53

3. Materials

and Methods

Bacterial

Lipase

Lipase

Bacterial

Lipase

Lipase

54

55

3. Materials and Methods

3.1 Microbial strains

Bacillus sp. DVL-1, Bacillus sp. DVL-2 and Bacillus safensis DVL-43 were isolated from

the soil samples collected from different districts of Haryana, India. The stock cultures of

these microbial strains are being maintained on nutrient agar (peptone 0.5%; beef extract

0.5%; agar 2%, w/v) at 4 C by periodic sub- culturing after every two weeks.

3.2 Chemicals

During this investigation the chemicals namely acrylamide, bis-acrylamide, tris base,

SDS, ammonium sulphate, glycine, bromophenol blue, protein molecular weight markers

for native and SDS- PAGE, bovine serum albumin, p-nitrophenyl palmitate, oleic acid and

Phenyl Sepharose were purchased from Sigma-Aldrich Chemical Company, USA. Tributyrin,

gum acacia, p-nitrophenyl acetate, peptone, beef extract, sodium chloride, yeast extract,

sucrose, agar, sodium hydroxide, calcium chloride, sodium dihydrogen phosphate,

disodium hydrogen phosphate, sodium acetate, acetic acid, HCl, fast blue RR, stearic acid

and palmitic acid were purchased from Himedia Pvt. Limited Bombay, India. The chemicals

procured from E. Merck, India were hexane, toluene, methanol, ethanol, chloroform, acetic

acid, acetone, DMSO, DMF, ethyl acetate and acetonitrile. All other chemicals were of high

quality analytical grade obtained from Glaxo (Qualigens) and Ranbaxy Laboratories, India.

3.3 Composition and preparation of various media used

The composition of various media used in various experiments is given in tabulated form

in their respective sections. The constituents of each medium (as given in respective tables) were

weighed, dissolved in ultra pure water, adjusted to the required pH and autoclaved at a pressure

of 1.05 kg/ cm2 (15 lbs/inch

2) for 20 min.

3.4 Sample collection and processing

Samples were collected from decaying wastes of local dairies, restaurants, kitchens,

oil /fats industries, city common garbage site from different districts (Karnal, Kurukshetra,

Yamuna Nagar and Gurgaon) of Haryana, India. The samples were processed for isolation

56

of esterase/lipase producing microorganisms. One gram of each sample was suspended in

10 mL sterile distilled water. This suspension was subjected to serial dilutions and 100 L

of each dilution was spread on nutrient agar (NA) plates. The microbial colonies, which

appeared on nutrient agar plates, were purified.

3.5 Qualitative screening for isolation of lipolytic microorganisms

The microbial colonies on NA plates were subjected to qualitative screening for

identification of lipolytic (lipase/esterase- producing) microorganisms on tributyrin agar

(TBA), rhodamine olive oil agar (ROA), tween 20 and tween 80 agar media. The pH of each

of these media was set at 7.0. The composition of these media is shown in Table 3.1.

3.5.1 Tributyrin agar (TBA) plate assay

TBA media plates were prepared according to the composition given in Table 3.1.

Lipase/esterase producing microorganisms produced a zone of clearance (hydrolysis) when

their appropriate dilutions were spread on the TBA media plates and incubated at 37 °C.

The zone size was measured after 12, 24, 36 and 48 h of incubation.

3.5.2 Rhodamine olive oil agar plate assay

A sensitive and specific plate assay for detection of lipase- producing bacteria makes

use of rhodamine olive oil agar medium. Rhodamine olive oil agar media plates were

prepared according to the composition given in Table 3.1. The bacterial colonies were

inoculated on to these plates and incubated for 48 h at 37°C. Lipase- producing strains were

identified on these spread plates by the formation of orange fluorescent halos around the

bacterial colonies visible upon UV irradiation. The halos were formed due to hydrolysis of

olive oil by lipase produced by the culture.

57

Table 3.1 Composition (per liter) of different media used for screening of lipolytic micro-

organisms

3.6 Qualitative screening for xylanase production

Lipolytic bacteria were qualitatively screened for xylanase production with the help

of Congo red dye (Teather and Wood, 1982). The purified lipolytic colonies were grown on

wheat bran media or xylan agar media as per Table 3.2 for 24 h. The colonies were then

flooded with 1.0% (w/v) aqueous Congo red dye for at least 1 h followed by destaining

with 1.0 M NaCl. The plates were examined for the appearance of yellow zone of hydrolysis

around the colonies. The presence of the yellow zone of hydrolysis indicated the

production of xylanase by the culture.

3.7 Simultaneous screening of lipase and xylanase- producing microorganisms on

common synthetic medium

Samples were serially diluted with sterile distilled water and spread on the common

synthetic medium named viz. tributyrin wheat bran agar medium (TWB Agar) prepared as per

Table 3.2. The culture was streaked on TWB agar plate and incubated for 24-48 h at 37°C for the

S. No. Components Nutrient agar

(NA)

Tributyrin

agar (TBA)

Rhodamine

olive oil agar

Tween20/

Tween 80 agar

1 Peptone 5.0 g

2 Beef extract 3.0 g

3 Tributyrin - 10.0 g - -

4 Olive oil - - 31.25 mL -

5 Agar- agar 20.0 g

6 Rhodamine dye

(1mg/mL)

- - 10.0 mL -

7 NaCl - 3.0 g 4.0 g -

8 Tween 20/

Tween 80

- - - 10 mL

9 CaCl2. 2H2O - - - 0.1 g

58

growth of microorganisms. Firstly, lipase- producing colonies were detected by examining TWB

agar plate for zone of hydrolysis produced due to hydrolysis of tributyrin. Afterwards, xylanase

production was detected by staining the above TWB agar plate with 1.0% congo red dye for 1-2

h followed by dye decolourisation with 1.0 M NaCl and examining the plate for yellow zone of

hydrolysis.

Table 3.2: The composition (g/L) of wheat bran agar, xylan agar and Tributyrin wheat

bran agar media

Components Tributyrin wheat bran

agar medium

Wheat bran agar Xylan agar

Peptone 5.0

Beef extract 3.0 -

Yeast extract - - 2.0

Wheat bran 10.0 10.0 -

Xylan - - 5.0

Tributyrin 5.0 - -

Agar- agar 20.0

The pH of each medium was set at 7.0.

3.8 Effect of different production media and incubation time on esterase/lipase

production

Basal production media (BM) and other three production media (PM1, PM2 and

PM3) were prepared as per their composition given in Table 3.3. Each production medium

was inoculated with 18 h old culture of DVL-1, DVL-2 and DVL-43 separately and incubated

at 37°C for different time periods (24, 36 and 48 h). Fermentation was carried out using the

above mentioned production media (pH 7.0) inoculated with 2% (v/v) inoculum as these

were generally optimum for bacterial growth. After fermentation, lipase/esterase was

extracted.

59

Table 3.3: Composition (g/L) of different production media (PM1, PM2 and PM3) used for

lipase/esterase production

S. No. Components Basal Medium PM1 PM2 PM3

1 Peptone 5.0 5.0 5 .0 -

2 Beef extract 5 .0 3 .0 - 3.0

3 Yeast extract - - 3.0 -

4 Olive oil 1.0 1.0 - 1 .0

5 Tributyrin - - 1.0 -

6 Tryptone - - - 5 .0

7 Glucose - 3 .0 - -

8 Tween80 - - 2.0 -

9 Sucrose - - - 3.0

The pH of each of these media was set at 7.0.

