1

CLONING OF Candida antarctica LIPASE A GENE IN

Kluveromyces lactis EXPRESSION SYSTEM

A PROJECT REPORT

Submitted by

KARTHIKEYAN.T

RAJARAJAN.P

RAMKUMAR.M

In partial fulfillment for the award of the degree

of

BACHELOR OF TECHNOLOGY

in

INDUSTRIAL BIOTECHNOLOGY

CENTRE FOR BIOTECHNOLOGY

ANNA UNIVERSITY
CHENNAI – 600025

DECEMBER 2009 – MAY 2010

ANNA UNIVERSITY: CHENNAI 600 025
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BONAFIDE CERTIFICATE

Certified that this project report “Cloning of Candida antarctica Lipase A gene in

Kluveromyces lactis expression system” is the bonafide work of Karthikeyan.T

(Reg.No.20062735), Rajarajan.P (Reg.No.20062760) and Ramkumar.M

(Reg.No.20062761) who carried out the project work under my supervision.

SIGNATURE SIGNATURE

Dr.P.Kaliraj Dr.S.Meenakshi Sundaram

Professor and Head Professor,

Centre for Biotechnology, Centre for Biotechnology,
Anna University, Anna University,
Chennai-25. Chennai- 25.
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ACKNOWLEDGEMENT

We sincerely thank our project guide Dr.S.Meenakshisundaram, Professor,

Centre for Biotechnology, Anna University for allowing us to do our project in the
field of molecular biology in bioprocess. He gave us full freedom to try new
techniques and to conduct our own experiments.

We would like to thank Ms.A.K.Prasanna Vadhana for assisting us

throughout the project. We really appreciate her effort in trying to help us in our
project.

We are happy to get acquainted with other research scholars in bioprocess

lab. They were extremely helpful and enabled to create a comfortable working
atmosphere.

We also extend our thanks to other fellow students whose guidance and
views have gone in to the completion of this project.

Karthickeyan T Rajarajan P Ramkumar M

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TABLE OF CONTENTS

CHAPTER NO. TITLE PAGE NO.

ABSTRACT 6
LIST OF FIGURES 7
LIST OF TABLES 8
LIST OF SYMBOLS 8

1 INTRODUCTION 10

1.1 LIPASES 10
1.2 BIOCHEMISTRY OF LIPASES 10
1.3 LIPASE ACTIVITY ASSAY 12
1.4 REVIEW OF LITERATURE 13
1.4.1 Candida antarctica LIPASES 13
1.4.2 INDUSTRIAL APPLICATIONS 14
OF LIPASES
1.5 E.coli MAINTANENCE HOST 16
1.6 K.lactis EXPRESSION SYSTEM 16

2 MATERIALS AND METHODS 19

2.1 HOST STRAINS 19

2.2 VECTORS 19
2.3 ANTIBIOTICS 19
2.4 CULTURE MEDIUM 20
2.5 GENE OF INTEREST 20
2.6 POLYMERASE CHAIN REACTION 20
2.7 AGAROSE GEL ELECTROPHORESIS 22
2.8 RESTRICTION 23
2.9 PURIFICATION 25
2.10 STOCK PREPARATION 26
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2.11 INOCULATION 26
2.12 PLASMID EXTRACTION 26
2.13 RESTRICTION 29
2.14 DEPHOSPHORYLATION 30
2.15 PURIFICATION 31
2.16 LIGATION 32
2.17 COMPETENT CELLS PREPARATION 36
AND TRANSFORMATION
2.18 TRANSFORMATION 38
2.19 PATCHING 40
2.20 LYSATE PCR 40
2.21 CONFIRMATION PCR 41

3 RESULTS AND DISCUSSION 43

3.1 PCR AMPLIFICATION 43

OF C.ANTARTICA LIPASE A
ENCODING GENE
3.2 RESTRICTION AND PURIFICATION 44
OF LIPASE A GENE
3.3 pKLAC2 – EXTACTION AND RESTRICTION 45
3.4 CLONING 47
3.4 TRANSFORMATION 48

4 CONCLUSION 51

REFERENCES 52
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ABSTRACT

Lipase A is an enzyme from Candida antarctica strain isolated from Lake

vanda in Antarctica. The gene coding for Lipase A is CALA gene. Lipase A is the
most thermostable lipase known and is used as a biocatalyst in food and
pharmaceutical industry. Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are
ubiquitous enzymes of considerable physiological significance and industrial
potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free
fatty acids. Lipases are serine hydrolases. Lipases display little activity in aqueous
solutions containing soluble substrates.

Lipase A gene was from Candida antarctica was amplified using gene
specific primers Xho1 restriction site. The gene was cloned unidirectionally into
Xho1 site in pKLAC2 vector. pKLAC2 with lipase A gene insert was transformed
into E.Coli TOP 10F’ strain and the transformed colonies were screened for
positive transformants. Future prospects involve expressing lipase A gene in
Kluveromyces lactis expression system and conducting expression studies in a
fermenter.
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LIST OF FIGURES

CHAPTER NO TITLE PAGE NO

1 1.1 Mechanism of lipase catalysis 11

1.2 pKLAC Vector Map 18

2 2.1 Plasmid Extraction 29

2.2 A pictorial example of how a 33

ligase works (with sticky ends)

2.3 A simple mechanism 34

representing the role of DNA Ligase
2.4 Artificial Transformation 39

3 3.1.1 Analysis of PCR amplified 44

lipase A DNA of Candida antarctica

3.2.1 Analysis of purified sample 45

of Lipase A gene

3.3.1 Analysis of PKLAC2 plasmid 46

extracted by standard kit method
3.3.2 Analysis of pKLAC2 restricted Plasmid 47
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3.4.1 Analysis of ligation mixtures 48

of Lipase A gene with PKLAC2
plasmid using pKLAC2 forward
and lipase reverse as primers.

