It leads to the loosening of the chemical

bonds linking the components of the substrate.

As the reaction is completed the
is split into end products and enzyme is substrate
released.
V. Microbial Production of
Enzymes
Awide class of organisms are used as
prOC
ess-organisms to extract enzymes in industries.
These organisms include bacteria, fungi and
actinomycetes and the cultures of plant and ani
mal cells. They utilize the nutrients present in
the culture medium and
accumulate enzymeS
either in the cells or in the medium. The en
zymes are isolated and put into mnany uses in
industries.
The microbial production of enzymes is
more advantageous than usual enzyme prepa
ration:
1. The microbes grow easily in the me
dium containing the cheap raw materials.
2. The genetic manipulation of microbes is
casy to carry out under laboratory condition. It
helps to produce a large quantity of enzymes.
3. The microbes grow well even in the fluc
tuating climatic conditions. They can resist the
minor changes in the culture environment.
4. The culture and maintenance of micro
bial cells are easier than those of plant and ani
malcells.
Microbial production of enzymes involves
the following steps:
1. Selection of Process-Organisms
The proper strain of micro-organism must
be selected from natural habitate for the indus
trial production of enzymes. The selected strain
must have the following features; then only the
isolation of a large amount of the desired en
zyme from the spent medium willbe possible.
a. Theorganisms must accumulate the de
sired enzymes in the medium.
b. They must produce a large proportion of
desirable enzyme within a limited time.
c. They must produce the desirable en
zymes steadily.
 are incalled cillium, a i.
traction culturingmicroorganisms ing genes to production
tions moremolecule Some tation for amounts. quantity
inavoiding tothe substrates, of
etc.dium
suchascal for 700
made trays commercial Cultivation
Substrate Solid produce
more the make
In This enzymes.Generally three Cultureof 3. the microbes 2. producing and
tray by In are copies Some mutants Mutation Generally the They
must t e. a.
this causes
synthesis Improvement chemical organisms
whichAspergillus, of Submerged
of method . i.Production
cultivationiii. i Solid Enzymes of using recent used inducer-dependent them of TheyTheThey
or Deep of
enzyme
during
the the Production
method, thinenzymes
woodcultivation mutation enzymes must
are2x40 scale. They years, for a of show requirement
the favoursthe microorganisms are temperature, the must
genetic
enzymes particular the to
or bed
is substrate this production of manipulated themselves
properties adjust grow
metal. the from used are genes, resistance enzymes. produce enzyme. be
etc. It cultivation Microbes for
methodsare engineering. purpOSe. many induce in non-pathogenic
microbes
cms is to the of in
Thifungi,
s adopted
produce cultivation witha by enzyme. production
enzyme
of of cheap
The in introducingmicrobes responsible biosynthesis. culture.Organism pH, of
the enzyme of inducer
This
enzymes Enzymes
properly
layer
trays size. culture. view to more produce the the
method such production the raw
are enzymes
for adopted for Point type Genetics availabilityculture physt
the to
The
arecultured to the are synthesis.
repressor enzymesmolecule materials.
is
asthe
produc proper made muta for of in in a for organism
filled travs largeorder small
also Peni ex mu
in the of by of me
microbes,
nducible repression.
amount
For thus induce SOme is Dy
otherSynthesis the fermenter.
andacration, tained ThepreparedThe tinuous Submerged
the Cultivation
tured lated
iii.them.
from Then tculture
ure substrates.
bialadded bran,qulture Cultivation
Deep-bed microbial
extraction
enzymes.
i . of bation, thover
e a anarelatedturing with
xample, two uscd semi-solid
Jacob used microbial or incubated
surfaceincubator
produced specific Some in In microbes finoculum
rice Here, raw
the when processes. 4.mediumproperly pH, culture. athis vessel
a into vessels onthe
are of
Regulation for medium fermenter few thematerials
production enzymes (1961)
Monod
and the etc. mcthod, th e Any bran,microbes mat ofdense t of
starch iscalled the enzyme is the inocy.are
for microbes.
aresubstances. Biosynthesis inoculum form ar e
days isvessel upt o with is solid
in
microbjal isolation After is culturalby incubated poured potato
one
named
enzymes harvested is The till then solid mat aa an
few
One adjusting pumped or inthe isolated the of or
an
inducers. are in getting medium of are separatcd air-conditioned medium.
of cells in order culare microbial over flakes, size medium. result
days.As nutrient
inducer specific induction conditions is microorganism th e the cultured microbes
culture
Such pr and js of of then at
to and the above
aheight. 2 of BIOTECHNOLOGY
produced controlledcanbeEnzymecnzymes. from intoliquid
tshaaty suficient the is
establish suitable 18x cmedium
The
i substances,
