25

CHAPTER 2

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Strains and plasmids

The yeast strain Candida antarctica JCM 10316 was purchased

from Japan Collection of Microorganisms, RIKEN BioResource Centre.

The Lipase B gene was amplified from the genomic DNA of this strain by

polymerase chain reaction (PCR).

The Pichia pastoris expression kit was purchased from Invitrogen

(San Diego, CA). The vectors used for CALB cloning were pPICZ B,

pGAPZ B and pPIC9k. The vector maps are given in the Figures 2.1 to 2.3

and features of these plasmids in Table 2.1 to 2.3. The E. coli strain Top 10F’

was used for genetic manipulation of the shuttle vector and for propagation of

the expression vector. Pichia pastoris strains GS115 (his4) and KM71H were

used to express the recombinant C. antarctica lipase B.

2.1.2 Biochemicals and Reagents

PCR reactions were performed by using PCR Master Mix kit from

Bangalore Genei. Restriction enzymes, T4 DNA ligase and RNase A were

purchased from MBI Fermentas (Canada). The DNA gel - extraction kit and

the plasmid extraction spin mini - prep kit were bought from Qiagen

(Germany). Media components such as yeast extract, bacto peptone, bacto

26

tryptone and agar were obtained from HI-MEDIA (India). The chemical

reagents were obtained from Merck or SRL (India).

2.1.3 Culture media

The following media were used.

Low salt LB agar medium: tryptone 10 g/L; yeast extract 5 g/L;

sodium chloride 5 g/L; agar (low melting) 15 g/L; pH 7.0

YPD agar medium: yeast extract 10 g/L; peptone 20 g/L;

dextrose 20 g/L; agar (low melting) 20 g/L

Tributyrin agar medium: yeast extract 10 g/L; peptone 20 g/L;

tributyrin 10 mL/L; agar (low melting) 15 g/L; methanol

5 mL/L

MDH agar medium: yeast nitrogen base 13.4 g/L, dextrose

20 g/L; biotin 0.4 mg/L; histidine 40 mg/L; agar 20 g/L

MMH agar medium: yeast nitrogen base 13.4 g/L, methanol

5 ml/L; biotin 0.4 mg/L; histidine 40 mg/L; agar 20 g/L

YPG medium: yeast extract 10 g/L; peptone 20 g/L; glycerol

20 g/L

Synthetic minimal glycerol (SMG) medium: yeast nitrogen base

13.4 g/L, glycerol 10 g/L; biotin 0.4 mg/L; histidine 40 mg/L

Synthetic minimal methanol (SMM) medium: yeast nitrogen

base 13.4 g/L, methanol 5 ml/L; biotin 0.4 mg/L; histidine

40 mg/L

Fermentation basal salts medium: phosphoric acid 85%

27 ml/L; calcium sulfate 0.9 g/L; potassium sulfate 18 g/L;

27

magnesium sulfate.7H2O 15 g/L; potassium hydroxide 4.13 g/L;

glycerol 40 g/L; PTM solution 4.0 ml/L

PTM1 trace elements solution: cupric sulfate.5H2O 6.0 g/L;

sodium iodide 0.08 g/L; manganese sulfate.H2O 3.0 g/L; sodium

molybdate.2H2O 0.2 g/L; boric acid 0.02 g/L; cobalt chloride

0.5 g/L; zinc chloride 20 g/L; ferrous sulfate.7H2O 65 g/L ;

biotin 0.2 g/L; sulphuric acid 5.0 ml; water to 1.0 litre.

2.2 MOLECULAR BIOLOGICAL METHODS

2.2.1 Yeast genomic DNA extraction

The yeast genomic DNA was extracted by the ‘smash and grab’

protocol as described in Ausubel (2005) which involves lysis of the yeast cells

using glass beads. The clones were grown at 25 ºC, 200 rpm for 48-72 hr, in

50 ml YPD medium. The cells were harvested by centrifugation and 100-150

mg of the pellet was resuspended in 200 µl lysis buffer containing 2 % Triton

X100, 1 % SDS, 0.1 M NaCl, 0.01 M Tris.HCl, pH 8.0 and 1 mM EDTA.