3.5.1 Lysate PCR analysis of E. coli 49

transformants (7-10) having the plasmids
with the inserts CALA using pKLAC2
forward and Lip A reverse as primers.

3.5.2 Analysis of plasmids extracted 50

by manual method from patches of
7, 8, 9, and 10 Top 10 E.coli
transformants

LIST OF TABLES

CHAPTER NO TITLE PAGE NO

1 1.1 Characeristics of CALA 14

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LIST OF SYMBOLS

CALA Candida antarctica Lipase A

⁰C Degree celcius

DNA Deoxyribo Nucleic Acid

mg milligram

ml milliliter

rpm Rotation per minute

PCR Polymerase chain reaction

ul microlitre

LB Luria Bereni

kb kilobase pair

bp base pair

M molar

CaCl2 Calcium chloride solution

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CHAPTER 1

INTRODUCTION

1.1 LIPASES

Lipases (triacylglycerol hydrolases EC 3.1.1.3) belong to the family of

hydrolases which catalyze the hydrolysis of long chain triglycerides. These
enzymes are found in a vast species of plants, animals and microbes. Fungal lipase
is of much importance due to its significant application as biocatalyst. They are
used widely across food, flavor and biopharmaceuticals industry. Though
conventional techniques for commercial production of lipase incur higher costs,
this can be overcome by novel molecular biological techniques.

1.2 BIOCHEMISTRY OF LIPASES

Lipase family include structurally and functionally homologous group of

enzymes. The tertiary structure of lipases exhibits the hydrolase fold pattern
(Schrag and Cygler 1993). The lipase structure is composed of a core of upto eight
parallel beta strands, connected and surrounded by alpha helices. The active site is
formed by a catalytic triad consisting of a serine residue as the nucleophile,
histidine as base and aspartic (or glutamic) acid as the acidic residue. The active
site residues are placed inside a hydrophobic pocket termed as ‘nucleophilic
elbow’ and the pocket is covered by a lid like structure, composed of one or two
amphiphillic alpha helices. The activation of lipases requires the opening up of the
 11

pocket by the displacement of the lid and this process is known as interfacial
activation.
The catalytic mechanism of lipase hydrolysis consists of four subsequent
steps: i) the absorption and activation of the lipase at the interface between aqueous
and organic phase and then binding of the ester substrate within the hydrophobic
pocket; ii) in the second step, the nucleophilic oxygen of the serine side chain
attacks the carbonyl carbon atom of the ester bond leading to the formation of a
tetrahedral intermediate and this is stabilized by one or two hydrogen bonding with
amide nitrogen atom of the amino acid residues located in the region called as
‘oxyanion hole’. iii) The ester bond is then cleaved liberating an alcohol and leaves
behind the acyl-enzyme complex. In the last step, the acyl-enzyme is hydrolyzed,
when a water molecule (sometimes another alcohol) enters the active site, thereby
liberating the free fatty acid (or a new trans-ester with the alcohol) and the enzyme
is regenerated. Example of lipase catalysis mechanism is given in Figure 1.1.

Figure 1.1 Mechanism of lipase catalysis

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1.3 LIPASE ACTIVITY ASSAY

A wide range of assay protocols have been developed for the estimation of
lipase activity (Beisson et al. 2000). Based on the general triacylglycerol
hydrolysis reaction, the lipase activity is assayed by the release of either fatty acids
or glycerol from triacylglycerols or fatty acid ester. The various procedures used
for lipase analysis are summarized below.

i) PLATE ASSAY
Agar paltes supplemented with triacylglycerides such as tributyrin and
triolein are used in this assay. Lipolysis of triacylglycerides produces a clear
halo or colour change of Phenol Red / Victoria Blue / NileBlue Sulphate, or
fluorescence with Rhodamine B under UV light due to dye-Free fatty acid
complexation.

ii) TITRIMETRY
It involves potentiometric determination of free fatty acids liberated upon
hydrolysis by lipase. Simple titration by neutralization reaction of these free
fatty acids with NaOH to constant end-point pH can also be used to estimate
lipase.

iii) SPECTROPHOTOMETRY
Estimation of the lipase hydrolyzed yellow-colored p-nitro phenol at 420 nm
and 2,4-dinitrophenol at 360 nm gives the lipase activity .Also precipitation
of free fatty acids with calcium or copper and measurement at 500 nm gives
the lipase activity.
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iv) FLUORESCENCE
Lipolysis of fluorogenic substrates such as ω-linked pyrenic acyl-glycerol
derivatives gives lipase activity quantified in terms of increasing
fluorescence intensity with time.

