolymer,392
markergenes,182
Microsatellite Mutations,34, 258
Microbialbiofilm,694
1BB,652
Microsatellites, Muteins,192, 198
Microbialculturesystems,
259
Microsporeculture,539 Myocardialinfarction,195
Microbialenhancedoil
recovery,382
528
Miniprotoplasts, Mycophage,'l 77
Mismatch repair,37 Mycoprotein,379
Microbial enzyme production-
geneticengineering,286 Missensemutations,35 Mycolhizas, 646

Biotechnology [55]
866 BIOTECHNOLOCY

Ovariancancer,236
N o Ovarianhyperstimulation
syndrome,236
Ovary culture,542
Na r b o m ycin , 3 .10 Obesitygene,183
Ovule culture,542
NASA bioreactor, 455 Octopines,575
Oxidationditch, 706
Na tu r a l cu ltu r e m e d ia , 4 1 5 Okazakipieces,25
p-Oxidationof fatty acids,804
Ne m a to d e r e sista n ce ,6 0 6 3
Old biotechnology,
Ne o m ycin p h o sp h o tr a n sfe r a se5, 8 8
Ozone,732
63.1
Oleosinpartitiontechnology,
Ne ste d PCR, 1 1 5 Ozone depletion,733
O l e o s i n - h i r u d ifnu s i o n
nif genes, 642 protein,63.l Ozone hole, 733
NIH g u id e lin e s, 9 0 Oleosins,624, 631
Nitr ifica tio n , 6 9 2 , 7 OO Oligomers,783
Nitr ifyin g b a cte r ia , 7 5 6 rected
Ol igonucleotide-di
Nitr o g e n cycle , 6 3 9 130
mutagenesis,
Nitr o g e n fixin g b a cte r ia , 6 3 9 TT2
Oligosaccharides,
P
Nitrogen-fixing genes, 642 Oligospermia,23l
.l
Nitr o g e n a se ,6 4 l chip, i-08
Oligonucleotide ps3 gene, 8.1
nod genes, 644 Oncomouse,486 Packed bed bioreaclors, 242
No d u la tio n g e n e s, 6 4 4 Oocyte recovery,230 P acked cel l vol ume, 505
No n - a u to lo g o u s ce lls, 16 4 Oocyteretrieval,233 P acked tow ers, 677
No n - co m p e titive in h ib itio n , 7 9 0 Operon concept,60 Packed-bed reactors, 694
No n - fe ca l b a cte r ia , 6 8 5 P aki l o,378
Opines,574, 591
No n - h o m o lo g o u s r e co m b in a tio n ,3 2 Palatability of foods-genetic
591
Opine synthase,
No n - m e th a n o g e n ic b a cte r ia , 6 9 6 engi neeri ng, 6'l 8
30-l
Optical biosensors,
No n se n se m u ta tio n s, 3 5 P arasi tes,755
761
Optical isomerism,
No p a lin e , 5 7 5 P arki nson'sdi sease, 183
Optical isomersof amino
No r m a lity, 7 6 3 P arti cl e bombardment, 58 4
acids,778
No r th e r n b lo Vb lo ttin g , 5 9 , 9 9
791
Opticalspecificity, P arti cl e B un, 584
Nuclear transfer, 492
Organicacids-microbial P arti cul ateai r pol l utants, 670
Nuclease protection assay, 69 production,3lB P atenting bi otechnol ogy
Nu cle a se Sl m a p p in g , 6 9 Organcultures,468 i nventi ons, 744
Nu cle a se s,7 9 222, 821 Pathogenesis-relatedproteins, 605
Organtransplantation,
Nu cle ic a cid b lo ttin g
Organicair pollutants,
620 P athogeni corgani sms i n
te ch n iq u e , 9 7
Organic fertilizers,647 sewage, 684
Nu cle ic a cid h yb r id iza tio n , - 1 7 3
Organicslurries,708 pB R 322 pl asmi d, 83
Nu cle ic a cid p u r ifica tio n , 9 4
Organicsolvents-microbial P C R -ampl i fi edol i gonucl e oti de-
Nu cle ic a cid th e r a p y, 1 7 0 di rected mutagenesi s,132
- l.l production,3.11
Nu cle ic a cid s- co m p o n e n ts,
Organicwater pollutants,680 P C R -appl i cati ons,l 19
Nu cle ic a cid s- fu n ctio n s,11
553, 556
Organogenesis, P ecti n methyl transferase,6.13
Nu cle o lu s, 7 5 0
irect,556
Organogenesis-d Pekilo, 378
Nu cle o p la sm , 7 5 0
ndirect,557
Organogenesis-i P eni ci l l i ns,33l
Nu cle o sid e a n tib io tics, 3 4 2
Organotypiccullures,472 Peptide bond, 78.l
Nu cle o so m e , 1 9
Orthocleavagepalhway,722 P epti de bond formati on, 53
Nu cle o tid e e xcisio n r e p a ir , 3 6
611
Osmoprotectants, P epti de vacci nes, 2O2
Nu cle o tid e s, i.l
Nu cle u s, 7 5 0 Osmosis,764 Peptidyltransferase,53