The is added the used growth
enzyme mediumant
dh e etc. and The incu-
is of ara
molecule enzyme the
temperature, in
treated in
fermenter arfermenter.
e form. substrate
The are 200",
rectangular used formed iroomculture
enzy into either tempera well-grown
thbeio growth, a appears. for
which in wiul large a mai n con is mi c ro. used Whezt forthe trays
the a the the The in iso or cul-
is as in
 tacellular
Tatedgiven
heheir enzymes a.
difficult
cell.tsihedrather
e in- BuSmpl t y any of depends
amount enzymes the
glucose
undesirable
environment.
cellular y Extraction
Enzymes thmaie innly as
the extraction glucose hirepressions,
methodThe
cellular from simplified the
extractive The The
Tact iseculhiioessynthesiIns oftrepressione back Thereare the Such theby
contofsaurlpiaminhursenzyme.
enzymes, cells, catabolic
extraction by of galactosidase
iynntnghoesis of cultulorcek.ed sponse eSnoammzeyylasemejpdu(ca)nes. CH.
ne extra in
living 5. generallyrepress by to e 50:
the washing enzyme the Extraction the two
biosynthetIincfeed. bioSomesynttimheneesizsymthses the,
stances
contents cultured present enzyme. of
example, the
cellular nature process. the s back This of ns
cells ENZYME
culture process, process. etc. upon culture. cells protease ty pes addition tthhe e,addiotrigoannisenzymes
: are
to
of ofthe
repression,
biosynthesis
pH].
used to catabolic an d process
isth re- in a-of
dryingTheup below:1S to in
This of repression,
pathway
further of; called culturethe TECHNOLOGY
Sowing microbial Intraintracellular
be ofthe are of areBacillus
cellsenzymes enzyme
the For finducers
selves.by and as
They extracted, process of block addition
in For fcertain continuously
constitutive
the Cellular they with
location The ThiSmade medium, is
allowed extraction Enzymes cxample,
process
Bacilli.
the acidsinhibits called sps
to lofextraction are
cells the proper the the without
cellsof cells are separated
enzymes
do
organisms, of releaseto repressed isenzymes. substrate repression.substances
of to produceto
not the the synthesis inhibits the endrepression.
namely enzyme the
utilization of to present is in enzymes. produced
are solvents. requirestability enzyme processknown product culture. any
y E.coli,
reduces drsepa
of their mo] the feed
in in- 1S out the by of
a a
tinuoustype materials
by nate
Separation of
b. tion. solvents
ath e branes. used shouldsolution in
Solution with water. molecules treated
owing
become due large of water
are
types
centrifuges, the structed
tinuously. capacity solids
ous quantity small the pestle
continuous centrifugation.
type i. in
Broth cell 4. to
available Some Enzymes Sometimes to cell an endosmosis. quantity
with
In The 3.and 2.
centrifuges, from Removal the quantity Sometimes
lysate. have releases alkali The to The
forcentrifuges are release may turgid.
the
industries, and
a
are offound
following lysate. enterdistilled buffer
properly
Such
centrifuges
releasing enzymes,
the used homogenate
range absorption mortar. cells
or
fsharples usedcentrifuges resistance solution. of
extraction homnogenate. are But the the are
instrumentations in of of detergents to [But the The cells. solution.
industrial for separated
theSolid enzyme theselysozymes membrane from Due Dynomill
cells waterhomogenized
bowl-scroll with Inmethods: release enzymes th e dried
super the this homogenate
th e
order Enzymes from turgid Sometimes to
have
been methods towards
enzyme 11.5 The thus of and in
ofsolidspurpose. 60 for Material:
industries endosmosis,
still cells
use. enzymes. to during the like to
obtained as order
centrifuges, Generally
continukg the from and from pH more cells a is
and of produce enzymes bound 12.5. result used are
centrifuges, tritonX-100
some this to of to
specially con
are solid-holding separation are the produce break vwith treated
liquids adopt the th e be The this amount break the to
helpful removed Thehomoge enzymes
range
isolated is the the cellsbreak the
Disc-type organic treated
basket a solid extrac from
mnem alkali
alkali water
down cells therm
con conlarge only help with 701
etc for of is of of re
rgillus
niger.
lucosidase cipitant
photransferase. precipitation
is precipitatesettling
tion ofsolids metabolites,
enzymes
ods have Thesecontains solid terfere
centrifugation.
atremovedby
clease
Polyethylencamine,tate they nucleic ally rationmealstrifugation, The move 702
calledprecipitations. several
a.and 1. are The nucleic
Streptomycinare
Protamine,
sulphate, two vacuum-drum i.
with been
components After 6. iii. ofand some
in Positive chromatography Membrane
Column
electrophoresis filtration
Continuous 5. 4.
of Adsorption 3. given components with
enzymes added Remnoval
PolyacrylicAmmonium
" positive
sulphate negativePrecipitation: Precipitation2. 1. from Purification a homogenate
acidsRemovalsolids
methods
the inlo w
solid adopted removal
large purificationsites. starchy
of th e below: the amino acids. from fraction the
in