Equal volumes of phenol/chloroform mixture (1:1) and the culture were added

in a micro centrifuge tube and mixed well by pipetting and incubated for

2 min at room temperature. The lysate was then centrifuged at 10,000 rpm for

5 min and two immiscible phases were obtained. The top phase having the

genomic DNA was transferred to another micro centrifuge tube and two

volumes of 100 % ethanol was added to precipitate the DNA. The pellet was

collected by centrifugation and dissolved in TE buffer (10 mM Tris.HCl,

pH 8.0, 1 mM Na2EDTA) containing 10 µl (concentration 30 g / mL) RNase

A, at room temperature. After incubation for 1 hour, the genomic DNA was

precipitated at room temperature by addition of 2 volumes of ethanol and

18 µL of 5 M ammonium acetate (per ml ethanol) and centrifuged for 10 min

at 13,000 rpm. The pellet was finally reconstituted in a convenient volume TE

28

buffer and the quality of genomic DNA extracted was analyzed in 0.5 %

agarose gel electrophoresis.

2.2.2 Plasmid DNA extraction

The plasmid DNA isolation from Escherichia coli was done

according to the instructions in the QIAprep Spin Miniprep Plasmid kit

(Qiagen). E. coli Top 10 F’ cultures transformed with the required plasmids

were grown in 15 ml low salt LB medium with zeocin antibiotic for about 6 to

8 hours and were used to extract the plasmids. The QIAprep plasmid isolation

procedure consisted of three basic steps: i) preparation and clearing of a

bacterial lysate, ii) adsorption of DNA onto the QIAprep membrane and iii)

washing and elution of plasmid DNA. The extracted plasmids were analyzed

using agarose gel electrophoresis.

2.2.3 Agarose gel electrophoresis

The agarose gel electrophoresis was carried out for analysis and

separation of DNA fragments based on the molecular sizes. Agarose gels

were cast with concentrations of agarose (low melting, USB) ranging between

0.6 - 1.2 %, depending on the molecular weight of the DNA fragments to

separate. The buffer TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) was

used as electrophoresis buffer, and a mixture of glycerol 30 % (w / v),

bromophenol blue (BPB) 0.2 % (w / v), EDTA 25 mM, pH 7.5 was used as

6X - DNA loading buffer. The constant DC voltage of 100 V was applied

using Medox-Bio 1250 Power pack. After electrophoresis, the separated DNA

fragments on the ethidium bromide stained gel were visualized under UV

light using a Molecular Imager Gel Doc XR+ System (Bio-Rad Corporation).

29

2.2.4 DNA cleanup from agarose gels

The QIAquick gel extraction kit (Qiagen) is used for the cleanup of

upto 10 µg of DNA fragments from agarose gels. The kit uses a spin - column

technology which has a selective DNA binding silica membrane. As

mentioned in the kit protocols, the agarose gel was solubilized and added to

the column. The DNA from the gel was adsorbed to the silica membrane

while the contaminants do not bind but flow through the silica column. The

pure DNA was eluted with Tris buffer (10 mM Tris-Cl, pH 8.5) or water and

was analyzed by agarose gel electrophoresis.

2.2.5 Transformation into E. coli

E. coli transformation was done according to the modified method

given by Sambrook and Russell (2001). The competent E. coli cells were

grown to an OD600nm of 0.4 – 0.6 in low salt LB medium. The culture was

transferred asceptically into sterile oakridge tubes and centrifuged at

6000 rpm for 10 minutes at 4 °C. The pellet was resuspended in 25 ml of ice

cold 0.1 M calcium chloride and incubated in ice for 30 minutes. The cells

were centrifuged again at 6000 rpm for 10 minutes at 4°C. The cell pellet was

resuspended in 10 ml of 0.1 M calcium chloride and incubated in ice for

15 minutes. The cells were centrifuged again at 6000 rpm for 10 minutes at

4°C and the pellet was resuspended in 500 µl of 0.1 M CaCl2. To 100 µl of

the competent cells, less than 50 ng of plasmid DNA was added and incubated

in ice for 30 minutes. The transformation into E. coli was done by giving a

heat shock at 42 ºC for 90 seconds. The tubes were transferred again to (ice)

4 °C and added 1 ml of sterile low salt LB medium, mixed well and incubated

at 37 °C for 30 minutes. The tubes were centrifuged at 6000 rpm for

5 minutes at room temperature (37 ± 2 °C). The pellet was resuspended with

100 µL of low salt LB medium and the transformed E. coli colonies were

30

plated on appropriate antibiotic containing LB agar plates and incubated at

37 °C incubator for 12 hours to appearance of colonies.