Nunc cefl factory, 456 Osmoticpressure/

527, 764 Perfused monolayer cullure, 457

Nu n clo n ce ll fa cto r y, 4 5 6 O s m o t i c u m5, 2 7 P eri odontal di sease, 178

INI,EX 867

anemia,355
Pernicious Plantcell culture,502 mRNA,50, 60
Polycistronic
752 biotransformation, 516 P o l y c l o n aal n t i b o d i e s2, 1 3
Peroxisomes,
measurement of growth,505
Perstraction,277 Polyenemacrolides, 340
measurement of viability,505
Petvaporation,277 secondarymetabolites, 507 P o l y e t h y l e ngel y c o l - m e d i a t e d '
suspension cultures,502 transformation,587
Pestresistance,599
s y nc h r o n i z a t i o n , 5 0 4 6.13
Polygalacturonase,
Petroleumplants,399
Plantcell culturesand 823
Polygenicdisorders,
Petroselenicacid, 626 5.16
biotransformation, P o l y h a p l o i d s5,3 8
pH, 762
Plantc ell - m q s cs u l t i v a t i o n5, 1 1 Polyhedrin g e n e ,1 4 1
Phagedisplay,70 752
Plantcefl-structure, kanoate
Polvhydroxyal
PhageM,., 84 Plantgrowthregulators,513,520 627
co-polymers,
Phagemiddisplay,72 Planttissueculturemedia,517 386, 626
Polyhydroxyalkanoates,
PhageI, 83 Planttissueculture- tyate, 387, 626
Polyhydroxybu
Pharmanimals,490 appli c a t i o n s , 5 0 6 Polyketide
antibiotics,339
Phasidvectors,84 Planttissueculture-basic chain reaction,112
Polymerase
Phe nylala ni ne,
353, 809 t ec hn i q u e , 4 9 9
Polymerases,
81
497
Planttissueculture-benefits,
Pho sp ha te
so lubiliz ing Polyphenoloxidase,616
bacteria,646 499
Planttissueculture-history,
Polyribosome,
49
Phosphatestripper,702 Pfantvariety tights,746
382, 774
Polysaccharides,
Phosphinothricin,
609 Plant ibo d i e s , 6 3 4
Pofyunsaturatedlatty acids, 776
acetyl-
Phosphinothricin 628
Plantsas bioreactors,
Pomatoes,533, 536
transferase,
589 Plantsas proteinfactories,628
Pond treatmentprocesses,
696
609
resistance,
Phosphinothricin 596
Planttransformation,
Populationdoublingsof cells,435
Phospholipids,
776 572
Plant t!'ansgenesis,
Positioneffects,490
method,109
Phosphoramidite Plasmidcopy number,95
linkage,l 71
Phoslhorothioate
pollution,665
Phosphorus PlasmidDNA purification,95
Post-harvest
spoilage,615
Phostrip, 7O2 Plas m id s8,3
gene
Post-transcriptional
242, 664
Photobioreactors, 753
Plastids, silencing,593
Photometry,767 Plastome,
594 ptional
Post-transcri
394, 664, BO3
Photosynthesis, Platetowers, 677 modifications,43
Plaquelift technique,127 Post-translational 56
modifications,
bacteria,398
Photosynthetic
Platingefficiency,466 Potentiometricbiosensors,299
Photosystems,
803
p-Pleatedsheet,783 Powderedactivatedcarbon, 699
Physicalgene(plant)'transfer,
583
262
Plugflow bioreactors, Pre-embryonicdetermined
Phytase,633
Plug-flowin-vessel
composting cells,558
514, 606
Phytoalexins,
reactors,113 genetic
Preimplantation
661, 667
Phytochelatins, 236
diagnosis,
Pluropotency,490
520
Phytohormones, Point mutations,34 treatmentof
Preliminary
Phytopathogens,
644 sewage,687
Pollenculture,539
666
Phytoplanktons, fermenter,24.1
Pressure-cycle
Pollulan 3
, 86
T28
Phytoremediation, Pollutants,659, 669 Pribnorvbox, 41
Piezoelectric 302
biosensors, Pollutioninducedpeptides,661 Primaryair pollutants,
670
Pig in organfarms,489 Pollut io nm o n i t o r i n g6, 5 9 Primarycell culture,437
785
Pigments, Polly,49'l Primaryexplanttechnique,441