purification enzymes molecular
components to from resulting
precipitation: filtrate. amount Hencewhich the
the precipitation. precipitatio.They filtrate. with of of The homogenate. are materials of
omogenate acid acids,
include homogenate, ete. ofthe filter
of Fine
low nucleic cut is employed
in are weight a of of of treated the precipitate are homogenate.
Nucleic containingsolids solution
is Precipitation
the positiveThere The view molecular
etc. enzymes the DNA the polycations.
used of solution in proteins, Enzymes acids,
is from Solids:
is the important
meth Many to enzymes. DNA Acids: uscd
nucleoside-4
oblained to used The are are with They to is
filtrate. isolating does they
in remove sand, the
filtercd
precipitate two
precipita allowed and the randomly in After
asselective
(filtrate) methods weights. endonu them
precipi- are the
solution.
a types
cellular filtrate
some not When
from pre The the is the are
Gener sepa wood
tore cen
to in the
colun,
enzymes. ods umn the towards
tratedstopped. odetively
anode.thedepending and different Zyme. cnzymes
is gel zyme Therefore, other porus Thecomplex
method,the removed sulphonatedsorb withtowards enzyme
cipitationthe isare chemicals materialsmethod,
are
passed
the polycthylene zyme 0s
chromatography
used appropriate After and otherbetween eluted used used
5.for charged mixture 4. solids 3. on
substances 2. b.
membrane. Ammonium pullulanase
ss-linked the Electrophoresis: proteins
molecules. Adsorption: as
in further The proteins
throughenzymes. Membrane
to solution by in "Cetyl-trimethyl
Negative the armmoniumbromide
Sephadex tColumnhe proper on negative
to to
enzy1nes negatively two resultingnever pass filtrate adopting
polystyren. lpH.oweffected unwanted
the precipitant a
molecules their is be glycolprecipitated
separation portion
use. electrophoresed
electrodes. through and such
romatography: separation, the pass is
contains isolated sulphate, solution. in
cellulOse is electric move It The
passed The form
column, is filtrate column as precipitation. (PEG) the
one electrodes, through at precipitation:
ofare charged move towards poured membrane
Filtration:
its hydroxypatre, agarose, low
proteins filtrate.
the then
complex highThese The must to
and ot charges. pores. through enzyme and ten.he
colunn,DEAE the When contains chromatography. molecular pH sodium addingby precipitate BIOTECHNOLOGY
puritication gel t
separated particleshe towards the filtrate
comon mel int0 toThe substances havepolyacrylic and
electricity
and the the
separate Proteins molecules (5-4).
electrodes an Usually some
CelluloSe
sephadex electricity acrude membrane. allows molecules.
Theenzymes well pure isotropic resistance
is
concen the In sulphate.
trom weight treated Hence certain In
Co
move cath pos0
in and the this other this
ol is the are ad pre acid
a en en
er megaterium
Escherichia
iae
siella cereus
Bacillus
coliBacillus
licheniformis
-amylase Bacteriaenzymes.theirand
Bacillus
IndustrialOrganism
coagulansBacillus table.Ing Czymes ng
enzymes useful
ducing ronment deredextentenzyme move ammonium
ture stored an
suspension.
the formformstoredare
of purpose.thforis
lableMicro-organis11s and active
VI. enzymes for the The for enzymeEnzyme enpackagi The
zymes ng commerciymesgarafplorhic columnm, aterials H
medicine. fIndustrial or for getting thusenzyme long a at leither ini
7.Storage
Enzymes
nurified of
solvent a
state. polyacrylamide
Lowe colugenl
50. Enzymes
Microbial
use aobtained suitablesulphate with substances a separation column,
1: long time. suspension stable
The 1(1981)
in are
powdered 1he 3.4 inenzymnes
Some industries, Some time.
stored fraction. solution the
form.
is
pHsulphate.
maintains
ammonium The M: I chromato-
gi v stated Ultragel
es A44,
provide
Applications enzymes in use.
micro-organisms
industrial are in a then under
nase Penicillinase
Penicillin
acylase
osidase
andPenicillin
acylase a-amylaseproduced
Enzyme 1s order are chemically
pureen- that
listed enzyme. is prepared thecolumn, lAc,
protease baking, conditioned heated spray The ofpowder
several a the purified mixed tO
in concentrated suspension low
enzymes maintain etc.
agarose
organisms the soap to dried by with
and important The a tempera enzymes are
follow- of certain mixing or
pro mak envi pow to in some used
re the the gel
is in
low: ment Actinomycetes Treesei
and
viridae
Saccharomyces
cerevisiae
sps.Trichoderma
intouses. and Actinoplanes Rhizopus notatum
boidinii
Candida
funiculosum
sps. Penicillium
lipolytica
Candida Aureobasidium
Penicillium
Neurospora micheli
crassaMucor Aspergillus
Aspergillus
flavus oryzae Aspergillus
niger Fungi
tional tion Medicinal
others pullulans
and * Some
of Industrial 4.
Manipulative
uses
Analytical 3.
uses 2. four
1. Enzymes
In
spepsia, uses
Therapeutic Applications
uses general, Enzymes of
O-amylase Fungal
fermentative discases categories.
Therapeutic
usesenzymes Uses 1. have and
isolated S.fragilis|
ofdue uses been
scence diastase is some