Lipase/esterase activity was determined in three different fractions viz.

extracellular, cell pellet and intracellular. Extracellular lipase/esterase was extracted from

the production medium after the desired incubation period (24, 36 and 48 h) by its

centrifugation at 10,000 x g for 30 min in a refrigerated centrifuge followed by collection of

the supernatant, which contained the enzyme. The pellet was also collected to calculate total

cell biomass. This cell pellet was used for the assay of cell bound lipase/esterase. It was

stored at –20oC for further use. To release the intracellular enzyme, 0.2g of the cell pellet

was suspended in 1.0 ml of lysis buffer (0.05 M phosphate buffer, pH 7.0) and subjected to

five rounds of cell disruption (1 min each) with the help of a sonicator (MSE Manor Roya

Crawley RH 10 2QQ) at 15 KHz. The sonicated cell suspension was centrifuged (15,000 x g

for 30 min) and the cell free extract (intracellular lipase/esterase) was collected for enzyme

assay.

3.9 Enzyme assay

3.9.1 Titrimetric method for the assay of esterase/lipase

The titrimetric method for the assay of lipase/esterase is based on the measurement

of free fatty acids liberated from enzyme catalyzed hydrolysis of triglycerides by titration

60

against alkali under suitable incubation conditions (Beisson et al., 2000). A mechanically

stirred emulsion of tributyrin (1%, v/v, in 1% gum acacia solution) was prepared for use as

substrate. The assay mixture contained 14.0 mL of the above mentioned emulsion of

tributyrin and gum acacia, 0.5 mL of 2% (w/v) CaCl2 and 0.5 mL of 1.0 M NaCl and its pH

was adjusted to 7.0. The enzyme extract (0.1 mL) was then added to the reaction mixture.

The addition of enzyme resulted in hydrolysis of tributyrin to produce butyric acid which

lowered pH of the reaction mixture. The change in pH was recorded using a pH meter. The

pH of the reaction mixture was maintained at 7.0 for 3 minutes by adding 0.01 M NaOH for

neutralization of the free fatty acids released from the hydrolysis of tributyrin by the

enzyme. The volume of NaOH added was noted and from this enzyme activity was calculated

using the following formula:

Enzyme activity =Volume of NaOH consumed mL Molarity of NaOH

Volume of enzyme (mL) Reaction time (min)

One International Unit (IU) of esterase/lipase activity was defined as the amount of enzyme

that liberated 1μmol titrable butyric acid from tributyrin min-1 at 30C and pH 7.0 under the

assay conditions.

3.9.2 Assay for lipase activity

The activity of lipase was determined spectrophotometrically using p-nitrophenyl

palmitate (p-NPP) as substrate according to the method of Nawani et al. (2006) with some

modifications. The reaction mixture containing 0.3 mL of 0.05M phosphate buffer (pH 8.0),

0.1 mL of 0.8 mM p-NPP and 0.1 mL of enzyme extract (lipase) was incubated at 37 C for 10

min. The reaction was then terminated by adding 1.0 mL ethanol. A control was run

simultaneously, which contained the same contents but the reaction was terminated prior

to the addition of enzyme. The absorbance of the resulting yellow colored product was

measured at 410 nm in a spectrophotometer. One International Unit (IU) of lipase activity

was defined as the amount of enzyme catalyzing the release of 1 μmol of p-nitrophenol min-1

from p-NPP under the specified assay conditions.

61

3.9.3 Assay of xylanase activity

The activity of xylanase was determined according to the method of Bailey et al.

(1985) by measuring the amount of reducing sugars (xylose equivalent) liberated from

xylan using 3, 5-dinitrosalicylic acid (Miller, 1959). The reaction mixture containing 490µL

of 2% birch wood xylan (Sigma) as substrate and 10µL of appropriately diluted enzyme

extract was incubated at 50°C for 10 min. The reaction was then terminated by adding 1.5

mL of 3, 5-dinitrosalicylic acid reagent. A control was run simultaneously that contained all

the reagents but the reaction was terminated prior to the addition of enzyme. The contents

were placed in a boiling water bath for 10 min followed by cooling in ice- cold water. The

absorbance of the resulting orange-red color was measured against the control at 540 nm

in a spectrophotometer. One IU of xylanase activity was defined as the amount of enzyme

that catalyzed the release of 1 µmol of reducing sugar as xylose equivalent min-1 under the

specified assay conditions.

3.9.4 Assay of cellulase activity

The activity of cellulase (carboxymethyl cellulase and filter paper hydrolyzing

activity) was determined according to the method of Ghosh (1987). The reaction mixture

for carboxymethyl cellulase (CMCase) activity containing 0.5 mL of 2.0 % carboxymethyl

cellulose (Sigma) and 0.5 mL of enzyme solution was incubated at 50°C for 30 min. The

reaction mixture for filter paper hydrolyzing activity (FPase) containing Whatman No.1

filter paper strip (1x6 cm), 1.0 mL citrate buffer (pH 4.8) and 0.5 mL enzyme solution was

incubated at 50°C for 60 min. In both the cases, the reaction was terminated by adding 3

mL of 3,5-dinitrosalicylic acid reagent. The reaction mixture was then boiled for 10 min in a

boiling water bath, cooled and added 20 mL of distilled water to it. A control was run

simultaneously that contained all the reagents but the reaction was terminated prior to the

addition of enzyme. The absorbance of the resulting color was measured against the

control at 540 nm in a spectrophotometer. One unit (IU) of cellulase activity was defined as

the amount of enzyme that catalyzed the release of 1 µmol of reducing sugar as glucose

equivalent min-1 under the specified assay conditions.

62

3.9.5 Assay of pectinase activity

The activity of pectinase was assayed by measuring the amount of reducing sugars

liberated from pectin using 3, 5-dinitrosalicylic acid. The reaction mixture containing

450µL of 1% pectin (Sigma) as substrate and 50µL of enzyme extract was incubated at

50°C for 10 min. The reaction was then terminated by adding 1.5 mL of 3, 5-dinitrosalicylic

acid reagent. A control was run simultaneously that contained all the reagents but the

reaction was terminated prior to the addition of enzyme. The contents were placed in a

boiling water bath for 15 min followed by cooling in ice- cold water. The absorbance of the

resulting color was measured against the control at 540 nm in a spectrophotometer. One IU

of pectinase activity was defined as the amount of enzyme that catalyzed the release of 1

µmol of reducing sugar as galacturonic acid equivalent min-1 under the specified assay

conditions.

3.10 Bacterial identification

3.10.1 Morphological and biochemical identification of selected isolates

The bacterial isolates DVL-1, DVL-2 and DVL-43 were identified on the basis of their

morphological characteristics (like cell shape, surface, Gram staining, spore staining and

motility) and biochemical tests viz. Voges Proskaur test, Citrate utilization, Gelatin

hydrolysis, Nitrate reduction, Ornithine decarboxylase, Lysine decarboxylase, Catalase test

and Tween (20, 40, 60, 80) hydrolysis, Indole test, Starch hydrolysis, H2S production, and

Gas production from glucose. The utilization of different sugars was studied using Hi-

chrome bacterial identification kit from Himedia.