1.4 REVIEW OF LITERATURE

1.4.1 Candida antarctica LIPASES

Among the widely used enzymes for biocatalytical purposes are the lipases
produced by different strains of genus Candida sp. In the late 1960’s the yeast
strain Candida antarctica was isolated from Lake Vanda in Antarctica and was
found to produce two different lipases (CALA and CALB) The two lipases of C.
antarctica were characterized and the amino acid and the DNA sequences
encoding these lipases were sequenced at Novo Nordisk A/S, by Patkar, Hoegh
and others. The lipase B was later crystallized and its structure was also
determined by Uppenberg et al. (1994).

C. antarctica lipase A and B were found to exhibit varying physio-chemical

characteristics. Lipase A is the most thermostable lipase known, being able to work
efficiently at >90°C. Such thermo stable enzymes are useful for industrial
applications such as pitch control in the paper industry, in the pulp and wax
industries and for asymmetric biocatalysis. The lipase B has become one of the
most prominent enzymes, in organic synthesis, especially for the kinetic resolution
of race mates. Currently, lipase B is the widely targeted enzyme for protein
 14

engineering so as to improve and optimize its substrate specificity and

enantioselectivity (Lutz 2004).

As both C. antarctica lipases have gained significant commercial

importance and as the expression levels in the native organism are too low,
recombinant over-expression is needed for the large-scale production of these
biocatalysts.

Table 1.1 Characeristics of CALA

Properties Value
Molecular Weight (kDa) 45
Isoelectric point (pI) 7.5
pH stability 6-9
Thermostability at 70° C 100[100]

1.4.2 INDUTRIAL APPLICATIONS OF LIPASES

The physical and chemical properties of Lipases that contribute to their

importance in industrial applications are i) stability in organic solvents ii) high
specificity iii) high enantio and regioselectivity and iv) no necessity for cofactors.

i) LOW CALORIE FAT PRODUCTION

Low calorie sunflower oil can be produced by inter-esterification with lipase
and behenic acid. Low calorie lipids are also obtained by interesterification
of tristearin with tricaprin or tricaprylin using an immobilized lipase.
 15

ii) MANUFACTURE OF COCOA BUTTER SUBSTITUTE

Cocoa butter which is used in confectionery industry has a high production
cost. Cocoa butter alternatives with similar flavor can be manufactured by
interesterification of less expensive fats like illipe fat, sal fat and shea butter.

iii) ACCELERATION OF CHEESE RIPENING

Lipases catalyze lipolysis in milk and therefore accelerating the cheese
ripening process.

iv) DETERGENT INDUSTRY

Lipolase, a lipase based proteolytic detergent has an optimal pH 10.5 – 11.0
and is active over a broad range of temperatures.

v) PHARMACEUTICAL INDUSTRY
Lipase is used in pharmaceutical industry as a biocatalyst as it avoids
isomerization, racemization, epimerization and rearrangement reactions in
chemical synthesis of drugs.

iv) RESOLUTION OF RACEMIC MIXTURES

The enantioselective nature of lipases is used in resolution of racemic
mixtures of molecules, thereby producing only one enantiomer in higher
concentration.
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1.5 E.coli MAINTANACE HOST

Genotype: TOP10F´: F'{lacIqTn10 (TetR)} mcrA Δ(mrr-hsdRMS-

mcrBC) Φ80lacZΔM15ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK
rpsL endA1 nupG

a) TOP10 F’ E.Coli strain is ideal for high-efficiency cloning and plasmid

propagation. They allow stable replication of high-copy number
plasmids. It has transformation efficiency greater than 1*109 /μg DNA.
b) hsdR for efficient transformation of unmethylated DNA from PCR
amplifications.
c) IrecA1 for reduced occurrence of non-specific recombination in cloned
DNA.
d) F´ episome carries the tetracycline resistance gene and allows isolation of
single-stranded DNA from vectors that have an f1 origin of replication.

1.6 K.Lactis EXPRESSION SYSTEM

Kluyveromyces lactis is a yeast commonly used in genetics research and

could potentially be used to produce pharmaceuticals or other compounds
Kluyveromyces lactis is a yeast species commonly used for genetic studies and
industrial applications.

Kluyveromyces lactis(formerly Saccharomyces lactis) is a yeast which has

the ability to assimilate lactose and convert it into lactic acid. Kluyveromyces lactis
and other organisms ie, Aspergillus niger var awamori and Escherichia coli K12
 17

are grown in fermenters to produce chymosin (rennet) on a commercial scale; this

rennet, which replaces the conventional form obtained from slaughtered animals, is
now widely used in cheese production.

ADVANTAGES:

a) High level and scaleable expression of recombinant proteins.

b) Rapid high cell density growth.
c) Simultaneous expression of multiple proteins possible.
d) No background gene expression during E. coli cloning steps.
e) Easy and fast cell transformation procedure.
f) No expensive antibiotics required.
g) Attractive commercial sublicensing.

REASONS FOR USING pKLAC2

• Variant of LAC4 promoter found in K. lactis

• No background transcription

• Even genes toxic to E. coli can be cloned

• Presence of multiple cloning sites

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Figure 1.2 pKLAC Vector Map

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CHAPTER 2

MATERIALS AND METHODS

2.1 HOST STRAINS

E. coli TOP10F’ – Maintenance Host

K. lactis – Expression Host

2.2 VECTORS

pKLAC2 vector system integrated in E. coli DH5α is used.