given They ofput from
dyspepsia. metabolic toare
enzymes employed enzymes isomerase
GlucoseCellulase Dextranase
Glucose invertase
oxidase Trysinase Esterase
Rennet oxidase
andprotease
Urate pectinase
oxidase
glucose
|Amylase, and
lipase,
li|Amylase,
is are in Invertase Lipase
LipaseFormate
for given microbial
several dehydrogenase
indigestion, -
and insutticiency,
are are
for in
colic given the practical
divided cultures
indiges cand
treat 703
pain. func be
 testing drawbacks. come
these latory erty cannotenzymes is ing.promote to streptodornase
used lergy.enzymes clean oflymphocytic low into ingpregnancy.
used Uonturbances, wormns. enzymes of fig)proteins. containing
richinfoods.fat 704
Properties However, thereare in "Collagenase
The Rhodonase after are
the system sensitive be
that * is* * wounds. * *and the * to * with * * Protease
to Streptokin..se stop given Concen- * Lipase *
Enzymes Some Sincefollowing
distributed as cure B-lactamase speeds Enzymes Asparaginase
hence body, Thrombin
tooth Pepsin Pancreatin Papain
resence
Anrlytical Uses 2. potent blood pai n
flatulence digest
of enzymes enzymes ulcers
post-operative to
of patients. Cleaning tumour the extraction. promoting digestionand is renmove
cells
some man. have clotting up leukaemia. (from intois
drugs are wound L-asparagine
are becomes
such level isolated given is given
of used and is of givenfor
certain short maytarget
toare
some
on given
and is cells is
enzymes few the in given the as for given worms, intestinal papaya)
elicit large hydrolyses
- collagen
haemophilia healing. as used dyspepsia. for
skin. urokinase
for wounds trypsin,cannot When bleeding from abdominal
methods to life cell/organ limitations
disinfectants
for for indigestion indigestion
stances molecules,
antigenic
span cyanide in blood leading
are penicillin it the digestive worms. and
grow and ficin
used in patients. are
with ficin treatment is
to poison- injected of to
over circu propeasily. they used these fast. bleed- pigs duringdisten death These(from offoods that ofhigh-
in in use and
al to is dis
Organisms Usedfor taking mine
uric mentation measures
dm in freshnessdeterminc dase brothsin6 ures glucosemeasures onds. Zymes:
dmsucrose media. in following
mechanicalThese electric cal or ments. urement. uringtration presence the in
samples.
acid minutes. 2 measure
bothend
Many *penicillin * * measures * sucrose reactions
place. cholesterol Biosensor * It In the reaction.
manipulation
Manipulative concentration Biosensor Biosensor Biosensor Biosensor in can
Biosensor signals biosensors, is
through enzymesUses 3. broth cholesterol intheir minutes. the glucose are light ratecalled substrate isA
It It minutes. 4 measure devices Enzymes are
measures
concentration in can amines
concentration sample. examples with Estimation
in freshness. are signals endpoint of the of
ecombinant isolated containing which samples.
having measure having containing containing
invertase level
containing reaction of
contents It meas attached detected the the point the
of of can substancewhile
penicillin in 10-
2x in
of enzymes enzyme
genetic urine it
penicillinase cholesterol mcat measure used protuct
converted a
from in blood analytical and
measurement. is
10-3x It catalyzing
uricase 25 in
in ofmeat mcasures monoamine of glucose
toand
biosensors
the
to in BIOTECHNOLOGY
DNA seconds. blood and 3x10 the kinetic
microbes
make 30 production fermentation within generate
in product
kinetics responsible
minutes. 10-2x enzyme. readouts the
technol measures fleshes uses Imeasure-
and and
10oxidase mol oxidase
20 biochemi
up deter their
analvte .
are fer oils oxi dm: ! en. of ionic meas-Meas-
o is mol to 10 sec The hy
 Inindustry
Sdairyindustries.ceutical
The
Of
brevwing
industry. in Methylase ligase Polynucleotide
DNA Transfer
kinase Alkaline nucleotidyl
Terminal endonuclease
Tag
dairy used enzymes the transferase lendonuclResteaseriction Sl enzyme
Klenow CH.
.*
Rennin * a Microbial DNA Ribonuclease Re-vH
Lactase dairy 50:
y. cheese in Dairy
erse Table.
Industrial
are industry. Uses 4.
phosphatase
polymerase|Amplification
Enzymes
transcriptase
:
50.2ENZYME
Industry
hereunder
listed
are given enzvmes
is
It from
usedobtained
making. industry,
es Manipulative
TECHNOLOGY
below:
as detergent groupmethyl|Joining
ofDNA group phosphate group generate
nucleotides
from5 |DNA. Addition
to ofCutting
recognition method
sitesDNAs DNA.
atPCR|Degradation
via stranded
portionsingleof
mentary
|templatestrand
ofstrand on
hybrid.
|Synthesis
comple |Selective
digestion
portions
stranded
DNAmRNA
of single mRNA
offrom
a
Common industry,have |DNA.|Addition OH 3
of Synthesis
rating
agei Uses : endRemoval
distillery group -OH
ose wide to trom of Function
of ofcut
ofphosphateDNA. joinable use
nucleotides ofgroup of
applications
Mucorenzymes-industrial and ofATP teninal
from ends DNA of DNA enzymes. of
to of ends. to DNA
michei
pharma-starch
milk uses of of 5
in -