3.10.2 Molecular identification using 16S rDNA sequencing

Two bacterial isolates (DVL-2 and DVL-43) were identified using 16S rDNA

sequencing. DNA was isolated from these bacterial isolates and its quality was evaluated

on 1.2% agarose gel. The 16S rDNA gene was amplified by PCR from the above isolated

DNA and the PCR amplicon was purified to remove contaminants. Forward and reverse

DNA sequencing reaction of PCR amplicon was carried out with 8F (50-

AGAGTTTGATCCTGGCTCAG-30) and 1492R (50-GGTTACCTTGTTACGACTT-30) primers

using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence

63

of 16S rDNA gene was generated from forward and reverse sequence data using aligner

software. The 16S rDNA gene sequence was used to carry out BLAST with the nrdatabase of

NCBI genbank database. Based on maximum identity score first ten sequences were

selected and aligned using multiple alignment software program Clustal W. Distance matrix

was generated using RDP database and the phylogenetic tree was constructed using MEGA

4.

3.10.3 Electron Microscopy

Electron Microscopy of the isolates DVL-2 and DVL-43 was also done. Bacterial cells

were grown in TSB (tryptone-soya broth) medium for 24 h at 30 °C and collected by low

speed centrifugation (10,000 x g, 10 min at 4°C). The pellet was washed thrice with 50mM

phosphate buffer (pH 7.0). The samples were fixed in 3% glutaraldehyde (protein fixation)

in 0.1 M phosphate buffer (pH 7.0) for 3 h, post fixed in 1 % osmium tetraoxide (lipid

fixation) in 0.1 M phosphate buffer for 4 h. The sample was subjected to critical point

drying using liquid CO2 in acetone for dehydration. The sample was mounted on stubs

coated with gold in a sputter coater and examined in electron microscope (ItoL 100 C*2)

with ASID at 40KV and then photography was done.

Optimization of Lipase Production

3.11 Lipase production under submerged fermentation (SmF)

Lipase was produced in submerged fermentation from the isolate Bacillus sp. DVL-2.

The enzyme production was optimized using one variable at a time approach and by

statistical methods.

3.11.1 Inoculum preparation

A colony of the bacterial isolate Bacillus sp. DVL-2 was transferred to an Erlenmeyer

flask (250 mL) containing 50 mL nutrient broth (peptone 0.5% and beef extract 0.3%, pH

7.0) with the help of an inoculation loop under aseptic conditions and incubated in an

incubator at 30°C for 18 h with an agitation of 200 rpm.

64

3.11.2 Determination of CFU (Colony forming units)

CFU (Colony forming units) for Bacillus sp. DVL-2 was determined on nutrient agar

plate. Different dilutions (10-1, 10-2, 10-4, 10-6 and 10-8) of the inoculum (18 h old) were

prepared in sterile nutrient broth. Then, 1 mL (or 0.1 mL) of each dilution was spread on

sterile nutrient agar plate and incubated at 37 °C for 24 h. At the end of the incubation

period, all of the petri plates containing between 30 and 300 colonies were selected and the

colonies on these plates were counted. The CFU/ml in the inoculum was calculated as:

CFU/ml = Number of colonies counted x dilution

3.11.3 Lipase production

The basal production medium (Table 3.3), autoclaved at 1.05 kg/cm2 for 20 min,

was inoculated with 3.0 % (v/v) of 18 h old inoculum (1.5×107 cfu/mL) followed by

incubation at 30 °C for 24 h under agitation at 200 rpm. After incubation, the contents of

the flasks were centrifuged (10,000 x g for 20 min at 4 °C) and the pellet (biomass) was

collected and weighed. The harvested cells were suspended in lysis buffer (0.05M

phosphate buffer, pH 7.0) at a concentration of 0.2 g/mL and subjected to 5 rounds of cell

disruption (1 min each) with the help of a sonicator (MSE Manor Roya Crawley RH 10 2QQ)

at 15 KHz to release intracellular lipase for recovery of maximum enzyme. The sonicated

cell suspension was centrifuged (15,000 x g for 30 min) and intracellular lipase (cell free

extract) was collected and assayed for its catalytic activity using standard procedure as

described under section 3.9.

To maximize the lipase production by DVL-2, the following parameters were

optimized using one variable approach:

3.11.4 Inoculum age and inoculum size

To study the effect of inoculum age on lipase production, 1 mL inoculum of different

age (6, 12, 18, 24, 30 and 36 h) was added separately to 200 mL of production medium

(taken in 1 L conical flasks) and the flasks were incubated at 30 C for 24 h under shaking

at 200 rpm. The intracellular lipase was extracted (as mentioned in section 3.11.3) from

the fermented medium by centrifugation and lipase activity was determined.

65

The effect of inoculum size was studied by adding different levels of inoculum (1.0,

2.0, 3.0, 4.0, 5.0 and 6 %) from 18 h old bacterial broth to the production media and

incubated at 30ºC for 24 h under shaking at 200 rpm followed by enzyme extraction and

determination of its activity.

3.11.5 Incubation time

To study the effect of incubation period on lipase production, conical flasks each

containing 200 mL of production medium were inoculated with 3.0 % inoculum and

incubated at 30ºC for various time intervals (12, 24, 36, 48 and 60 h) with constant shaking

in a rotary shaker at 200 rpm. Then the enzyme was extracted and its activity was

determined.

3.11.6 Agitation rate

Conical flasks each containing 200 mL of production medium were inoculated with

3% of 18 h old inoculum and incubated at 30 °C for 24 h in a rotary shaker incubator at

different agitation rates (50, 100, 150, 200 and 250 rpm). One flask was also kept under

stationary conditions. Then the enzyme was extracted and its activity was determined.

3.11.7 Temperature and pH of production medium

To investigate the effect of temperature on lipase production, conical flasks

containing 200 mL initial production media were inoculated with 3% inoculum and

incubated at different temperatures (25, 30, 35, 40, 45 and 50 °C) for 24 h in a rotary

shaker incubator at 200 rpm. The lipase activity was then determined.

The effect of pH on lipase production was investigated by varying the pH of the

production medium in the range of 3.0–11.0. The production media (200 mL each) of

different pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0) were inoculated with 3.0 %

of 18 h old inoculum and incubated at 30 °C for 24 h in a rotary shaker incubator at 200

rpm. The activity of lipase was then assayed.

66

3.11.8 Carbon source

The enzyme production was carried out as described under section 3.11.2 except

that the carbon source was varied. Various carbon sources (each at 1.0 %, w/v) used for

lipase production were sucrose, fructose, maltose, xylose, mannitol, lactose, glucose,

galactose, starch, xylan, olive oil tween 20, tween 40, tween 60, and tween 80. A control

devoid of carbon source was also kept. Further, effect of the selected carbon source on

enzyme production was investigated at its different concentrations.

3.11.9 Nitrogen source

The enzyme production was monitored using various inorganic (KNO3, NaNO3,

NH4NO3 and NH4Cl) and organic (peptone, yeast extract, corn steep liquor, casein and

tryptone) nitrogen sources at 1 % (w/v or v/v) individually and also in combinations of 0.5

% each (peptone + CSL, peptone + yeast extract, peptone + KNO3 and peptone + beef

extract). A control devoid of nitrogen source was also kept. Further, effect of the selected

nitrogen source on enzyme production was investigated at its different concentrations.

3.11.10 Statistical methods for screening and optimization of medium constituents

for lipase production

3.11.10.1 Plackett-Burman (PB) design

Initially 11 variables (Table 3.4) were selected at two levels (-1 and +1) for lipase

production. A set of 12 experiments (Table 3.5) was constructed using the Design Expert

(version 7.1.2) software (Stat-Ease Corporation, USA). The level of each variable was

selected on the basis of literature available for lipase production. The constituents of

production media were selected as per PB design followed by incubation at 30 °C for 24 h

with agitation of 200 rpm. Response was measured as lipase activity and biomass in the

periodically withdrawn samples.