Pituitarydwarfism,192 Primarylymphoidorgans,B-l7
Poly-Atail, 44
254
Primarymetabolism,
Plant breeders'ti1hrs,746 167
Poly-L-lysine,
gel electro- 254
Primarymetabolites,
498,
Plantbreeding-conventional, Polyacrylamide
) / z^ phoresis,93 of
Primarystructure protein,781
a68 BIOTECHNOLOCY

Primarytranscript,39, 43 718
Pseudomonas, tPA, 195
Recombinant
Primarytreatmentof sewage,688 Pseudomonassyringae,612, 742 vaccines,199
Recombinant
Primase,24 8',,
Pseudomonas-vitamin vacciniaviruses,211
Recombinant
production,357 194
Recombinate,
Prematuremenopause,236
Primaryexplanttechnique,441 Pulsed-fieldgel electrophoresis,
93
Recombinationof DNA, 31
Primerextension,69 Pump and treat technique,728 31
Recombination,
Primerextensiontechnique,104 P u r i n e sl,l
Recombivax,202
11
Pyrimidines, (plant cells),499
Primers,24, 114 Redifferentiation
107
Pyrosequencing,
Primerwalking,104 Regulatablepromoters,138
Prions,56 synthesis,327
Reichstein-Crussner
Procrit,198 Rennet,287, 364
Prograrnmedceli death, 435 Replicaplating,126
Prokaryotes,749 a bubble,24
Replication
Prokaryotichosts,82, 137 Replicationfork, 25
Promotersof transcription,592 Replicationin eukaryotes,27
Quantitativetrait loci, 652
310
Prostaglandins,
Quaternarystructureof Replicationin prokaryotes,24
Prostatemouse, 487 protein, 783 33
transposition,
Replicative
Proteaseinhibitors,601 Quaylecycle,376 Reportergenes,590
ProteinDNA interactions,65 553
Quiescentmeristems, 76, 136
endonucleases,
Restriction
133
Proteinengineering, Quinoid pigment,785 Restrictionenzymes,76
Proteinengineering-disulfide Quorn, 378
bonds , 133 Restrictionfragmentlength
polymorphisms, 185, 649
ProteinenBineering-
asparagine,
l33 Reteplase,196
57
Proteinlocalization, 182
Retirroblastoma,
32
Retrotransposition,
Proteinproductionin plants,628 ll
Proteinpurification-use
of Retroviralvector method,481
MAbs, 243 Retrovirusvector system,165
R A C E ,1 1 8 , 1 2 3 , 7 2 1
Proteinsorting,57 46,757
Retroviruses,
Radioactiveisotopes,765
Proteintargeting,57 Reversemiceller systems,277
pollutants,
Radioactive 681
Proteinase
inhibitors,601 46, 115
Reversetranscriptase,
769
Radioimmunoassay,
ProIeins,777 Reversetranscription,46
225
Radioimmunotherapy,
783
Proteins-classification, Reverse PCR,115,
transcription
Randomamplifiedpolymorphic
781
Proteins-structure, 125
DNA, I16
P_roteome,
38 of cDNA
Rapidamplification Rheologicalproperties,383
Proteomics/
38 ends,70, 1'lB Rheology,382
Protocatechuate,
722 Real-timequantitative
PCR,116 Rho dependenttermination,41
Protoclonal
variations,
546 Recalcitrant 720
xenobiotics, termination/42,
Rho independent
Protoplastcoculture,527 Recemicmixture,761 123
Protopfastculture, 523, 526 DNA Advisory
Recombinant Rhodopsinknockout mouse,487
Protoplast
fusion,529 Committee,90, I58 Ri plasmids,577
isolation,523
Protoplast DNA, 75
Recombinant Riboflavin,357
Protoplastregeneration/527 Recombinant
toods,742 P, 22
Ribonuclease
Protropin,193 hCH, 192
Recombinant RibosomalRNA, 22, 45
ProvitaminA, 618 insulin,191
Recombinant nactivating
Ribosome-i
A enrichedrice, 618
Provitarnin Recombinant 197
interferons, proteins,605
Pseudobactin,
645 proteins,144
Recombinant Ribosomes,49
 INDEX 869