2.2.6 Lysate PCR confirmation of E. coli clone

The E. coli colonies obtained after transformation were then patched

on to another low salt LBagar plates and incubated at 37 °C for 12 hours. The

patches obtained were then scraped out and transferred into 1.5 mL micro

centrifuge tubes and lysed by incubating the cells at 100°C (boiling water

bath) for 10 minutes and immediately quenched into ice. PCR was carried out

with CALB forward and reverse primers to check the presence of CALB

insert.

2.2.7 Electroporation of vector in Pichia Pastoris

The P. pastoris cells were grown in a 50ml YPD medium, 30 °C,

200 rpm until the OD600nm reached 1.0 - 1.5. The cells were pelleted by

centrifugation at 6000 rpm, 4 °C, 10 minutes. The pelleted cells were then

made competent by washing twice with 1.0 M ice cold sorbitol. The

competent cells were electroporated with the linearized plasmid using BTX

electroporator at 1500 V. The cells were kept for two hours incubation at

30 °C in orbital shaker (~ 200 rpm). The cells were then plated onto screening

agar plates. The plates were incubated at 30 °C for 4 – 5 days. The individual

colonies obtained were patched on YPD agar plate.

2.2.8 Screening of Pichia transformants for the expression of

recombinant lipases

Screening of Pichia transformants for the secretory expression of

functional lipases was done by stabbing the clones on tributyrin agar plates

31

containing 0.5 % methanol. Lipase secreting clones were identified by a

clearance zone of hydrolysis of the tributyrin around the colony.

2.2.9 Determination of Mut Phenotype of recombinant Pichia clones

The methanol utilisation phenotype is determined to confirm the

Mut+ phenotype of the recombinant Pichia strains and differentiate those

MutS phenotype of Pichia strains which show slower growth on methanol

medium. The recombinant Pichia transformants are patched in a regular

pattern using sterile toothpicks on an MMH plate first, and then on MDH

plate. The plates are incubated for 2 or more days in a 30 C incubator.

2.3 FERMENTATION AND RECOMBINANT EXPRESSION IN

PICHIA PASTORIS

2.3.1 Expression analysis in shake flask cultivations

Expression study of selected Pichia clones GS115 / pPICZ B / CALB,

KM71 / pPICZ B / CALB and GS115 / pGAPZ B / CALB was done in

shake flask. The experiment was carried out in 500 mL conical flasks with the

YPG complex media and a defined SMG media. The shake flasks containing

100 mL of the media were inoculated with 1 ml of Pichia culture, which were

grown in 3 ml test tubes for 24 hours. The shake flasks were kept incubated

for approx. 36 – 48 hours at 30 C in a shaker and after the cultures attained

stationary OD600nm (around 40), methanol was added to the flasks at a

concentration of 0.5 % for every 24 hours. Samples of the induced cultures

were withdrawn for every 12 hour intervals and were analyzed for the growth

and recombinant expression profiles for upto 4 days post - induction. Samples

were collected for further analysis like activity profile, amount of protein

expressed after induction by SDS - PAGE and protein estimation by Bradford

method.