67

Table 3.4 Design summary for Plackett–Burman design

Range of components: % (w/v or v/v)

Table 3.5 Plackett–Burman design for 12 experiments to screen 11 medium components

A: Corn steep liquor (CSL), B: Peptone, C: Tween 80, D: Glucose, E: Lactose, F:

Tributyrin, G: KNO3, H: MgSO4, I: ZnSO4, J: K2HPO4, K: NH4Cl

3.11.10.2 Response surface methodology

The significant variables obtained from PB design were applied to central composite

design (CCD). A 24 factorial CCD design using the Design Expert software was used to

Factor Name Low Actual High Actual Low Coded High Coded

A CSL 0.50 2.5 -1 1

B Peptone 0.50 2.0 -1 1

C Tween 80 0.20 0.8 -1 1

D Glucose 0.20 1.0 -1 1

E Lactose 0.20 1.0 -1 1

F Tributyrin 0.20 1.0 -1 1

G KNO3 0.01 0.1 -1 1

H MgSO4 0.05 0.1 -1 1

I ZnSO4 0.50 1.0 -1 1

J K2HPO4 0.02 0.1 -1 1

K NH4Cl 0.03 0.1 -1 1

Std Run A B C D E F G H I J K

1 10 1 1 -1 1 1 1 -1 -1 -1 1 -1

2 5 -1 1 1 -1 1 1 1 -1 -1 -1 1

3 9 1 -1 1 1 -1 1 1 1 -1 -1 -1

4 6 -1 1 -1 1 1 -1 1 1 1 -1 -1

5 3 -1 -1 1 -1 1 1 -1 1 1 1 -1

6 11 -1 -1 -1 1 -1 1 1 -1 1 1 1

7 2 1 -1 -1 -1 1 -1 1 1 -1 1 1

8 12 1 1 -1 -1 -1 1 -1 1 1 -1 1

9 4 1 1 1 -1 -1 -1 1 -1 1 1 -1

10 7 -1 1 1 1 -1 -1 -1 1 -1 1 1

11 8 1 -1 1 1 1 -1 -1 -1 1 -1 1

12 1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1

68

optimize the concentration of above four significant factors (Table 3.6) yielding a set of 30

experiments (Table 3.7). These experiments were conducted in 1L Erlenmeyer flasks each

containing 200 mL production media (pH 8.0) prepared according to the design. Inoculum

size of 3 % (v/v) was used for each experiment. The incubation was done at 30ºC and 200

rpm. Lipase activity and growth were recorded as response at the end of 24 h. Response

data were analyzed for optimum concentrations of the variables.

Table 3.6 Experimental range and levels of each variable studied using Central Composite

Design in terms of actual factors for the production of lipase by Bacillus sp. DVL 2

Factor Name Units/ 100

mL

Low

Actual

High

Actual

Low

Coded (-α)

High

Coded (+α)

Mean

(0)

A CSL mL/100mL 1.0 2.5 -1 1 1.75 B Peptone g/100mL 0.5 2.0 -1 1 1.25

C Tween 80

g/100mL 0.5 1.0 -1 1 0.75

D MgSO4 g/100mL 0.02 0.1 -1 1 0.06

Purification and Characterization of lipase

3.12 Purification of lipase

Cell free extract obtained after sonication of the cells of Bacillus sp. DVL-2 grown

under optimized SmF conditions for 24 h at 30 °C was used for purification. To prepare the

cell free extract, the above bacterial strain was grown under optimized SmF conditions and

the cells harvested after 24 h incubation were suspended in lysis buffer (0.05M phosphate

buffer, pH 7.0) at a concentration of 0.2 g/mL and subjected to 5 rounds of cell disruption

(1 min each) with the help of a sonicator (MSE Manor Roya Crawley RH 10 2QQ) at 15 KHz.

The sonicated cell suspension was centrifuged at 15,000 x g for 30 min and supernatant

(cell free extract) was collected. It was analyzed for lipase activity and protein content

using standard procedure as described earlier. The enzyme was purified in two steps by

ammonium sulfate fractionation and Hydrophobic interaction chromatography using

Phenyl Sepharose CL-4B. The purification was carried out at 4 C.

69

Table 3.7 Experimental design for RSM using central composite design

Std Run CSL Peptone Tween 80 MgSO4

1 29 1.00 0.50 0.50 0.02

2 11 2.50 0.50 0.50 0.02

3 30 1.00 2.00 0.50 0.02

4 7 2.50 2.00 0.50 0.02

5 18 1.00 0.50 1.00 0.02

6 5 2.50 0.50 1.00 0.02

7 8 1.00 2.00 1.00 0.02

8 2 2.50 2.00 1.00 0.02

9 27 1.00 0.50 0.50 0.10

10 17 2.50 0.50 0.50 0.10

11 19 1.00 2.00 0.50 0.10

12 26 2.50 2.00 0.50 0.10

13 28 1.00 0.50 1.00 0.10

14 13 2.50 0.50 1.00 0.10

15 4 1.00 2.00 1.00 0.10

16 22 2.50 2.00 1.00 0.10

17 20 0.30 1.30 0.80 0.06

18 23 3.30 1.30 0.80 0.06

19 1 1.80 -0.30 0.80 0.06

20 15 1.80 2.80 0.80 0.06

21 9 1.80 1.30 0.30 0.06

22 12 1.80 1.30 1.30 0.06

23 25 1.80 1.30 0.80 -0.02

24 24 1.80 1.30 0.80 0.14

25 3 1.80 1.30 0.80 0.06

26 14 1.80 1.30 0.80 0.06

27 21 1.80 1.30 0.80 0.06

28 6 1.80 1.30 0.80 0.06

29 16 1.80 1.30 0.80 0.06

30 10 1.80 1.30 0.80 0.06

70

3.12.1 Ammonium sulphate fractionation

Cell free extract from Bacillus sp. DVL 2 was subjected to (NH4)2SO4 fractionation in

the range 0-30 % and 30-70 %. The crude extract containing lipase was subjected to

(NH4)2SO4 fractionation by gradual addition of solid (NH4)2SO4 with constant stirring to

bring it to 30 % saturation and kept it for 1 h in a refrigerator to allow the protein to

precipitate. Then, the suspension was centrifuged at 10,000 x g for 20 min to collect the

supernatant and the pellet. The resulting supernatant was further subjected to 70 %

saturation by adding solid (NH4)2SO4 gradually with stirring and kept it overnight. The

suspension was centrifuged at 10,000 x g for 20 min to collect the supernatant and the

pellet. Both the pellets were dissolved in a minimum volume of 0.05M phosphate buffer

(pH 8.0) and dialyzed against 0.01M phosphate buffer (pH 8.0) overnight with change of

buffer twice. The lipase activity was determined in the dialyzed pellets as well as

supernatant obtained after 70 % (NH4)2SO4 fractionation. The fraction containing lipase

activity was analyzed for protein content by the Lowry’s method. Specific activity, %

recovery and fold purification were also calculated. This fraction was loaded on Phenyl

Sepharose CL-4B column for further purification.