Ribostamycin,
310 SCPfrom methane,375 Shikimicacid pathway,608
Ribozymes,
22, 53, 172, 604 SCPfrom methanol,377 Shine-Dalgarno sequence,
51
Rice actin promoters,592 SCPfrom sewage,379 Shoottip culture,553
Ricin,2 23 SCPfrom wastes,378 Short interspersed nuclearelements
Risksof biotechnology,740 SCPfrom wood, 379 ( S l N E s )3,3
RNA plant virusesas vectors,582 Screeners,
687 Shotgunapproach,120
RNA polymerases,
42 Scrollcentrifuge,
273 Shreddar,'687
RNA primer,24 air pollutants,670
Secondary Shuftlevectors,86
RNA structure,19 Sickle-cellanemia,48, 179
Secondarymetabolism,254
type s.21 Siderophores,
644
Secondarymetabolites,254
RNA vaccines,207 Signalpeptide,57, 146
Secondarymetabolites-el icitor
Roller bottle culture,456 in d u c t i o n5, 1 4 Silentmutation,35
Rotarydrum vaccum filler, 271 Secondarystructureof Siliconcarbidefibres,587
Rotatingbiologicalcontactors,
693 protein, 782 Siliconcarbidemediated
Secondarymetabolites(of plant transformation,
587
Roughingfilters,693
cultures),507 Simplecopy segments
of
Roundspermidnucleus
Secondarytreatmentof genomes,65l
injection,235
sewage,689 Simplesequencelength
Round up, 608
SecretorylgA, 634 polymorphisms,
651
Rubberplant,399
Sedimentation
coefficient,768 Simpletandemrepeats,1BB
Rubisco,626
Sedimentation
tanks, 688 Singlecell clones(of plants),505
Single-cell
oils, 391
Seedterminatortechnology,621
Singlenode culture,554
Selectablemarkergenes,588
Singlenucleotide
Selectivebreedingmethods,480
s Semi-aridplant biotechnology,
653
p o l y m o r p h i s m s1,5 1 , 1 7 9 , 1 8 8
S i n g l e - c eol li l s , 3 9 1
Semi-aridregion,653
protein,373
Single-cell
Saccharin,
367 Semi-continuous
culture/
protein-use
Single-cell of
Saccharification,
368 fermentation,
261
microorganisms,374
Saccharomycescerevisiae,137, Senescence,
31, 435 Sisterchromatidexchange,652
140, 365, 368 Senescence
and cancer,3 l Site-directedmutagenesis,
129
Safeagriculturalpractices,602 Sept a g e , 7 1 6 Skimmingtanks,687
Salmonella, 210 Septagedisposal,716 Slopeleaching,402
Salvagepathway,2'14 Septictanks,707 Sludge,708
SatelliteRNAs,603 Sequencecharacterizedamplified Sludgeconcentration
Sauekraut,365 regions,652 (thickening),
709
Scaffoldaftachmentregions,593 Sequencetaggedsites,651 Sludgeconditioning,7'l4
Scale-up,453 Sequencingbatch reactor,691 Sludgedisinfection,
714
Scale-upin monolayer,
455 Serum-free
culturemedia,421 S l u d g ed i s p o s a l7, 1 5
Scale-upin suspension,
453 Severecombined Sludgestabilization,
710
Scale-upof fermentation,268 im m u n o d e f i c i e n c y6, 2
'l , 2 1 7
Slurry-phase
bioreactors,
728
Scale-upmonitoringof cell Sewage,682,686 Small sewagetreatment
growth, 458 Sewagesludge,708 systems,706
Scleroglucan,
385 Sewagetreatment,686 Solidphasesoil reactor,728
Schistosomiasis,
493 Sex chromosomes,823 Solid substrate(state)
SCID mouse,217, 487 Sex-linkedinheritance,
824 fermentation, 247
SCPfrom alkanes,375 Shelf-lifeof fruits-extend.O, S o l u b l eR N A , 2 1
Ur,
SCPfrom COz, 379 :. _
ot) >o t u u o n s , . / b 2
870 BIOTECHNOLOCY