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2.3.2 Bioreactor setup

Fermentation experiments were performed in Bioengineering KLF

2000 stirred vessel fermenter with 3.7 litre total volume and control modules

for pH, dissolved oxygen, temperature and rpm. The fermenter is an in - situ

sterilizable glass bioreactor, and has a bottom mounted motor for driving the

agitation. An Ingold sterilizable pO2 electrode based on the amperometric /

polarographic principle was used in determining the dissolved oxygen

concentrations. The pH measuring unit is based on the Ingold sterilizable

combination electrode and the temperatures were measured by Pt100

temperature measuring element. Peristaltic pumps from Watson-Marlow

(Wilmington, MA) were used for pumping the inoculum and for feeding of

other nutrients. The following parameters were monitored and controlled

during the fermentation process:

Temperature (28 ºC) Controlled by chill water supply from chilling

unit and heating by a heater rod inside the

vessel.

pH (4.8) Adjusted using 28 % NH4OH / 10 % H3PO4

Agitation (600 – 1500 rpm) To maintain DO above the set point

Dissolved oxygen (> 20 %) Airline supplemented with pure oxygen after

agitation reaches maximum

Antifoam Polypropylene glycol was added to prevent

excessive foaming

33

2.3.3 Inoculum preparation

The starting inoculum was prepared from frozen glycerol stocks

stored at – 80 °C. The frozen cells were thawed and inoculated in 3 mL YPD

medium in a sterile 9 mL test tube and grown for 24 hours at 28 °C. This

3 mL pre - inoculum was then transferred aseptically to 50 ml sterile

inoculum medium in 250 ml Erlenmeyer flasks. The culture was grown for

36 hours at 28 °C and 200 rpm in a gyratory water - bath shaker and then

inoculated aseptically into the fermenter with 1.8 litre Basal salts medium.

2.3.4 High Cell Density Fermentation

The fermentations of P. pastoris were started in batch mode by

sterilizing 1.8 litre of basal salts medium in the fermenter. After sterilization

and cooling to 28 °C, the medium was adjusted to pH 4.80 with 28 %

ammonium hydroxide, and 4 mL PTM1 trace salts / litre of basal salts medium

was added aseptically. The fermenter was then inoculated with 50 mL culture

from shake flasks and the biomass concentrations were monitored with time.

As the culture grows, the DO decreases and when the glycerol in the medium

is completely consumed the DO rises sharply. This indicated the end of the

initial batch phase and the second step of glycerol fed-batch was started.

A glycerol feed (100 % (v/v) glycerol) was then maintained for 4 hours at a

growth - limiting rate of 15 mL / L / h (9.5 gm / L / h of glycerol). After the

limiting glycerol fed batch period, the culture was induced with a methanol

feed (100 % methanol + 4 mL PTM1 L-1

methanol) at 1 mL / L / hr. After the

culture was fully adapted to methanol utilisation, which was indicated by zero

residual methanol concentrations, the methanol feed rate was gradually

increased over a period of 6 hours to 6 mL / hr and the fermentation was

continued for a further 48 to 72 hours. 1 mL culture samples were withdrawn

and centrifuged at 12,000 rpm for 5 minutes and the supernatants were

collected and frozen for further analysis.

34

All the bioreactor studies were carried out on the basis of dissolved

oxygen levels in the bioreactor. A study on production of lipase after

induction at different cell densities was carried out. In the KM71 strains

containing the pPICZ B - CALB clone, the bioreactor strategies were

followed as that used for the GS115 strains and the one particular study was

carried out where in 5 % H2O2 (hydrogen peroxide) was fed along with 95 %

methanol during the induction phase to see the effect of increase in

productivity of lipase B as exogenously added H2O2 induces effective

breakdown of formaldehyde by inducing catalase and cytochrome c

peroxidase (CCP).

2.4 ANALYTICAL METHODS

2.4.1 Biomass analyses

Cell biomass was monitored by the method of turbidometric

measurement or light scattering. Optical density (OD600) was used to

represent the cell concentration which is proportional to the amount of light

scattered by the culture broth. The turbidity or light scattering was measured

at the wavelength of 600 nm where the absorption by the broth components is

minimum. The spectrophotometer (Hitachi U-2000) was used to quantify the

amount of turbidity of culture broth.

2.4.2 Lipase Assay

The pH-stat method is a general method used in the lipase assay.

The principle of the assay is that the lipase enzyme acts on triacylglycerols to

liberate free fatty acids with time, is neutralized by titration with the addition

of sodium hydroxide to maintain pH at a constant end point value for fixed

time intervals.