3.12.2 Hydrophobic interaction chromatography (HIC)

The dialyzed enzyme obtained after salt fractionation was loaded on to Phenyl

Sepharose CL-4B column (6 cm x 2 cm) that had been pre-equilibrated with 1.0 M

ammonium sulphate dissolved in 0.05 M phosphate buffer (pH 8.0). The bound lipase was

eluted by applying a negative linear gradient of 1.0 M to 0 M ammonium sulphate in 0.05 M

phosphate buffer (pH 8.0). The fractions were collected and analyzed for protein content

by measuring the absorbance at 280 nm. The lipase activity was determined in those

fractions which contained protein. The fractions containing lipase activity were pooled. The

pooled fraction was analyzed for lipase activity and protein content. To assess the progress

of purification, parameters such as specific activity, % recovery and fold purification were

also calculated. Its purity was then checked through electrophoresis.

71

3.12.3 Testing of enzyme purity

The enzymatically active fraction obtained after HIC lipase was tested for its purity

through electrophoresis using Native-PAGE and SDS-PAGE.

3.12.3.1 Native polyacrylamide gel electrophoresis (Native-PAGE)

Native-PAGE was performed using anionic system of Davis (1964). The following

reagents were prepared:

Acrylamide-bis-acrylamide solution (30:0.8)

Dissolved 30.0 g acrylamide and 0.8 g methylene-bis-acrylamide in distilled water

and made up the volume 100 mL. Filtered the solution through Whatman No.1 filter paper

and stored in a brown bottle at 4 °C.

Resolving gel buffer stock (3.0 M Tris-HCl, pH 8.8)

Dissolved 36.3 g of Tris base in 60 mL distilled water and adjusted its pH to 8.8 with

1.0 N HCl and the final volume was made 100 mL with distilled water. The solution was

filtered through Whatman No.1 filter paper and stored at room temperature.

Stacking gel buffer stock (0.5 M Tris buffer, pH 6.8)

Dissolved 6.05 g of Tris base in 60 mL distilled water and adjusted the pH to 6.8

with 1N HCl and made up its volume to 100 mL with distilled water. The solution was

filtered through Whatman No.1 filter paper and stored at room temperature.

Ammonium persulphate solution (1.5 %, w/v)

The solution was prepared by dissolving 15.0 mg in 1.0 mL water. This solution was

unstable and prepared fresh just before use.

Reservoir buffer

3.0 g Tris base and 14.4 g glycine were dissolved in distilled water to a final volume

of 1.0 L. Adjusted the pH of the solution to 8.3 and stored it at 4 C.

72

Staining solution

Dissolved 0.25 g Coomassie brilliant blue R-250 dye in 250 mL of water, methanol

and glacial acetic acid (105: 105: 40). It was filtered to remove any undissolved material

and stored at room temperature.

Destaining solution

Mixed 30 mL methanol with 10 mL glacial acetic acid and made up its volume to 100

mL with distilled water.

Sample preparation

The protein sample was prepared in sample buffer (1.0M Tris-HCl, pH 6.8

containing 5 % glycerol and 0.02 % bromophenol blue).

Procedure

Properly cleaned and dried glass plates were tightly held with the spacer bars on both sides.

Resolving and stacking gel solutions for polymerization were mixed just before use as given in Table 3.8. The

solution of resolving gel was poured into a vertical slab and a few drops of distilled water were layered on top

of the gel solution to ensure the production of a flat gel surface and to exclude oxygen. The gel was allowed to

polymerize for half an hour. After polymerization of the resolving gel, the water layer was removed and

soaked off with a filter paper. The stacking gel solution was then poured and immediately the comb was fixed

at the top to make the wells for sample application. The stacking gel was allowed to polymerize for half an

hour. The comb was removed and the wells were cleared thoroughly with reservoir buffer using a syringe so

that no unpolymerized acrylamide was left in the wells.

The spacer fixed on the lower side was removed and the lower and upper chambers of the

apparatus were filled with reservoir buffer in such a manner that no air bubble was formed

between gel and buffer system. After this, pre-electrophoresis was carried out at 10 mA for

15 min. Protein samples dissolved in sample buffer were loaded into the wells using a

Hamilton syringe. The electrodes were connected to an electrophoretic power supply unit

and run at 10mA till the dye reached the end of resolving gel as indicated by the trackng

dye. After the electrophoresis, the gel was taken out and stained in coomassie brilliant blue

R-250 staining solution with constant shaking for 8 h to visualize protein bands. After

staining, the staining solution was removed and the gel was transferred to the destaining

73

solution. The gel was destained with gentle shaking on a gel rocker by changing the

destaining solution several times till the gel background was clear. After destaining, the

gels were photographed and preserved in destaining solution with 10% glycerol in dark

and cool place.

Table 3.8 NATIVE polyacrylamide gel preparation using discontinuous buffer system

Solutions Resolving gel

(12 %)

Stacking gel

(4.5 %)

Acrylamide-bis-acrylamide 12.00 mL 3.0 mL

Resolving gel buffer stock 3.75 mL --

Stacking gel buffer stock -- 4.50 mL

Ammonium per sulphate 1.90 mL 1.00 ml

TEMED 0.015 mL 0.018 mL

Distilled water 12.335 mL 11.482 mL

3.12.3.2 Zymogram analysis

Lipase/esterase activity was detected in NATIVE- polyacrylamide gel according to

the method of Gabriel (1971). The enzyme sample was run on polyacrylamide gels (10 %)

under non-denaturing conditions in a cold room. After electrophoresis, the gel was washed

with 0.1M Tris-HCl, pH 8.0 and then incubated it in the staining solution [containing β-

naphthyl acetate (8 mg/mL of absolute alcohol), fast blue RR salt (2 mg/mL) and 0.1M Tris-

HCl, pH 8.0] for 10-15 min at 37 C till red coloured bands appeared. The β-naphthol

released from the substrate β-naphthyl acetate couples with diazonium salt present in the

incubation mixture to form an insoluble red colored product visible as discrete bands.

3.12.3.3 SDS-PAGE

SDS-PAGE was carried out by the method of Laemmli (1970) with slight modifications.

Acrylamide-bis-acrylamide solution, resolving gel buffer, stacking gel buffer, ammonium

74

persulphate, staining and destaining solutions, and bromophenol blue were the same as

used for Native- PAGE. The following additional solutions were prepared for SDS-PAGE:

SDS (10%, w/v)

Dissolved 1.0 g SDS in 10 mL of distilled water.

Reservoir buffer

Dissolved 3.0 g Tris base, 14.4 g glycine and 1.0 g SDS in distilled water and adjusted

its pH to pH 8.3. The volume was made to 1.0 L with distilled water.

Sample buffer (2x)

It was prepared by mixing 2.5 mL of 1M Tris-HCl buffer (pH 6.8), 2.0 mL glycerol

(20%), 0.4 g SDS, 1.0 mL β-mercaptoethanol and 0.4 mL of 1% bromophenol blue and its

volume was made to 10.0 mL with distilled water.

Sample preparation

Protein sample was mixed with equal volume of sample buffer (2x), boiled for 5 min,

cooled and loaded into sample wells in the slab gel.

Molecular weight markers

A pre-stained mixture of SDS-PAGE molecular weight markers (Fermentas) viz. β-

galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate

dehydrogenase (35 kDa), REase Bsp98I (25 kDa), β-lactoglobulin (18.4 kDa), lysozyme

(14.4 kDa) was used as supplied by the firm.

Procedure

SDS-PAGE was performed using 12 % resolving and 4.5 % stacking gel, the

compositions of which are given in Table 3.9

. The procedure adopted for preparation of gels, carrying out electrophoresis and

visualization of protein bands in gels was same as described for Native-PAGE. The standard

SDS-PAGE molecular weight markers were co-electrophoresed with samples.