rocesSS,
tation merase perature favourable
atAspergillus glucoamylase ase. syrup.
maltodextrins,
of
B. toseenzymes before gives is washing
high are dyeing. cottonfor cloths.
dirts from gests0.4-0.8%
licheniformis andIus proteinaceous
lipases
and applications sweetened lk. 1S
Kluyveromyces
mifragilis and cheese.ing milk and
65°C used used
* * blood* non-ionic
* * * b. Lactase Catalase
When If maltodextrinIf When c. softness *and * is*andwhey.
and starch StarchAmylase as Cellulase Alpha Serine Proteases, areDetergents Detergent
made
from in Lipase sweet
of 55°C, starch dehairing Alkaline soil the
for stains of used It
B.coagulans
all pH oryzae starch particles. of is
to the
detergents dirts manufacture
these 6.5- is it isthis used
Industry: leathers. amylase protease in enzymes is milk. also
hydrolyzed isprotease and weight Industry used
hydrolyzedgives reaction. is used agent obtained the on
at hydrolyzed
licheniformis is
a-amy
and contaminated fabrics
with amylases, used used
a 8.5,
reactions
mixture
ctose, of maltose
syrup in B.amyloliquifaciens di milk manufacture cloths manufactured
high pH formed. to
it or the add to for removes isolated ofand in add to to
In of dirt detergent of concentrate
gives 5.0 with leather
bacteria from detergents.soaps. enzymes
remove n AspergilluS
The : niger make
Streptomyces maltose
with manufacturestarch from and flavour
starch to cellulose ice-creams
occur
fructose - pH with fungi starchy textilefollowing
glucose a-amylase7.0 from lactoselow
5.5 industries,
fruc
and and and cloths. They of industry
in syrup. and dextrose anionic fibres. to
- cloths skin. is Bacil milk.
syrup.a 7.0 fungi used dirts form from ripen
te m and and 705
fer sps. iso of are
of is It -
 PenicillinGlase ,verted Vchain. acylase
side penicillin lowing
into industries, way -
ceutical tries.
galacturonase,
polygalactur-onase, proved
polymethyl crease
into enzymes starch. of stages lose mentation for rate. -
posestries, Sweeter
ture $2% 706
into G volume
" " * f. saccharification
into 6APA 6-amino They e. * *and Bacterial
* and * *
enzymes d. of gluCOse
Penic:ilin Penicillin Pharmaceutical
are Increasing pectin Wine the fo r fruity
s-sVnihetic Bve: Penicillin Shortening
Enhancing Reducing
cloudiness
Avoiding quality. are Fungal Papain Cellulases
barley Protease Brewing than
used rate
maturation of
i'illin G andV are the
ynatic penicillaric seini-synthetic
penicillins. esterase Industry:
are employed syrups.
in used and B-glucans. products are
glucose. and
V the glucoamylase is is
side
acylase Gacyias G
the enzymes the the the Pectin amount used are Industry:
used 6%
adtilion, acylase for of used
6 bioconversion fermentation flavour rate viscosity of fungal It
penicillins. cihain. Industry - to In th e usedstarch. and dextrins is
APA in to fo r
converts Onvertsacid are of transeliminase, used get wine
of improve used
the6APA and fermentation hig h product.
post-fermentation to
amylases to the In
+i (6APA) used of of in saccharification hydrolyse
Penicillin fruit making, is
increase following brewing in is
: wine wine yield the
formed.
side penicillin penicillin of time
in In used texture
is and natural pharma
the juices indus are the manufac
chain andpectic
to
con- fol im cellu used and purindus It
G V in fer-
is
ilising
carrier. the
the Methods living
cells. ofproducts. cent trate ucts time. unit tion
enzymes
the avoided.enzymestachedsubstratebility vantages tant zymes Fusarium
model a pure enzyme
arresting them oxysporum. aroideae,
isolated
NCIM-2400 nyl 6
APAPenicillin V
specific free VIl.
The Tne 6. to 5. with 4. of 3. 2. advantages