35

Figure 2.1 The pH-stat setup for lipase assay

Courtesy: Beisson (2000)

Activity of expressed clone was analyzed by pH titration method.

Tributyrine was used as a substrate for Lipase B enzyme.

One unit (U) of lipase activity was defined as the amount of enzyme

that liberates 1 µmol fatty acid per minute under assay conditions.

ft

d1000CV)mL/U(Activity (2.1)

where V – Volume of titrant NaOH added (ml)

C – Concentration of NaOH (molarity M)

1000 – Conversion of milliequivalent to microequivalent

d – Dilution factor

t – Time of titrant addition (min)

f – Volume of enzyme added (mL)

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2.4.3 Glycerol and Residual methanol analysis

Glycerol and residual methanol levels in the fermentation broth

were determined in High Performance Liquid Chromatography (HPLC -

SHIMADZU CORPORATION, JAPAN) using Aminex column (Bio-Rad).

Tripple distilled water containing 5 mM sulphuric acid was used as the mobile

phase at a flow rate of 0.7 ml / min for sample analysis. Refractive index

detector was used to detect and column temperature was maintained at 50 °C

throughout the sample analysis. Chromatogram data were collected and

analyzed by the PC - based VT - VHS software package. Glycerol and

methanol standards were prepared at concentrations ranging from 0.1 % to

5 % and used for comparison with samples.

2.4.4 Total protein analyses

Total protein in culture supernatant was quantified by Bradford

method for protein estimation from Bio - Rad, USA. The basis of color

change of Coomassie Brilliant Blue G - 250 dye due to its binding with

primarily basic (arginine) and aromatic amino acid residues of the protein.

The protein concentrations were quantified in the micro plate format, and the

absorbance readings were measured at 595 nm using an Enzyme Linked

Immunosorbent Assay (ELISA) reader (Labsystems Multiskan MS).

2.4.5 SDS-Polyacrylamide Gel Electrophoresis

The recombinant lipases were analyzed by SDS - PAGE, prepared

using 12 % resolving gel and 5 % stacking gel as follows:

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Table 2.1 Composition of resolving and stacking gel for SDS-PAGE

Components Resolving gel

(20 mL)

Stacking gel

(5 mL)

Water

30 % (w / v) acrylamide in water

with 0.8 % bisacrylamide

1.5 M Tris (pH 8.8)

1.0 M Tris (pH 6.8)

10 % SDS

10 % ammonium persulfate

TEMED

6.6 mL

8.0 mL

5.0 mL

-

0.2 mL

0.2 mL

0.008 mL

3.4 mL

0.83 mL

-

0.63 mL

0.05 mL

0.05 mL

0.005 mL

(Courtesy: Methods in molecular biology, Sambrook and Russel)

The samples of culture supernatants were mixed in sample loading

buffer containing 50 mM Tris - Cl (pH 6.8), 100 mM dithiothreitol, 2 % SDS,

0.1 % bromophenol blue and 10 % glycerol and boiled for 10 min to denature

the proteins. These samples were then applied on vertical SDS-PAGE slab

gels and subjected to electrophoresis in Tris-glycine electrophoresis buffer

containing 25 mM Tris base, 250 mM glycine (pH 8.3), 0.1 % SDS. The

resolved proteins were stained 25 % Coomassie brilliant blue R - 250 in

destaining solution (50 % water, 40 % methanol, 10 % acetic acid) for 1 hour

and then destained with 50 % water, 40 % methanol, 10 % acetic acid for 1

hour.

2.5 CLONING OF CANDIDA ANTARCTICA LIPASE B (CALB)

GENE IN pPICZ B

The primer for the PCR amplification of lipase gene was designed

without the native signal sequence of the C. antarctica lipase B. Forward

primer for lipase B was flanked with Xho I restriction site and the reverse

primer was flanked with Xba I restriction site. The C. antarctica JCM

38

10316 genomic DNA was extracted and used as the template for PCR

amplification of gene encoding CALB. The optimization of PCR conditions

were carried out by varying the denaturing temperature between 95 ºC to

98 ºC, the annealing temperatures from 50 ºC to 65 ºC, and the extension time

between 90 seconds to 120 seconds.