S o m a clo n a l va r ia n ts, 5 4 7 Streptomyces-Benetic S yntheti c vacci ne,202

342
manipulations,
S o m a clo n a l va r ia tio n s, 5 4 6 1B 3
C X ,-S ynucl ei n,
S o ma clo n a l va r ia tio n s-
Streptomycin,
337
ap p lica tio n s,5 4 9 Stroma,753
S o ma clo n a l va r ia tio n s- 760
Structuralisornerism,
l im ita tio n s,5 5 0 StufferDNA, 84
S o ma clo n e s,5 4 7 528
Sub-protoplasts,
T
S o m a tic ce ll g e n e th e r a p y, 1 5 7 Subcellular 768
organelles,
S o m a tic e m b r yo g e n e sis,5 5 3 , 5 5 7 Subculture,445 T-DNA,575
S o m a tic e m b r yo s, 5 5 8 monolayercultures,445 T o l y s o z y m e1, 3 3 , 1 3 5
cultures,446
suspension 281
Takadiastase,
S o m a tic h vb r id p la n ts, 5 3 2
Substratespecificitv, 792 Tandemgenearrays,138
S o m a tic h yb r id iza tio n , 5 2 8
straintheory,794
Substrate TATAbox, 42
S o m a tic h yb r id iza tio n -
ap p lica tio n s, 5 3 6 S u b t i l i s i n1,3 5 Tautomericforms,12
S o m a tic h yb r id iza tio n - im
l ita tio n s, Subunitvaccines,201 30
1'elomerase,
537 Subzonalinsertion,235 Telomere,30
Sorghum beei', 370 Svedbergunit,768 codons,46, 53
Termination
Sour-doughbread, 365 water
Sugarindustry-waste Terminators of transcription,624
S o u th e r n b lo Vb lo ttin g , 6 9 , 9 8 treatment,705
Tertiarystructureof protein, 783
S p a wn , 3 8 1 Suicidegenetherapy,168
Tertiarytreatmentof sewage,698
Spectrophotom eter, 7 67 S u i c i d ei n h i b i t i o n 7
, 91
Testtube babies,233
S p er m se xin g , 2 2 8 S u l f o n y l u r e a6s1, 0
Tetrac),clines,33B
S p he r o id s,4 T l Sulfonylureas 610
resistance,
45
B-Thalassemia,
S p i n o ce r e b r a la ta xia , 1 8 0
S u l f u ra m i n o a c i d s ,8 1 1
Thaumatin,367
Spirulina SCP, 379
Superbug,
725
oxygendemand,683.
Theoretical
S p l i ce o so m e ,4 4
Supercoils,26
300
Thermalbiosensors,
Sporophytes, 755
Supermouse,481
Thermal incinerator,677
S p r ay to we r s, 6 7 3 , 6 7 7
fluid extraction,276
Supercritical
297
Thermistors,
Superovulation,22S
S p r i n klin g filte r s, 6 9 2 300
biosensors,
Thermometric
18'l
dismutase,
Superoxide
S t a b ility o f p r o te in s, 1 3 9 ELISA,301
Thermometric
Surfacecondense,671
Starch, 622, 774 of plants,560
Thermotherapy
Surfaceimmobilizedplant cell
S t e m ce ll cu ltu r e s, 4 4 7 Thiobacillus ferroxidans, 402
t e c h n i q u e5, 1 1
S t e m ce ils, 4 4 7 T h i o n i n s6
, 06
Surfacetension,765
Stem cells-charactetization, 448 Threedimensionalcultures,47-1
Suspensioncloning,467
S t e re o iso m e r ism ,7 6 0 Threonine,352
Suspension cultures .l
Stereospecificity, 791 (of plants),502 Thrombosis,95
S t e r iliza tio no f b io r e a cto r , 2 4 5 cultures,446
Suspension T h y l a k o i d sT, 5 3
S t e r iliza tio n o f cu ltu r e m e d ia icrobialproduction,U., T i p l a s m i d 7
Sweeteners-m , 0, 574
( o f fe r m e n ta tio n ) ,2 5 0
Tissueengineered peripheralnerve
>terotost / / / Symbaprocess,378 i m p l a n t s4, 7 6
S t i cky e n d s, 7 8 Symbaproduct,378 Tissueengineered skin,475
Stirred tank reactors, 240 Symbioticnitrogenfixers,646 urothelium,476
Tissueengineered
S t i r r e r cu ltu r e , 4 5 3 Symbioticnitrogenfixing,640 472
Tissueengineering,
S t i r r e r fla sk, 4 5 3 Symmetrichybrids,534 Tissuemodelling,477
Stratagene, 'l 23 Synchron ization Tissueplasminogen
activator,135,
S t r e p to kin a se ;19 6 (of plant cultures),504 194
 471