than1.The The
Immobilized The maximum
The The to are into Free aceticPenicillin
immobilization system
cell-free reach nearly enzymes. The immobilization. Immobilization
The is during the into the immobilized he a
tenzymes from Side
additionchain
reactions of solidenzymes reaction
more immobilized immobilized
than often
the the solidproducts free semi-solid
free Pseudomonas acidgrown Penicillin
different Enzvme for the acylase V
zymes immobilized materials cent the are named movement Bacillus (PAA). G
reaction amount when
So materials. acylase
between
studying enzymes
percent produce potential enzymes. given free
are Semi-synthetic in
penicillins
they extraction is enzymes.
enzymes a
methods
are of compared enzymes higher.enzymes imnobilized carTier. protected medium
Immobilization
enzymes freely catalyseof below: sphaericus,
Penicillin Is 6APAt
given system products Such of obtained
the
the produce purity. only So of BIOTECHNOLOGY
enzyme enzyme. the of of So enzymes This acidovorans
available
enzyme allow the the the have
have Some protected Enzymes containino
beloW: depends desired immobilized are
tum by V
behaves the with product
wastage process acylaseis Vfrom
cent the within fimly enzymes.
more inserting side
actiou produc that over more impor is Erwini
1o1 a ol per sub prod called E.cok chain
as a of is of at ofsta ad en of and n
 Enzyme trapment. This Iaround is
hx mattrhiex Matrix: AmyloglucOSIdase
tions ning
enzymehigh, carrier.theoning tion. Sometimes
enzymes.
d lize riers.ecules CH.
EnzvDIe
50.6:Fig. immobilizes
immobilization mixed 2. sephadex exchange
anresins,
in Physical
problems. Amino Electrostatic
bringVander 's
Waalcharcoals 1.50:
adsorption.
Fig.50,5:
Phsical
with Enzyme ndustrics are
Polymer Polyacry leakage about GenerallyPhysical
ENZYME
adsorbed
an acylase
adsoptionpolymer
the enzme. However, isi adsorption
physical areporus
glass |
cnAmemolecules. smalande
ThiEntrapment +() because hydrophobic
forces, also polymers
adsorption:
en/Nmewithin
nmcthod is torces on:TECHNOLOGY
adsorbed is solid
ment which for
isadsorbed used
it is it
simple are
is foms a ofhas
loimmobilization.
andcellulose-based
as
materials, materials
called polvmer. w causeS
on hydrogen carriers. immobi-usedto
several Enzyme
the a in cost. to
charcoal.
enyme thread-like Enzyme perform on of called
in polymer. Polymer Polymer many silica
polmer When applica- DEAB enzymebond-interac
run- gels ion car- mol
en but
it
zymea enzyme 20enzyme
uctssmall face microcapsules.
membrane
called entrapped immobilization.
for lon lizing
Canada immobilizing
forctate one
surrounds as
lope. enzymes flask
K aresult membrane
4.and in for Hollow the Encapsulation: 3.
orAbout
mal
0.8:
ent. This orand molecules immobil1zation.
encapsulating
molecules. m encapsulation enzymes. first calcium
Liposomal
oftor tube
the aphospholipid Encapsulation.
Fig.50.7: capsules in
out. and ) fibre adopted
a
lipid proper
enzyme being and semi-permeable
or In gram l
bodies such they membranes collodion this
alginate gram
inserted
mixing. shaken containing this of
and Entrapment: as
They do more They of method, polymer
of
are enzymes. technique is
are not membrane
Collodion enzyme.
calledforms The into wellmixedare substrates amount
permeableallow provide are Enzyme membrane Chang
TM.S. in used
the membrane. the
lipid the to available enzymes for as is
Cellulose
required
Lipid
layer liposomes. a lipidinduce
lipid together leakage enzyme The a
-Enze completel and enzyme. of large immobi polymer
The only
enve bodies prod small used is 707
the sur for are Nyare acfor
in en to of
 708