75

Table 3.9 Composition of resolving and stacking gels for SDS-PAGE

Solutions Resolving gel

(12 %)

Stacking gel

(4.5 %)

Acrylamide-bis-acrylamide solution 12.00 mL 3.00 mL

Resolving gel buffer 3.75 mL --

Stacking gel buffer -- 4.50 mL

SDS (10%) 0.30 mL 0.20 mL

Ammonium persulphate solution 1.50 mL 1.0 mL

TEMED 0.015 mL 0.015 mL

Distilled Water 12.435 mL 11.285 mL

Total volume 30.00 mL 20.00 mL

3.13 Characterization of Lipase

The purified enzyme was characterized for its molecular weight and effects of pH,

temperature, substrate concentration, and additives.

3.13.1 Determination of molecular weight

Molecular weight of the purified enzyme was determined by SDS-PAGE which was

performed as described earlier. The mobility of each protein band was calculated as

follows:

Distance moved by a protein band Relative mobility (R) = Distance moved by the tracking dye

A standard curve was prepared by plotting log molecular weight of marker proteins

(on Y-axis) versus corresponding relative mobility (on X-axis). After calculating the relative

mobility of lipase protein in the gel, its molecular weight was determined from the

standard curve.

76

3.13.2 Effect of temperature on enzyme activity as well as stability

The lipase activity was measured at different temperatures (25, 30, 37, 40 and 45

°C) under standard assay conditions using p-NPP as substrate to determine the optimum

temperature of the enzyme action. Relative activity (%) was calculated relative to the

maximum enzyme activity taken as 100%. The results are shown in the form of a graph

between lipase activity (on Y-axis) and temperature (on X-axis).

Thermo stability was determined by pre-incubating the purified enzyme at different

temperatures (30, 40, 45, 50, 55 and 60 °C) in 0.05 M phosphate buffer, pH 8.0 for various

time intervals (30, 60 and 120 min) followed by keeping the samples in ice. The lipase

activity was then determined in each sample at the optimum temperature using the

standard assay. The residual activity (%) was calculated with respect to that recorded at

the optimum temperature.

3.13.3 Effect of pH on enzyme activity as well as stability

The effect of pH on the purified lipase was studied by determining its activity at

different pH values (3.0-12.0) using sodium citrate (pH 3.0–6.0), sodium phosphate (pH

7.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine-NaOH (pH 10.0-11.0) buffers each at 0.05 M.

The relative activity (%) was calculated relative to the maximum enzyme activity (pH 8.0)

taken as 100%. The results are shown in the form of a graph between % residual activity

(on Y-axis) and pH (on X-axis).

The pH stability was investigated by mixing equal aliquots of the purified enzyme

and different buffers (0.05 M each) viz. sodium citrate (pH 3.0–6.0), sodium phosphate (pH

6.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine-NaOH) (pH 9.0–11.0) in micro centrifuge tubes

followed by pre-incubation at 30 C for 30, 60, 120 and 240 min. The lipase activity was

then determined in each sample at the optimum pH using the standard assay. A crude

enzyme without addition of any buffer was placed as control. Lipase residual activity was

calculated under standard assay conditions, after different time intervals starting from 30

to 240 min. The residual activity (%) was calculated with respect to control in which buffer

was replaced with distilled water.

77

3.13.4 Stability in organic solvents

Purified DVL2 lipase was treated with various organic solvents viz. butanol, iso-

propanol, xylene, methanol, ethanol, acetone, DMSO, chloroform, toluene, hexane,

dichloromethane (DCM), ethyl acetate and acetonitrile, each at 25% (v/v) concentration,

for different time intervals (12, 24 and 36 h) at 30 C followed by determination of lipase

activity using standard assay. The sample without any organic solvent was taken as control.

The lipase activity in treated samples was expressed relative to the control.

3.13.5 Effect of substrate concentration

In this experiment, the activity of purified lipase was measured at different

concentrations of the substrate tributyrin (2.5-40.0 mM) and Lineweaver-Burk plot was

drawn for calculation of Km and Vmax.

3.13.6 Effect of metal ions, EDTA and PMSF

The effect of various metal ions viz. Mg+2, Fe+3, Ca+2, Zn+2, Ba+2 , Mn+2, and Hg+2 ,

EDTA and PMSF at 1mM concentration on the purified enzyme was studied. The enzyme

was pre-incubated with various metal ions for at 30 °C for 2 h and then lipase activity was

measured under standard assay conditions. The sample without any metal ion was taken as

control. The residual activity (%) was then calculated.

3.13.7 Effect of bile salts and surfactant on lipase activity

To study the effect of bile salts like sodium deoxycholate, cholic acid, deoxycholic

acid, sodium taurocholate and surfactants like Tween 80 and Triton X-100 were added at a

final concentration of 0.1% (w/v) in the reaction mixture. A control devoid of surfactant

and bile salts was used as reference for determining the residual activity.

3.13.8 Stability of the purified lipase during storage

The stability (shelf-life) of the purified enzyme was determined by storing it at 4 °C.

Enzyme samples were withdrawn at 15 days intervals up to 60 days for determination of

78

lipase activity. The residual lipase activity in these samples was determined with respect to

the initial activity (at zero time).

Immobilization of Bacillus sp. DVL-2 lipase

3.14 Immobilization of Lipase

The partially purified lipase, produced from Bacillus sp. DVL-2, was immobilized on

glutaraldehyde-activated aluminum oxide pellets (Sigma). The immobilization parameters

were optimized statistically using RSM. The potential of the immobilized enzyme was

studied in esterification between oleic acid and ethanol in hexane.

3.14.1 Immobilization of lipase on aluminum oxide pellets

Aluminum oxide pellets were employed as support for immobilization of partially

purified lipase through covalent attachment. The pellets were activated by dipping in 2 %

(v/v) aqueous glutaraldehyde solution for 1 h at 30 C followed by addition of the enzyme.

The coupling of enzyme with the glutaraldehyde-activated pellets was allowed to occur at

30 C for 90 min. The pellets were washed with distilled water at each step to remove the

excess of glutaraldehyde and the unbound enzyme. The enzyme activity was determined in

the supernatant as well as in the enzyme bound pellets. Immobilization yield (IY %) was

calculated in the following manner:

Immobilization Yield (%) =Total activity immobilized on pellets

Total activity offered for immobilization x 100

Total activity immobilized on pellets refers to the difference in enzyme activity offered for

immobilization and activity recovered in the supernatant.

3.14.2 Assay of the immobilized lipase

Activity of the immobilized lipase was assayed by adding 0.1 mL of 0.8 mM p-

nitrophenyl palmitate (p-NPP) and 0.4 mL of 0.05 M phosphate buffer (pH 8.0) to the

enzyme bound aluminum oxide pellets in an eppendorf tube. After incubating the reaction

mixture for 10 min at 30 C, the pellets were removed by transferring the contents of the

reaction mixture to another eppendorf tube. The reaction was then stopped by adding 1.0

79

mL ethanol and intensity of the resulting yellow colored product was read at 410 nm in a

spectrophotometer. The pellets were washed with phosphate buffer and reused.