geneexpression,
Tissue-specific 65 method, 482
TK suic idegene,169 for human di seases,487
mi croi nj ecti on method, 481
u
To mat or ipening,613 retroviral vector method, 481
Topatoes,533 Transgeni cmosqui toes,493 Ubiquitinpromoter,592
Totalorganiccarbon,683 Transgeni cpi gs, 488 768
Uftracentrifugation,
Totipotency,490, 497 Transgeni cpl anl s, 572, 585, 622 Uniformresourcelocator,828
Totipotent,572 Transgeni cpl ants as bi oreactors, Urea biosensor,
300
Toxoids,200 622 Urea cycle,809
Tradesecrets,745 Transgenicplants-therapeutic Urokinase,196
Traditionalbiotechnology,3 proteins,638
'lransgenic
Traditionalvaccines,199 sheep,488
Transamination,
809 Transgenic
snails,493
Transcriptional
genesilencing,593 Transgenic
tsetseflies,493
Transcriptionfactors,43 Transgenic
veggievaccines,637 v
Transcription
inhibitors,45 Translation,
46
Transcription,
39 Transposable
elements,32 Vaccines,l98
in eukaryotes,
42 Transposition,
32 Vaccinesin plants,636
in prokaryotes,
39 Transposition
of genes,67 V a c c i n i av i r u s , 2 1 1
Transcriptome,
38 I'ansposons,
32 Vacuoles,753
Tra ns c r ipt om ic s , 3S Transposontagging, TO Variablenumber tandem repeats,
Transduction,88 fray towers, 677 186
TransferredDNA, 70 Trehalose,624 Vectorrecombinant
vaccines,211
Transfer
RNA, 21, 45 l r l ac y l gl y c er O l S/ / /t) Vectorvaccine,211
Transformation,
87 Tricklingfilters,692 Vector-mediated(plant)gene
Transformation
assays,462 Triosephosphate isomerase, 133 rransret5/J
Transformation
efficiency,87 Triple repeatdiseases,-180 VectorlessDNA transfer,583
Transformation
of cells, 463 Triple-stranded
DNA, 17 Vectors, 82
Transformedcells, 430 Trophophase,
254 Vegetablevaccines,636
Transformed
cells-characteristics, Trypsinization,438 Veggievaccines,636
463 Tryptophan, Vermicomposting,
714
353, 811
Transgene,480, 572 Viable celfs-separalion,
44'l
Tryptophanoperon, 62
Transgenesis,
480 Vinegar,325
Tryptophanrepressor,62
Transgenesis
in largeanimals,487 v r r a to r s e a s e s , , / 5 /
Tuberculosis-diagnosis,