Enzyme to be immobilized is mixed with a

solution of 1-6,diaminohexane and then the so
The linker molecule has
Eg. Cyanuric chloride, two BIreactOTECHiNvOe
lution is dispersed in chloroform containing cyanogen bromide.
diaminohexioic acid. Small droplets containing Immobilization by glutaraldehyde
cOvalent
runningpurbopncOstoses.
dingsveThihegsh.
enzyme are formed. They arecalled liposomes. strong and is used for specific
5. Covalent Bonding: Here covalent is no enzyme leakage but
bonds are generated between the atoms of en 6. Co-polymerization: is
zymes andthose of the polymer. The functional
groups of enzymes are not usually involved in
covalent bonding. The groups unavailable for the
polymers are often usedin the
lization. Each polymer Connects Multifuntional
enzymethe imnohj.
molecules and it also connects the eNzye,
ca,

are other poy-

enzyme reaction only take part in covalent bond mers. That is. the
polymers
ing. Penicillin-G-acylase is immobilized in
sephadex polymer by covalent bonding.
with one another and also with
Glutaraldehyde is one oftthe the
enzyMescom-.inter-connete
Synthetic polymers form effective linkers monly
in the immobilization of enzymes. For example, used in enzyme immobilization. co-polymers
acryl copolymer has aldehyde group, carboxy
methyl group and hyydrocyanate group. These
groups are involved in the establishment of cova
lent bonds with the enzymes.
Enzyme

Polymer Fig.50. 11: Co-polynerization.

Glucose isomerase is immobilized in cros
linked glutaraldehyde.
Table. 50.3: Important industrial uses of
Fig. 50.9: Covalent bonding. immobilized cnnes.
Sometimes, some small
as linkers to join the enzymes molecules are used
with the polymers.
Enzyme Product

CI
CI
Aminoacylase
|Aspartate ammonia-lyase
L-amino acids
L-aspartic acid
OH + CL?
Reactive N N+ HCI |Aspartate 4-decarboxylase L-alanine
CI Cyanidase Formic acid
group of CI
polymer Cyanuric Polymer-Cyanuric Glucoamylase |D-glucosefrom
starch
chloride
acid complex Glucose isomerase High-fructose
corn syrup
NHEI Histidine ammonia-lyase Urocanicacid
ON:NHHEH
Enzyne CI N= CI
N+ HCI Hydantoinase D-and L-amino
acids
Polymer-Cyanuric Invertase Invert sugar
acid complex Enzyme-pol
complex
ymer Lactase Lactose free
milkand whey
Fig.50.10: The
bycovalent immobilization of enzvme
bonding with the help of |Lipase
Oxynitrilase Cyanohydrines
Cocoa butter
linker molecule.
substitutes
 50: ENZY)
CH.S0 YME 1ECHNOLOGY
709
Nitrilehydratase
Penicillinamidase Acrylamide COvalently bound with zinc ions used in the treat
Penicillins ment ofleukaemias. Zinc ions andused to treat
Rafinase Raffinose free diabetics. Zinc increases the survivability and
solutions reaction potential of theinsulin.
Thermolysin
lGlycosyltransferase Aspartame 6. Immobilized enzyme reaction is used as
Oligosaccharide a model system to determine the characteristic
of cellular metabolism in living cells under dif
Applications of Immobilized ferent physiological conditions.
Enzymes
Immobilized enzymes have considerable Enzyme Engineering
practicalapplications
ap in;industries, medicine and The manipulation ofenzyme structure for
model studies. The important having maximumn catalytic activity and resist
thefollowing:
applications are ance to reaction environment is calledenzyne
1 Immobil1zed enzymes are used as ana engineering.
lytical agentsin enzyme electrodes. The en- Many enzymes are sensitive to heat. Some
zyme electrode is made up of aglass electrode enzymes catalyse reversible reactions. Some
surroundedby athin film ofimmobilizedlenzyme. enzymes are sensitive to fluctuation in the pH.
The reaction kinetics is very poor while using
Enzymeelectrodes are very sensitive. They de such enzymes. Geneticengineers have manipu
tect the presence of certain substances even if
lated the genes coding for the enzymes in such a
they occur in smallamounts. Now enzyme elec way as to have desired features. This manipula
trodes are available to detect drugs, pesticides, tion is said to be enzyme
toxins, glucose level in blood, ctc. engineering.
More number of disulfide bonds in enzymes
2. In food industries, immobilized enzymes increases the thermostability as well as resist
play the following important roles: ance against extreme pH changes. Nucleotides
a. Immnobilized enzymes are used to
coding for cystine are introduced into the gene
convert starch intoglucose. coding for the enzyme by site directed muta
b. Milk whey contains lactose. The genesis. The modified gene produces enzyme
immobilized enzymes are used to convert the with some more cystine residues and establishes
whey into simple sugars. someadditional disulfide bonds. The engineered
c. Immobilized enzymes are uscd in the enzymes are more stable towards high tempera
preparation of cottagechecse. ture and extreme pH changes. As the major
d. Immobilized glucose isomerase en portion ofthe enzyme remains the same, there
Zyme is used in the manufacture of fructose is no change in the catalytic activity.
Syrup. Another reason for poor stability towards
3. In dairy industry, the immobilized en high temperature is the presence of more as
ymes are used to coagulate the milk protein parag1ne in the enzyme molecule. One or two
during and to treat the waste asparagine residues are replaced by threonine
whey. cheese-making or isoleusine by site directed mutagenesis in the
4. Immobilized aminocylase enzyme is gene coding for thatenzyme. The resulting en
used in fluidised bed reactors or CSTR in phar- zymes are more stabletowards heat.
Free sulfhydryl group of cystine that does
maceut
acyl ical industry. This enzymee converts D.L-
acids into L-amino acids. The
L- not participate in disulfide bonds, reduces the
aninoamino
acids are used as ingredients in food stufts. catalytic activity of enzymes. Cystines that do
5. enzymnes are used in en- not participate in sulfhydrylbornding are changed
Immobilized
zyme therapy. The enzyme asparaginase is to serine by inducing site directed mutations in
 10