Oligonucleotides used as primers for the PCR:

CALBf - priming site 76-95 bp of CALB gene; XhoI restriction site

5’-CCGCTCGAGAAAAGACTACCTTCCGGTTCGGACC-3’

CALBr - priming site 1029-1011 bp of CALB gene; XbaI restriction site

5’-TGCTCTAGATTAGGGGGTGACGATGCCG-3’

–factorf - priming site 941-959 bp of pPICZ B vector

5’-CCGGAATTCATGAGATTTCCTTCAATT

The polymerase chain reaction (PCR) is used for the amplification

of defined fragments of DNA. The amplification reaction of CALB gene was

performed in PCR by using master mix which is a ready-to-use

2.5 X concentrate reagent mix, containing Taq DNA polymerase along with

the amplification buffer and dNTPs. The PCR reaction was setup in 0.2 ml

thermo tubes (ABgene, UK) and the following components were added:

Forward primer (10 pmol/µl) - 1 µL

Reverse primer (10 pmol/µl) - 1 µL

Template DNA - 10 ng - 50 ng genomic DNA

Water - 12 µL

Eppendorf HotMasterMix - 8 µL

39

The reaction tubes were loaded in Peltier Thermal Cycler

(Eppendorf personnel cycler). The PCR reaction products were finally

analyzed by agarose gel electrophoresis or stored at - 20 ºC until needed.

The PCR product of the CALB gene amplified from C. antarctica

genomic DNA was purified by Qiagen gel-extraction kit and then digested

with the restriction enzymes, XbaI and XhoI. The stock plasmid pPICZ B

provided in the Pichia expression kit (Invitrogen) was transformed to E. coli

Top10F’ host and then the plasmid was extracted in sufficient quantity.

Restriction digestions of the insert and the vector with the enzymes XhoI and

XbaI were set up and performed as suggested by the REases manufacturer.

After analysis of the restriction digest using agarose gel electrophoresis, the

DNA fragments were purified as described in QIAquick gel extraction kit.

Ligation reactions were carried out by incubating the reaction mix of the

restricted inserts and plasmids (1:3 molar ratio) and T4 DNA ligase for

16 hours at 16 ºC and the ligation mixtures were transformed to competent

E. coli Top 10F’ cells. Preparation of competent cells was done using calcium

chloride method and transformation was carried out by heat shock at 42 ºC.

The selection of E. coli transformants was done on low salt LB plates with

30 µg / mL Zeocin antibiotic. Colonies were screened by PCR. Plasmids were

extracted from the positive E. coli transformants. Lysate PCR and restriction

analysis was performed to check the correct insertion of lipase genes in the

vector. The plasmids having the expression construct for lipase B of

C. antarctica was labeled pPICZ B/CALB.

2.5.1 Generation of Recombinant Mut+ and Mut

SP. pastoris strains

Electroporation was used in the transformation and construction of

recombinant P. pastoris strains. Electroporation was carried out in P. pastoris

strains GS115 (Mut+) and KM71 (Mut

S). The plasmids with the insert

40

expression construct were extracted from E. coli in sufficient quantities and

were linearized with Sac I and purified with Qiagen cleanup kit. 8 - 10 µg of

linearized plasmid and 80 µL of competent cells were incubated for 5 minutes

at 4 ºC in a 0.2 cm cuvette and electroporation was performed at 1500 V,

250 and 50 µF at a time constant of 4.8 – 5.0 s. Immediately after pulsing,

1 mL of ice-cold sorbitol was added to the cuvette. Cells were allowed to

recover for 5 hours at room temperature, then pelleted by centrifugation and

plated directly on YPD agar. 20 – 100 µl of cells per plate containing

100 g/mL Zeocin for selection of recombinant clones. Plates were incubated

at 30 ºC incubator.

2.6 CLONING OF LIPASE GENE IN THE CONSTITUTIVE

EXPRESSING VECTOR pGAPZ B

The PCR product of the CALB gene amplified from C. antarctica

genomic DNA was purified by Qiagen gel-extraction kit and then digested

with the restriction enzymes, EcoRI and XbaI. The stock plasmid pGAPZ B

provided in the Pichia expression kit (Invitrogen) was transformed to E. coli

Top10F’ host and then the plasmid was extracted in sufficient quantity.