3.14.3 Statistical optimization of immobilization

To examine the cumulative effect of immobilization parameters viz. no. of beads,

glutaraldehyde concentration, enzyme dose, coupling time, response surface methodology

was employed using a statistical software package Design Expert 7.1.2, Stat-Ease, Inc. A 24

full factorial central composite design (CCD) with 16 trials for factorial design, 8 trials for

axial point and 6 replicate trials at the central point, leading to a set of 30 experiments was

designed. The range and levels of variables (-α, -1, 0, 1, +α) are presented in Table 3.10

and the experimental design is shown in Table 3.11. All the variables were taken at a

central value represented by “0”. The response value from each experiment of CCD was the

average of triplicates.

Table 3.10 Experimental range and levels of each variable for lipase immobilization on

aluminum oxide pellets

3.14.4 Operational stability of the immobilized lipase

To evaluate the recycling stability of the immobilized lipase, the enzyme bound

pellets were incubated with buffer and p-NPP for 10 min at 30 ºC followed by termination

of the reaction according to standard assay conditions. The bound enzyme was repeatedly

used to hydrolyze p-NPP up to 15 batch reactions. The residual activity (%) was

determined as:

Residual activity =enzyme activity in nth cycle×100

enzyme activity in 1st cycle

Factor Name Unit Low level (-) High level (+) Mean (0) S.D.

A No. of beads numeric 4.0 8.0 6.0 1.79

B Glutaraldehyde (% v/v) 2.0 6.0 4.0 1.79

C Enzyme dose mL 0.02 0.06 0.04 0.02

D Coupling period min 30.0 120.0 75.5 39.21

80

Table 3.11 Central composite design for lipase immobilization on aluminum oxide pellets

S.

No.

A:

No. of Pellets

B:

Glutaraldehyde

(% v/v)

C:

Enzyme dose (mL)

D:

Coupling period (min)

1 4 2 0.02 30

2 8 2 0.02 30

3 4 6 0.02 30

4 8 6 0.02 30

5 4 2 0.06 30

6 8 2 0.06 30

7 4 6 0.06 30

8 8 6 0.06 30

9 4 2 0.02 120

10 8 2 0.02 120

11 4 6 0.02 120

12 8 6 0.02 120

13 4 2 0.06 120

14 8 2 0.06 120

15 4 6 0.06 120

16 8 6 0.06 120

17 2 4 0.04 75

18 10 4 0.04 75

19 6 0 0.04 75

20 6 8 0.04 75

21 6 4 0 75

22 6 4 0.08 75

23 6 4 0.04 0

24 6 4 0.04 165

25-30 6 4 0.04 75

3.14.5 Effect of pH on free and the immobilized lipase

The effect of pH (4.0-12.0) on soluble and the immobilized lipase was studied by

determining the enzyme activity using different buffers (each of 0.05M) viz. sodium citrate

(pH 4.0–6.0), sodium phosphate (pH 7.0–9.0) and glycine- NaOH (pH 10.0-11.0). The pH at

which the enzyme displayed highest activity was termed as the optimum pH for catalytic

action of the enzyme. The relative activity (%) at each pH was calculated with reference to

the enzyme activity at the optimum pH (taken as 100%).

81

The pH stability of soluble as well as the immobilized lipase was investigated by pre-

incubating the enzyme in the above mentioned buffers of different pH (4.0-12.0) for 2 h

followed by measurement of its activity using standard assay at the optimum pH. The

residual activity (%) at each pre-incubating pH was calculated relative to the maximum

enzyme activity (taken as 100%). The results of the effect of pH on the immobilized enzyme

were compared with those of the free enzyme.

3.14.6 Effect of temperature on free and the immobilized lipase

The temperature optima of soluble and the immobilized lipase were investigated by

determining the enzyme activity at different temperatures (25-45 °C). The relative activity

(%) at each temperature was calculated with reference to the enzyme activity at the

optimum temperature (which was taken as 100%). The activation energy (Ea) of catalysis

for free and the immobilized lipase was determined from the slope of the Arrhenius plot

[log V (logarithm of % relative activity) versus reciprocal of absolute temperature in Kelvin

(1000/T)], using the following equation:

Slope = −Ea

R

Thermal stability of soluble and the immobilized lipase was studied by pre-

incubating the enzyme at various temperatures (25-60 °C) for 1 h followed by

determination of activity using standard assay at the optimum temperature. The residual

activity (%) was calculated relative to the maximum enzyme activity taken as 100%.

Thermal stability of the immobilized lipase was also investigated by pre-incubating it at

temperatures ranging from 30-60 °C for different periods (30, 60, 90 and 120 min). The

residual activity was calculated by taking the enzyme activity at 0 min incubation as 100 %.

The first order thermal deactivation rate constants (kd) were determined from the

regression plot of log relative activity (%) versus time (min). The activation energy (Ed) for

lipase denaturation was determined by a plot of log denaturation rate constants (ln kd)

versus reciprocal of the absolute temperature (K) using the following equation:

Slope = Ed

R

82

The half-lives (t1/2) and D-values (decimal reduction time or time required to pre-

incubate the enzyme at a given temperature to maintain 10 % residual activity) of the

immobilized lipase at each temperature were determined from the following relationships:

t1/2 =In 2

kd

D − value =In 10

kd

The changes in enthalpy (∆H°, kJ mol−1), free energy (∆G°, kJ mol−1) and entropy

(∆S°, J mol−1 K−1) for thermal denaturation of lipase were determined using the following

equations:

`

∆H° = Ed – RT

∆G° = −RT In (kd.h

kB.T)

S° =H° − G°

T

where T is the corresponding absolute temperature (K), R is the gas constant (8.314 J mol−1

K−1), h is the Planck’s constant (11.04 × 10−36 J min), and kB is the Boltzmann constant (1.38

× 10−23 J K−1).

The z-value (temperature rise necessary to reduce D-value by one logarithmic cycle)

was calculated from the slope of graph between log D versus Temperature (°C) using the

following equation:

Slope =1

z

3.14.7 Effect of organic solvents on the activity of the immobilized enzyme

To study the effect of organic solvents on the immobilized lipase, the enzyme bound

aluminum oxide beads were treated with organic solvents viz. isopropanol, xylene,

methanol, ethanol, dimethylsulphoxide (DMSO), toluene, hexane and acetonitrile to a final

concentration of 25% (v/v) for 12, 24 and 36 h at 30 °C. The residual activity of the

immobilized lipase was then determined under standard assay conditions. The enzyme

activity of the sample without adding any organic solvent was taken as control (100%). The

83

stability of the immobilized lipase in various organic solvents was also compared with that

of free enzyme after an incubation of 24 h at 30 °C.

3.14.8 Determination of Kinetic parameters

The apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) of free

and the immobilized enzyme were calculated using the Lineweaver-Burk plot. The

substrate (p-nitrophenyl palmitate) concentration used for enzyme assay ranged from 0.17

to 2.6 mM.

3.14.9 Application of the immobilized lipase in esterification of ethanol with oleic

acid

Both soluble and the partially purified lipase immobilized on aluminum oxide

pellets were used as biocatalyst for the esterification of oleic acid and ethanol in 1:1 (v/v)

ratio in hexane. The reaction was carried out at 37 °C with shaking at 100 rpm for 4, 8, 12,

16, 20 and 24 h with heat inactivated free enzyme as control. The ester content was

quantified by using the alkalimetric method of titrating unreacted acid with 0.1N NaOH

using phenolphthalein as an indicator. The percentage conversion in ester synthesis was

based on the amount of acid consumed (Bovara et al., 1995). Reusability of the immobilized

lipase in the above esterification reaction was also tested.