2.3 ANTIBIOTICS

Antibiotics are used for the selection of transformants. Here Ampicillin is

used. The working concentration of ampicillin is 100μg/ml which is used in the
selection process. Some bacteria exposed to penicillin survived because they
produced the enzyme β-lactamase that destroys penicillin’s structure. These
bacteria are known to contain antibiotic resistance gene.
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2.4 CULTURE MEDIUM

LB Medium (Luria Bertain Medium) .1000 mL of LB medium contains:

 1000 mL deionized water
 10 g Bactotryptone
 5 g Bacto yeast
 5 g NaCl several drops
 5 M NaOH several drops
 1 M HCl

2.5 GENE OF INTEREST

LIPA is the gene of interest that is isolated from Candida Antarctica.

2.6 POLYMERASE CHAIN REACTION

A Polymerase Chain Reaction (PCR) was done using gene specific
primers to amplify the gene of interest from the plasmid pPICZalpha B

REAGENTS REQUIRED

1. Master Mix (2X)

2. Forward primer
3. Reverse primer
4. Template DNA
5. Double distilled water
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REACTION CONTENTS

Template DNA (pPICZalpha B) 1.5 µl

lipA Xho1 forward primer 1 µl
lipA Xho1 reverse primer 1 µl
Double distilled water 6.5 µl
2X Master Mix 10 µl
REACTION VOLUME 20 µl

The reaction mixture for 20μl was prepared by taking water, DNA, master mix,
primer, & enzyme sequentially.

REACTION CONDITIONS

S.No Steps Temperature Duration

1 Initial denaturation 95˚C 5 min

2 Denaturation 95˚C 1 min
3 Annealing 60˚C 1 min
4 Extension 72˚C 2 min
5 Goto step 2 – 30cycles
6 Final extension 72˚C 10 min
7 Hold 4˚C
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2.7 AGAROSE GEL ELECTROPHORESIS

PRINCIPLE

Electrophoresis through agarose gel is the standard method used to separate,

identify and purify DNA fragments from 200 bp to 50 kb in length. When an
electric field is applied across the gel, the negatively charged DNA at neutral pH
migrates towards the anode. The rate of migration is determined by the number of
parameters such as molecular size of the DNA, agarose concentration,
conformation of the DNA (super helical circular, nicked circular and linear),
applied voltage direction of the electric field, composition of the electrophoresis
buffer.

REAGENTS REQUIRED
 Running buffer (TEB 0.5X buffer) for 100 ml
 Tris540mg,
 EDTA46mg,
 Boric acid275mg
 pH 8.3
 Gel loading dye
 40% sucrose (w/v) in water, 0.25% orange G & 10% v/v (1X) TEB
 Ethidium Bromide (EtBr)
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PROCEDURE
1. To the 40ml of 0.5 X TEB buffer, 320mg of agarose was added.
2. The solution was boiled for 50-60 seconds to dissolve the agarose.
3. When a bearable temperature was reached, 2.5μl of EtBr was added &
mixed
4. Properly.
5. The comb was placed on the gel tray in appropriate place.
6. The mixture was then poured into a gel tray containing comb.
7. It was allowed to solidify for 20 minutes.
8. The gel tray was kept into a gel tank containing 0.5 X TEB buffer. The
buffer
9. Level in a tank should be maintained above the gel tray.
10.The comb was then removed gently to avoid damage of the wells; the gel is
now ready for loading.
11.The DNA samples were loaded and the electrophoresis tank was connected
to the power pack & the voltage was set to 100V
12.The bands were observed under UV transilluminator.

2.8 RESTRICTION

The PCR product obtained is then restricted with Xho1 and

dephosphorylated for it to be used in ligation. Here the restriction is done with a
single enzyme Xho1.
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PRINCIPLE

Restriction endonucleases are enzymes that recognize & cut a specific

sequence of DNA. They are member of large group of enzymes called nucleases
which generally breaks the phosphodiester bond that link adjacent nucleotides in
DNA. Endonucleases cleave DNA at internal position. Restriction endonucleases
are categorized into three general groups (Types I, II and III) based on their
composition and enzyme cofactor requirements, the nature of their target sequence,
and the position of their DNA cleavage site relative to the target sequence. They
cut DNA at sites outside of their recognition sequence. Using ATP as energy
source, the enzyme move along the DNA molecule from recognition site to
cleavage site.

REAGENTS REQUIRED

1. Xho1 enzyme 1 µl
2. lip A gene 30 µl
3. Buffer 3 6 µl
4. 10X BSA 6 µl
5. Double distilled water 17 µl
REACTION VOLUME 60 µl

PROCEDURE

1. The reaction mixture for 40μl was prepared by taking water, DNA,
buffer, BSA solution and restriction enzymes sequentially.
 25

2. The mixture was incubated at 37˚C for 3hours for digestion to occur.

3. The digested sample was screened by 1 % Agarose gel electrophoresis

2.9 PURIFICATION

The restricted lip A gene PCR product was the purified before continuing on
to ligation.
REAGENTS REQUIRED
1. Sample ( gene)
2. Isopropyl alcohol (IPA)
3. QG buffer
4. Ethanol
5. PE buffer
6. MQ water
7. Columns

PROCEDURE

1. 80 µl of sample was added to 250ul of QG buffer and mixed well

2. 80 µl of IPA was then added to this mixture and mixed well
3. This is then poured onto a column and was centrifuged at 13000 rpm for 1
min.
4. The flow through from the column was discarded and then this step is
again repeated.
 26

5. 0.75ml of PE buffer and ethanol in the ratio of 1:3 was added to the
column.
6. Again centrifuge the column at 13000 rpm for 1 min.
7. Decant the flow through and dry spin the column.
8. The column is then placed in a new eppendorf tube and eluted it with 15 µl
MQ water. This is taken as elute1 (E1).
9. Again elute it with 15 µl MQ water and take it as elute2 (E2).
10.The E1 and E2 were run on 1% Agarose gel to confirm the purification of
gene.