Restriction digestions of the insert and the vector with the enzymes EcoRI and

XbaI were set up and performed as suggested by the REases manufacturer.

After analysis of the restriction digest using agarose gel electrophoresis, the

DNA fragments were purified as described in QIAquick gel extraction kit.

Ligation reactions were carried out by incubating the reaction mix of the

restricted inserts and plasmids (1:3 molar ratio) and T4 DNA ligase for 16

hours at 16 ºC and the ligation mixtures were transformed to competent

E. coli Top 10F’ cells. The selection of E. coli transformants was done on low

salt LB plates with 30 µg / ml Zeocin antibiotic. Colonies were screened by

lysate PCR. Plasmids were extracted from the positive E. coli transformants.

PCR and restriction analysis was performed to check the correct insertion of

41

lipase genes in the vector. The plasmids having the expression construct for

lipase B of C. antarctica was labeled pGAPZ B/CALB.

This plasmid was prepared in sufficient quantities, linearised with

SacI enzyme and electroporated into P. pastoris GS115 strain. Positive

colonies were screened on 100 µg/mL zeocin agar plates.

2.6.1 Generation of high copy P. pastoris recombinant strain

Plasmid pPICZ B - CALB was extracted in large scale by using

Qiagen plasmid extraction kit. The plasmid isolated is confirmed by PCR for

the presence of the gene. Large scale restriction digestion of plasmid

pPICZ B - CALB was done using high concentration Sac 1 restriction

enzyme (5X) for linearization of plasmid. The linearized plasmid is gel

extracted and purified using Qiagen kit. The transformation of the CALB

gene into P. pastoris strains GS115 was done by electroporation. P. pastoris

strain GS115. Here for electroporation higher ratio of plasmid to the

competent cells was used. 80 - 100 µg of linearized plasmid and 80 µL of

competent cells are incubated for 5 minutes at 4 ºC in a 0.2 cm cuvette and

electroporation is performed at 1500 V (BTX Electroporator 2510).

Immediately after pulsing, 1 mL of ice - cold sorbitol is added to the cuvette.

Cells are allowed to recover for 5 hours at room temperature, then pelleted

(20 – 100 µL of cells per plate) directly plated on YPD agar plates containing

1, 2, and 3 mg / mL Zeocin concentration for selection of high copy number

clones. Plates are then incubated at 30 ºC for 5 days for the appearance of

resistant colonies. The colonies which grow on plates are isolated and grown

on (YPD medium) medium and glycerol stocks are prepared to be stored at -

80 °C for further studies.

42

2.7 CLONING OF Candida antarctica LIPASE B (CALB) GENE

IN pPIC9K

The E. coli top 10 cells harbouring the pPICZ B - CALB gene was

grown in low salt LB medium until the OD600 reached 0.6, the cells were

pelleted from the medium by centrifugation at 8000 rpm, 4 °C, 10 minutes.

The pellet was extracted for the plasmid DNA using qiagen plasmid isolation

kit. The isolated plasmid was checked for its size in a 1.0 % agarose gel. The

expression cassette from the plasmid pPICZ B - CALB was amplified using

Sac 1 forward primer and Not 1 reverse primer.

Sac 1 Forward Primer: -

5’ GCC GAG CTC GCT CAT TCC AAT TCC TTC TAT TAG G 3’

Not 1 Reverse Primer: -

5’ ATA AGA ATG CGG CCG CTC AGG GGG TGA CGA TGC CG 3’

Gradient PCR ran at different temperatures to optimize annealing

temperature for amplification of CALB insert using forward primer flanked

with Sac 1 and reverse primer flanked with Not 1 enzymes. The amplification

temperature was tried between 55 °C - 65 °C to check the annealing

temperature.

The E. coli DH5 hosting the plasmid pPIC9K was grown in low

salt LB medium to 0.6 - 0.8 OD600, pelleted and extracted the pPIC9K

plasmid in large quantity by using Qiagen plasmid isolation kit. The isolated

plasmid was checked on 1 % agarose gel.