the gene. The resulting enzymes are somewhat

more powerful in their catalytic activity.
Catalytic RNAS are splicing
eukaryotic primary transcript RNAs
BIOTECHNOLOGY
products
of
In some enzymes, threonine at the active
site is changed into alanine or proline by site di
ter ligation of exons (coding
catalytic RNAs fold themselves in
fomed
at.
sequence).
The
rected mutation. It increases the catalyticac as to have autocatalytic activity. such a way
tivity. Eg. Tyrosyl-tRNA synthetase. In cells, they do cutting out. of
Ribozyme
sequence from the primary RNA
tra
joining cut t ends of exons. Heating
inscrntervipenit ng
and
RNA molecule that catalyses a specific cooling causes oligomerizationofcatalytic
o fol owedby
CRNAS,
biochemical reaction, is called ribozyme or but the catalytic activity is the same.
catalytic RNA. It was first discovered by Tho Ribozymes catalyse the breakdown of othe
masCech and Anthur Waug from the ciliated RNAs. They are used as potential pharmace).
protozoan Tetrahymena thermophilia in 1984. tical agents which attack viral RNAs.
Therefore, it is also known as Cech-Waug RNA.

Transcription
Intron I Intron II Intron III

ExonI Exon II Exon III Exon IV

Primary RNA
Splicing transcript or
Precursor RNA

Exon I Exon II Exon III Exon IV

Post transcr
Autocatalytic
iptional mod
activity ification

Mature RNA
Catalylic RNA

Active fom of catalyticRNA

(Ribozyme)
Fig.50.12: Formation of ribozyme from precursor RNA.
 CH. 50 : ENZYME 711
TECHNOLOGY
Ribozymes selectively destroy RNAS com
ing from oncogenes that they are used as
so Synzymes
anticancer agents. However, it is difficult to
introduce ribozymes into target cells, as the in ble Synthetic polymers or oligomers capa
of doing enzyme-like functions are called
etinal enzymes and drugs damage
them. Now synzymes or artificial enzymes.Asynzyme has
ibozymes are encapsulated in liposomes and then two important parts-a substrate binding site and
introduced into the body.
Ribozymes active site. Both these sites are designed and
are also employed in industries svnthesized
as catalysts andIin selecting separately and assembled together
Darwinian cloning. catalytic RNAs by to makeacomplete synzyme.
Synzymes are derivatives of certain pro
Abzymes teins. For example, when myoglobin is attached
with (Ru(NH,)J" to three histidine residues, a
An antibody which is capable of
cata
bsing a biochemical reaction iscalled abzvme. synzyme is formed; this synzyme, instead of
acting as an oxygen carrier, acts as an oxidase
It is a protein. Abzymes are produced against for oxidizing ascorbic acid. It is functioning
he transition state analogue of the substrate and
very similar to ascorbic acid oxidase. In gen
ISed for their catalytic property. eral, synzymes obey the Michaelis-Menten ki
Abzymes reduce the entropy ofbiochemi netics for enzymes.
cal reaction. So the speed of reaction increases
Polylysine, polyglutamic acid, cyclodextrins,
to6x10° times per minute. Therefore they can ctc. also act as synzymes under natural condi
eliminate competitive inhibition by inhibitors. tions. They are sensitive to denaturation, pH
Abzymes are very specific as the enzymes variation, oxidation and hydrolysis.
do. They catalyse esterase and peptidase type In synzymic cyclodextrins, hydroxyl group
reactions. The specificity of abzymesare use of B-cyclodextrin is covalently linked with
fil in biotransformation studies and in making pyridoxol coenzyme. This synzyme acts as a
biosensors. transferase but not reactive enough as natural
Complex drugs in the body can be destroyed transferases. However, synzyme obtained from
by abzymes. Abzymes selectively cleave com- polyethyleneamine is equally cffective as the
plex proteins of virus aid others. natural enzymes.