2.10 STOCK PREPARATION: (30% GLYCEROL STOCK)

1. Add 300µl glycerol to empty eppendorf

2. Add 700 µl of sample (pKLAC2 DH5α) to the tube and mix thoroughly
3. Seal it with parrafin

2.11 INOCULATION

1. 3 µl of ampicillin was added to 3ml of LB medium in a test tube

2. 50 µl of pKLAC2 DH5α (glycerol stock) to the tube
3. Keep at 37˚C for overnight

2.12 PLASMID EXTRACTION

After restriction the restricted gene has to be ligated in the pKLAC2

DH5α maintenance host for the cloning process. For this the plasmid has to be first
extracted and then restricted so that the lipase A gene can be incorporated in it.
 27

REAGENTS REQUIRED

1. P1 buffer
2. P2 buffer
3. N3 buffer
4. QIA prep spin column
5. PB buffer
6. PE buffer
7. Ethanol
8. Double distilled water

PROCEDURE
1. Aliquot the sample (pKLAC2 DH5α) in test tube into 2 eppendorf tubes
2. Centrifuge at 4000rpm for 10min to pellet it
3. Add 250 µl of P1 buffer to the pellet and resuspend it
4. Vortex for 1min
5. Add 250 µl of P2 buffer and mix thoroughly by inverting the tubes 2 times
6. Add 300 µl N3 buffer and mix immediately and thoroughly by inverting the
tubes 4-6 times
7. Centrifuge for 10min at 13000 rpm
8. Apply the supernatant to the QIA prep spin column by decanting or pipetting
9. Centrifuge for 30-60sec and collect the flowthrough
10.Wash the QIA prep spin column by adding 0.5ml PB buffer and centrifuge
for 30-60sec. Discard the flowthrough
 28

11.Wash QIA prep spin column by adding 0.75ml (200 µl PE buffer + 800 µl
ethanol) of PE buffer and ethanol in ratio 1:4 and centrifuge for 30-60sec.
Discard the flowthrough
12.Dry spin the column
13.To elute the DNA , place the QIA prep column in a clean 1.5ml eppendorf
tube and add the following:
i. 70 µl water in column for 1 min and spin. Collect flowthrough
E1
ii. 50 µl water in column for 1 min and spin. Collect flowthrough
E2
iii. 30 µl water in column for 1 min and spin. Collect flowthrough
E3

The DNA is the confirmed by running the elutes (E1, E2 and E3) in a 1% agarose
gel.
 29

Figure 2.1 Plasmid Extraction

2.13 RESTRICTION

The plasmid obtained is then restricted with Xho1 and

dephosphorylated for it to be used in ligation. Here the restriction is done with a
single enzyme Xho1.
 30

Small scale restriction large scale restriction

Xho1 1 µl 1 µl
10xbuffer 2 µl 4 µl
10x BSA 2 µl 4 µl
pKLAC2 E1 8 µl 20 µl
Double distilled water 7 µl 11 µl
REACTION VOLUME 20 µl 40 µl

PROCEDURE

1. The reaction mixture for 40μl was prepared by taking water, DNA,
buffer, BSA solution and restriction enzymes sequentially.

2. The mixture was incubated at 37˚C for 2hours for digestion to occur.

3. The digested sample was screened by 1% Agarose gel electrophoresis.

2.14 DEPHOSPHORYLATION

Antarctic Phosphatase catalyzes the removal of 5´ phosphate groups from

DNA and RNA. Since phosphatase-treated fragments lack the 5´ phosphoryl
termini required by ligases, they cannot self-ligate. This property can be used to
decrease the vector background in cloning strategies.
 31

REACTION MIXTURE
Antarctic Phosphatase enzyme buffer 1.5 µl
Antarctic Phosphatase enzyme 1.0 µl
pKLAC2 elute 1 15 µl

Incubate for 15 min at 37°C

Inactivate immediately at 65°C for 5 min.
The Antarctic Phosphatase has 90% efficiency that is out of 10 DNA molecules
only 9 are dephosphorylated. Antarctic Phosphatase buffer is added, so that overall
75% of plasmid DNA molecules are dephosphorylated.

Applications of antarctic phosphatase

 Removing 5´ phosphoryl groups from nucleic acids

 Preparing templates for 5´end labeling
 Preventing fragments from self-ligating
 Removal of dNTPs and pyrophosphate from PCR reactions

2.15 PURIFICATION

The restricted plasmid was the purified before continuing on to ligation.