The pPIC9K vector and the PCR amplified insert were double

restricted with Sac 1 and Not 1 enzymes. Both the restricted products were

cleaned up using qiagen gel clean up kit. The product obtained after clean up

is checked on 1 % agarose gel for sizes and ligation reactions are carried out

43

by incubating the reaction mix of the restricted inserts and plasmids

(1:3 molar ratio) and T4 DNA ligase for 16 hours at 16 ºC. The ligation

mixture was transformed into competent E. coli DH5 cells by calcium

chloride method. The transformed cells were plated onto low salt LB agar

plate containing the antibiotic kanamycin and incubated at 37 °C overnight

(12-16 hours). The colonies which obtained after incubation were patched

onto low salt LB agar plates and incubated for 12 -16 hours at 37 °C. Lysate

PCR was done to confirm the presence of CALB using forward primers

flanked with Sac 1 and reverse primers flanked with Not 1. The patch which

shows amplification of the gene product in lysate PCR was patched on LB

agar - Amp plate and restriction digestion with Sac1 and Not 1 restriction

enzymes were carried out for checking the pop out of the gene fragment.

2.7.1 Generation of Recombinant P. pastoris super clone

Large scale plasmid pPIC9K - CALB was extracted by using qiagen

plasmid extraction kit. The isolated plasmid was confirmed by PCR using

forward primer flanked with Sac 1 and reverse primer flanked with TT

(Transcription Termination). The isolated plasmid was then restricted with

Sac 1 enzyme (5 times concentrated). The plasmid was then gel extracted and

purified using qiagen gel extraction kit. The purified plasmid was used for

electroporation into Pichia pastoris high copy strain.

The high copy clone GS115 / pPICZ B - CALB, having ZeocinTM

as a antibiotic selection marker, was grown in a 50ml YPD medium and

competent cells were prepared. The competent cells were electroporated with

the linearized plasmid using BTX electroporator at 1500 V. The cells were

kept for two hours incubation at 30 °C in orbital shaker. The cells were then

plated onto YPD agar plates containing 0.5 and 1 mg / ml GeneticinR as an

antibiotic selection marker. The plates were incubated at 30 °C for 4 – 5 days.

The individual colonies obtained were patched on YPD agar plate.

44

HPLC-RID Glycerol Standards

HPLC Parameters:-

Flow rate: 700µl/min

Column temperature: 50 °C

Buffer: 0.005M sulfuric acid

Column specifications:-

HPLC Organic acid Column

Aminex HPX-87H inexclusion column

Size:- 300mm * 78mm

Table 2.2 Percentage glycerol concentration and the corresponding

area under the peak.

S.No. % Glycerol conc. Area under the peak

1 0.1 191197

2 0.5 886366

3 1.0 1798683

4 1.5 2857348

5 2.0 3637678

6 2.5 4741221

7 3.0 5765932

8 3.5 6602777

9 4.0 7529026

10 4.5 8408878

11 5.0 9462243

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HPLC-RID standard chromatograms of 1%, 2%, 2.5%, 3%, 4% and 5%

glycerol.

Figure 2.2 HPLC-RID chromatogram (Glycerol Standards)

46

Figure 2.3 HPLC-RID glycerol standard graph

47

Methanol standard using HPLC-RID

Table 2.3 Percentage methanol concentration and the corresponding

area under the peak.

S.No. % Methanol conc. Area under the peak

1 0.2 48841

2 0.4 71186

3 0.6 187911

4 0.8 243050

5 1.0 300000

6 2 520000

7 3.0 840792

48

HPLC-RID standard chromatograms of 0.8%, 1%, 2%, 3% and 4% methanol.

Figure 2.4 HPLC-RID Chromatogram (Methanol Standards)

49

Figure 2.5 HPLC-RID methanol standard profile

50

BSA STANDARD GRAPH BY USING BRADFORD DYE IN

MICROTITER PLATE

y = 0.0311x

R2 = 0.9943

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20 25 30

CONCENTRATION (µg/ml)

OD

@595 n

m

CONC. vs OD

Linear (CONC. vs OD)

Figure 2.6 Protein Standard Graph BSA