176
in plants,572
Transgenesis Virapap,178
Tubercu
losis-recombi
nant vaccine,
Transgene
stability,593 203 Virus coat proteins/602
Transgenic
animals,480 Tubularbowl centrifuge,
272 Virus resistanceof plants,602
Transgenic
bollworms,493 Tumornecrosisfactor,168 gene transfer,58'l
Virus-mediated
Transgenic
Btplants,743 Tumor-infiltrating
lymphocytes,168 Viruses-characteristics,
757
Transgeniccattle, 488 Tumor-suppressor gene, .l69 Viruses-importance,
757
Transgenic
ihickens,489 Tumorigenicity,
465 Virusesin gene therapy,159
crop plants,620
Transgenic TUNELassay,436 Viscosity,764
Transgenicfish, 49O Turbidbstatbioreactors,262 Vital force theory, 758
Transgenicgoats,486 Two-geneexpressionvector,144 V i t a m i nB r 2 ,3 5 5
Transgerric
medflies,493 Two-vectorexpressionsystem,144 VitaminC, 327
Transgenicmice, 2"17,481 Tyrosine,€09 Vitamins-microbial
production.355
applications,
485
embryonicstemcell TyrosylI-RNA synthetase,
136 Volatileorganiccompounds.618
6?2 BIOTECHNOLOCY

Water recycling,705 Xerodermapigmentosum,

36
w Westernblotting, 100 X y l a n a s e1, 3 3 , 6 3 3
Whey,249
Warm trypsinization, 439 Whiskers,587
Waste water, 682, 686 White buttonmushroom,381
Wastewater treatment,6B6 302
Whole cell biosensors, Y
Wastewater treatmentfor Windro system,7l2
dairies,703
Wines,370
Wastewater treatmentfor Yeastartificial chromosome,86,
distillery,T04 48
Wobble hypothesis, 1 4 0 ,4 8 4
Wastewater treatmentfor sugar Wood-richplants,399 Yeastexpressionsystem,141
indus t r y , 705 Yeastthree-hybridsystem,72
Wastewater treatmentfor Yeasttwo-hybrid system,72
tannery, 7O4
Yoghurt,365
Water blooms, 665
Water borne disases,680 x
Waterdeficitstress,610
Water deficit tolerance,6'11 Xanthan,383
Water hyacinth,396, 4O4,667
Xanthangum, 383 z
Water fettuce,4O4, 667
Xenobiotics,
718
Water pollution,679
Xenogeneiccells,473 Zinc finger motif, 66
Water pollution-measurement, 683
Xenograft,
821 Zoo-blotting,99
Water quality testingby DNA
.l Zygote intrafallopiantransfer,234
analysis, 84 493
Xenotransplantation,