REAGENTS REQUIRED

 Sample (plasmid)
 Isopropyl alcohol (IPA)
 QG buffer
 32

 Ethanol
 PE buffer
 MQ water
 Columns

PROCEDURE

1. 80 µl of sample was added to 250 µl of QG buffer and mixed well

2. 80 µl of IPA was then added to this mixture and mixed well
3. This is then poured onto a column and was centrifuged at 13000 rpm for 1
min.
4. The flow through from the column was discarded and then this step is again
repeated.
5. 0.75ml of PE buffer and ethanol in the ratio of 1:3 was added to the column.
6. Again centrifuge the column at 13000 rpm for 1 min.
7. Decant the flow through and dry spin the column.
8. The column is then placed in a new eppendorf tube and it is eluted with 15
µl MQ water. This is taken as elute1 (E1).
9. Again elute it with 15 µl MQ water and take it as elute2 (E2).
10.The E1 and E2 were run on 1.5% Agarose gel to confirm the purification of
plasmid.

2.16 LIGATION

PRINCIPLE
Ligation process involves the formation of four phosphodiester bonds i.e
two at each end of the molecule such bonds can be formed. The generation of
 33

phosphodiester bond between neighbouring 3’OُH end and 5’Pُ ends of double
stranded DNA chain is catalyzed by DNA ligase and they require coenzymes like
NAD+ & ATP.

Figure 2.2 A pictorial example of how a ligase works (with sticky ends)
 34

Figure 2.3 A simple mechanism representing the role of DNA Ligase

 35

REAGENTS REQUIRED

Reaction Volume: 20µl

pKLAC2 2 µl
Lip A gene 15 µl
10X buffer 2 µl
DNA ligase 1 µl
Doubled distilled water 0 µl
Vector control
pKLAC2 2 µl
Lip A gene insert 0 µl
10X buffer 2 µl
DNA ligase 1 µl
Doubled distilled water 15 µl

PROCEDURE

1. The ligation mixture for 20μl was prepared by taking water, insert (Lipase A
gene), and vector digest & ligase enzyme sequentially.
2. The mixture was incubated at 16˚C for overnight
3. The ligated sample was screened by agarose gel electrophoresis.

The ligated cells were then transformed in E. coli and then cloned.
 36

2.17 COMPETENT CELLS PREPARATION AND TRANSFORMATION

PRINCIPLE
E.coli cells take up only limited amount of DNA under normal
circumstances E.coli cells when soaked in an ice cold salt solution were more
efficient in DNA uptake than by unsoaked cells. CaCl2 causes the DNA to
precipitate onto the outside of the cells or perhaps the salt is responsible for some
kind of change in cell wall that improves DNA binding. E.coli cells and plasmid
interact productively in an environment of Ca ions & low temperature (0-5˚C). The
calcium ions destabilize the cell membrane and a calcium phosphate DNA
complex is formed which adheres to the cell surface and is resistant to DNases.
The DNA is taken up during a heat shock step where the cells are exposed briefly
to a temperature at 42˚C.

REAGENTS REQUIRED

1. Overnight bacterial culture

2. 0.1M ice cold CaCl2 solution
3. Plasmid suspension
4. LB medium
5. LB/amp+ agar plates
 37

PROCEDURE

1. From overnight inoculated culture of DH5α host 1ml of culture was

inoculated in 50ml LB medium
2. After 1 hour 45 min the flask was taken and kept at 4˚C
3. Transfer the culture in 2 centrifuge tubes equally and keep it in ice
4. Then centrifuge at 4000 rpm for 10 min
5. Discard the supernatant. Add small amount of CaCl2 solution and
resuspend the pellet by tapping. Then add CaCl2 solution to 3/4th of the
tube
6. Incubate for 30 min at 4˚C
7. Centrifuge at 4000rpm for 10 min
8. Discard supernatant. Add small amount of 0.1M CaCl2 solution and
resuspend the pellet by tapping. Then add CaCl2 solution to 1/4th of the
tube
9. Incubate for 20 min at 4˚C
10.Centrifuge at 4000 rpm for 10 min
11.Discard the supernatant. Add 400 µl of CaCl2 solution to one tube and
resupend it. Pool it to the other tube and add a little more according to
thickness of the pellet
12.Incubate for 25-30 min at 4˚C
 38

2.18 TRANSFORMATION

PRINCIPLE

In molecular biology, transformation is the genetic alteration of a cell

resulting from the uptake, genomic incorporation, and expression of environmental
genetic material (DNA). Transformation occurs most commonly in bacteria, both
naturally and artificially, and refers to DNA taken up from the environment
through their cell wall. Bacteria that are capable of being transformed are called
competent. New genetic material can also be transferred to cells through
conjugation or transduction. Conjugation involves cell-to-cell contact between two
different bacterial cells, with the DNA being transferred from one bacterial cell to
the other. In transduction, viruses called bacteriophages inject the foreign DNA
into their host. Introduction of foreign DNA into eukaryotic cells is usually called
"transfection". Transformation is also used to describe the insertion of new genetic
material into nonbacterial cells including animal and plant cells.

ARTIFICIAL COMPETENCE

Artificial competence is not encoded in the cell's genes. Instead it is induced

by laboratory procedures in which cells are passively made permeable to DNA,
using conditions that do not normally occur in nature. Calcium chloride
transformation is a method of promoting competence. Chilling cells in the presence
of divalent cations such as Ca2+ (in CaCl2) prepares the cell membrane to become
permeable to plasmid DNA. Cells are incubated on ice with the DNA and then
briefly heat shocked (e.g. 42 °C for 30–120 seconds), which causes the DNA to
enter the cell. This method works very well for circular plasmid DNAs. An
 39

excellent preparation of competent cells will give ~108 colonies per microgram of
plasmid. A poor preparation will be about 104/μg or less. Good non-commercial
preps should give 105 to 106 transformants per microgram of plasmid.

Figure 2.4 Artificial Transformation

PROCEDURE

1. 100 µl DH5α competent cells + 7 µl pKLAC2 lip A

100 µl DH5α competent cells + 7 µl vector (pKLAC2)
100 µl DH5α competent cells
2. Keep it in ice for 30 min
3. Then give heat shock at 42˚C for 90 sec
4. Then again place it in ice for 10-15 min
 40

After the above process the cells are to be grown in LB/amp+ (for negative
selection) agar plates in 37˚C for 16 hrs and then the plates are checked for growth
of transformed colonies.

2.19 PATCHING

After the plates are checked for the presence of colonies they are to be
patched in new LB/amp+ agar plates. This is done in the following way.
1. Check the plate for presence of colonies
2. Take a loop and with it take a colony form the transformed plates and
streak on a new LB/amp+ agar plates.
3. Mark the streaked places and allow it grow overnight
4. After 12 hours check for the growth of colonies.

2.20 LYSATE PCR

Lysate PCR is necessary for conformation of the presence of plasmid
in the transformed colonies.

REAGENTS REQUIRED
2X Master Mix 55 µl
pKLAC2 promoter forward primer 5.5 µl
pKLAC2 TT reverse primer 5.5 µl
10 colonies + 1 vector control 11 µl
Double distilled water 33 µl
REACTION VOLUME 110ul
 41

PROCEDURE

1. Take the 10 colonies from the patched and suspend it in 50 µl distilled

water
2. Heat at 100˚C for 10 min
3. Immediately place it in ice for 5 min
4. Centrifuge at 10000 rpm for 2 min and take only the supernatant for the
PCR
5. The PCR reaction was done ad explained before

2.21 CONFIRMATION PCR:

A confirmation PCR is done to check whether the required gene of interest is
inserted into the plasmid. This PCR is done to all the positively screened patches
(7, 8, 9 and10).

Negative control: pKLAC 2

Test : patches 7-10

2x MM : 25 µl
LipA forward : 2 .5 µl
KLAC TT reverse : 2 .5 µl
Template : 5 µl
Double distilled water : 15 µl
REACTION VOLUME : 50 µl
Here the template includes patch 7, 8,9,10 and a negative control.
 42

Positive Control : pPICZ alpha B lipA

Negative control : pKLAC 2
Test : patches 7-10

2x MM : 30 µl
LipA forward : 3 µl
Lip A reverse : 3 µl
Template : 6 µl
Double distilled water : 18 µl
REACTION VOLUME : 60 µl

Here the template includes patch 7, 8,9,10, a negative control and a positive
control.
 43

CHAPTER 3

RESULTS AND DISCUSSION

3.1. PCR AMPLIFICATION OF C.antartica LIPASE A ENCODING GENE

The PCR amplification of Lipase A was carried out using forward primer
with the Xho1 restriction enzyme site and reverse primer with Xho1 restriction
enzyme site. The cloning of lipases in the vector pKLAC2 was designed by
utilizing the XhoI site upstream of Kex2 cleavage site in vector so as to facilitate
the native expression of the recombinant protein. The forward primers also had the
sequences of the Kex2 cleavage sites downstream of the Xho I restriction site and
following this were the first 18 bases of the mature lipase A coding sequences. The
utilization of Xho I restriction site and Kex2 cleavage site enabled the expression
of recombinant lipase with their native N-terminus, after the cleavage of the signal
sequence from the expressed proteins by Kex2 proteinase. The reverse primers
utilized the ending 20 bases which code for the C-terminal regions of the lipases A
along with its stop codon.
XHO1 RESTRICTION SITE
KEX2 CLEAVAGE SITE

CALAf - 5’-CCGCTCGAGAAAAGAGCGGCGCTGCCCAACCCC-3’
CALAr - 5’-CTAGCTCGAGCTAAGGTGGTGTGATGGGGC-3’

XHO1 RESTRICTION SITE

 44

Agarose gel electrophoresis of PCR amplified lipase A gene is shown in the figure
3.1.1

Figure 3.1.1 Analysis of PCR amplified lipase A gene

1 2 3 4 5

1.4 kb

4 – 1 kb marker
2 – PCR amplified Lipase A gene from CAL A (1µl)

3.2 RESTRICTION AND PURIFICATION OF LIPASE A GENE

The PCR amplified Lipase A gene was restricted by Xho1 restriction

enzyme after three hours incubation. The restricted sample of Lipase A gene was
purified to remove excess salts and to increase the lipase A gene concentration.
The Agarose gel electrophoresis of purified samples – elute 1 and elute 2 of Lipase
A gene is shown in the figure 3.2.1.
 45

Figure 3.2.1 Analysis of purified samples of Lipase A gene

1 2 3 4

1.4 kb

1 – 1 kb marker
2 – First elution of Purified sample of lipase A gene (0.5 µl)
4 – Second elution Purified sample of Lipase A gene (0.5 µl)

3.3 pKLAC2 – EXTRACTION AND RESTRICTION

Agarose gel electrophoresis of the pKLAC2 plasmid extracted is shown in

the figure 3.3.1. The plasmid extraction was carried out by standard kit method.
 46

Figure 3.3.1 Analysis of pKLAC2 plasmid extracted by standard Kit method

1 2 3 4 5 6 7

1 – 1 kb marker
4 – First elution sample of pKLAC2 Plasmid
5 – Second elution sample of pKLAC2 Plasmid

Plasmid extracted was restricted with Xho1 restriction enzyme. pKLAC2

plasmid was completely restricted after two hours incubation. Agarose gel
electrophroesis of restricted plasmid is shown in the figure 3.3.2. The pKLAC2
plasmid which was restricted with single enzyme xho1 was dephosphorylated to
prevent self-ligation of sticky ends. Antarctic Phosphatase was used to
dephophorylate pKLAC2 plasmid. Antarctic Phosphatase catalyzes the removal of
5´ phosphate groups from DNA and RNA. Since phosphatase-treated fragments
lack the 5´ phosphoryl termini required by ligases, they cannot self-ligate. This
property can be used to decrease the vector background in cloning strategies.
 47

Figure 3.3.2 Analysis of restricted pKLAC2 plasmid

1 2

1 - Uncut pKLAC2 plasmid

2 - Restricted pKLAC2 plasmid

3.4 CLONING

The restricted lipase A gene was ligated with T4 ligase enzyme

Two Ligation mixtures containing lipase A gene and PKLAC2 vector in the
molar ratios of 3:1 and 5:1 were prepared and incubated at 16°C for 16 hours.
Agarose gel electrophoresis of two the samples is shown in the figure 3.4.1

Both the plasmid and lipase A were restricted using single restriction
enzyme Xho1. This kind of cloning is categorized as unidirectional cloning.
 48

Because of this, the ligation mixtures were confirmed for proper orientation
through PCR using pKLAC2 forward and lipase A reverse as primers.

Figure 3.4.1 Analysis of ligation mixtures of Lipase A gene with PKLAC2

plasmid using pKLAC2 forward and lipase reverse as primers.

1 2 3 4

1.8kb

1 – 1 kb marker
2 – First ligation sample (1 µl)
3 – Second ligation sample (1 µl)

3.5 TRANSFORMATION

pKLAC2 vector with lipase A gene insert was transformed into E. coli TOP
10 F’ strain by calcium chloride method and selected on LB medium with
amphicillin.. Among the transformants ten colonies for lipase A gene were
analyzed by patching the colonies on fresh LB agar plates with amphicillin. And
lysate PCR analyses of all these transformants were done. The plasmids of the
positive transformants were confirmed for correct orientation of inserts in the
vector and that screening of transformants were carried out using the vector
 49

forward(pKLAC2 forward) and the insert reverse(lipase A gene reverse) primers.

The presence of the lipase A gene inserts in the Patch 7, 8, 9, and 10 were
confirmed. A total of 4 positive transformants, having the plasmid with CALA
inserts were obtained.

The positive E. coli TOP 10 F’ strain transformants 7, 8, 9 and 10 were

cultured overnight in LB medium with amphicillin and the plasmid DNA was
extracted using manual methods. The plasmids were confirmed for the correct
orientation of the inserts in the vector by PCR using the vector forward (pKLAC2
forward) and the insert reverse (lipase A reverse) primers. Agarose gel
electrophoresis of these analyses is shown in figures 3.5.1. Here the genomic DNA
was used as positive control and the vector without insert was used as the negative
control.

Figure 3.5.1 Lysate PCR analysis of E. coli transformants (7-10) having the
plasmids with the inserts lipase A gene using pKLAC2 forward and Lip A
reverse as primers.
1 2 3 4 5 6 7

1.8 kb

1 – Mol wt marker (1 kb)

2, 3, 4 & 5 – positive E. coli transformants having lipase A gene insert (1µl)

6 – Negative control (1µl)

 50

The plasmid extracted from the positive transformants 7, 8, 9 and 10 by

manual method are shown in figure 3.5.2. Due to manual method of extraction,
contamination was there as shown in the way in the figure.

Figure 3.5.2 Analysis of plasmids extracted by manual method from patches

of 7, 8, 9, and 10 Top 10 E.coli Positive transformants

1 2 3 4 5 6

2, 3, 4 and 5 – plasmids extracted from Patches of 7, 8, 9 and 10 positive E.coli

positive transformants(1.5µl).
 51

CHAPTER 4

CONCLUSION

The CALA gene from Candida antarctica was successfully amplified by

PCR using forward and reverse primers with Xho1 restriction site. The lipase A
gene was purified and ligated with pKLAC2 plasmid restricted with Xho1
restriction enzyme. The recombinant plasmid pKLAC2/CALA was constructed
successfully. The recombinant plasmid was successfully transformed into E.coli
TOP 10F’ strain by chemical transformation. The transformant E.coli cells were
screened to verify successful transformation of recombinant plamid through Lysate
PCR.

The recombinant plasmid pKLAC2/CALA from the positive transformant

E.coli TOP10F’ cells can be linearised and transformed into the expression host
K.lactis for further expression studies. K.lactis is an ideal host for expressing of
heterologous proteins.
 52

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