3.14.10 TLC and 1H NMR

The formation of ester was visualized by Thin Layer Chromatography (TLC) and 1H

NMR. Analytical TLC was performed using pre-coated silica gel 60 F254 MERCK TLC plates

(20 cm x 20 cm). The spots were visualized by immersing the plates in 10 % H2SO4 in

ethanol followed by heating on hot plate. 1H NMR spectra were recorded with BUCKER 500

MHz NMR instrument. Chemical data for protons are reported in ppm downfield from

tetramethylsilane (TMS) and are referenced to the residual proton in the NMR solvent

(CDCl3: δ 7.26).

84

Applications of Bacillus sp.DVL-2 lipase

3.15 Applications of DVL-2 lipase

Application of the purified lipase from Bacillus sp. DVL-2 was studied in

esterification reactions, removal of chromophores from waste newspaper pulp and kinetic

resolution of benzoin. The protocols employed to perform these experiments are described

in this section.

3.15.1 Esterification of fatty acids (lauric acid, oleic acid, stearic acid and palmitic

acid) with ethanol

Partially purified lipase (specific activity 21.9 IU/mg protein; activity 70 IU/mL)

was used as biocatalyst for esterification of different fatty acids (lauric acid, oleic acid,

stearic acid and palmitic acid) with ethanol in 1:1 (v/v) ratio in hexane. The reaction was

carried out at 37 °C with shaking at 100 rpm for 6, 12, 18, 24 and 30 h with heat inactivated

free enzyme as control. The ester formation was identified by analytical thin layer

chromatography (TLC), 1H NMR and GC-MS (Gas Chromatography- Mass Spectrometry).

The spots of substrate, reaction and Cospot (substrate +reaction) were applied to one end

of the TLC plate with the help of capillary. After drying the spots, TLC was developed in ethyl

acetate/ hexane (20: 80) solvent system. After development with the mobile phase, the plate was

dried in a fume hood and heated to completely evaporate the mobile phase followed by charring

in 10% H2SO4 in ethanol or 3-5 % 2, 4- dinitrophenylhydrazine (dissolved in conc. H2SO4,

H2O and ethanol in the ratio 3:4:5) to visualize the separated components. 1H NMR spectra

were recorded with BUCKER 500 MHz NMR instrument. Chemical data for protons are

reported in ppm downfield from tetramethylsilane (TMS) and are referenced to the residual

proton in the NMR solvent (CDCl3: δ 7.26).

3.15.1.1 Statistical optimization of parameters for esterification reaction between

oleic acid and ethanol

Partially purified lipase (specific activity 21.9 Iu/mg protein; activity 70 IU/mL) was

used as biocatalyst for esterification of oleic acid and ethanol in 1:1 (v/v) ratio in hexane.

The reaction was carried out at 37 °C with shaking at 100 rpm for 6, 12, 18, 24 and

30 h with heat inactivated free enzyme as control. The ester content was quantified by

85

alkalimetric method in which the unreacted acid was titrated with 0.1N NaOH using

phenolphthalein as an indicator. The percentage conversion in ester synthesis was based

on the amount of acid consumed (Bovara et al., 1995). The ester formation was identified

by analytical TLC and 1H NMR as discussed above.

A 24 factorial design using CCD of response surface methodology (Design Expert

software 8.01) was used to statistically optimize the concentrations of four factors viz.

time, enzyme dose, temperature and shaking speed, yielding a set of 30 experiments for

esterification of oleic acid with ethanol. The design summary and experimental design was

presented in Table 3.12 and Table 3.13 respectively. These 30 experiments were

performed and the results were analyzed using software to find out the optimum values of

the parameters.

Table 3.12 Design summary showing the range of different factors choosen for RSM

optimization

Factor Name Units Minimum Maximum Mean Standard deviation

A Time h 6 30 18.4 10.03

B Enzyme dose mL 0.2 1.0 0.61 0.33

C Temperature °C 25 45 35 8.94

D Shaking speed RPM 50 200 125 67.08

3.15.2 Application of lipase in chromophore removal from waste newspaper pulp

The pulp was prepared by chopping waste newspapers to less than 2 cm2 size pieces

and dipping them in boiled distilled water for 48 h. The pulp was filtered through three

layers of muslin cloth and washed thoroughly with distilled water. The resulting pulp was

dried in an oven at 50 C and ground.

The waste news paper pulp of 10% consistency was treated with partially purified lipase of

Bacillus sp. DVL-2 lipase to measure the chromophores released by the enzyme.

86

Table 3.13 Experimental design for esterification using CCD of RSM

Std Run A:Time

(h)

B:Enzyme dose

(mL)

C:Temperature

(°C)

D:Shaking speed

(RPM)

1 2 6 0.2 25 50

2 24 30 0.2 25 50

3 5 6 1.0 25 50

4 18 30 1.0 25 50

5 6 6 0.2 45 50

6 21 30 0.2 45 50

7 16 6 1.0 45 50

8 22 30 1.0 45 50

9 23 6 0.2 25 200

10 30 30 0.2 25 200

11 14 6 1.0 25 200

12 27 30 1.0 25 200

13 19 6 0.2 45 200

14 9 30 0.2 45 200

15 10 6 1.0 45 200

16 11 30 1.0 45 200

17 1 6 0.6 35 125

18 25 42 0.6 35 125

19 12 18 0.2 35 125

20 8 18 1.4 35 125

21 29 18 0.6 15 125

22 28 18 0.6 55 125

23 13 18 0.6 35 -25

24 26 18 0.6 35 275

25 3 18 0.6 35 125

26 15 18 0.6 35 125

27 4 18 0.6 35 125

28 20 18 0.6 35 125

29 17 18 0.6 35 125

30 7 18 0.6 35 125

87

During pulp treatments, enzyme-mediated release of chromophoric material was

monitored in filtrates by measuring absorption spectra (UV/VISIBLE Spectrophotometer)

at wavelengths ranging from 260 nm to 360 nm. The experimental conditions i.e. amount of

pulp, amount of enzyme, incubation time and wavelength, were statistically optimized

using response surface methodology (RSM) for maximum release of chromophores from

the pulp by the enzyme. All the operations were carried out at 30 °C.

A 24 factorial design using CCD of response surface methodology (Design Expert

software 8.01) was used to statistically optimize the concentrations of four factors viz.

amount of pulp (g), enzyme dose (mL), incubation time (min) and wavelength (nm),

yielding a set of 30 experiments for removal of chromophores from newspaper pulp. The

design summary and experimental design was presented in Table 3.14 and Table 3.15

respectively. These 30 experiments were performed and the results were analyzed using

software to find out the optimum values of the parameters.

Table 3.14 Design summary showing the range of different factors chosen for RSM optimization

3.15.3 Kinetic resolution of Benzoin

The kinetic resolution of benzoin was carried out using 25 mg of commercial racemic benzoin,

100 μL of vinyl acetate and 450 μL of partially purified lipase ((specific activity 21.9 IU/mg

protein; activity 70 IU/mL) in 750 μL of toluene. The mixture was stirred at 30 C for 24 h

followed by HPLC analysis using chiral column (hexane /propanol, 90:10) to determine %

conversion and enantiomeric excess.

Factor Name Units Coded Values Mean Standard

deviation -1 1

A Amount of

pulp

g 0.50 2.00 1.26 0.653

B Amount of

enzyme

mL 0.50 1.50 1.00 0.447

C Incubation

time

min 30.0 200 116.83 72.485

D Wave length nm 260 340 300.00 35.777

88

Table 3.15 Experimental design for chromophore removal using CCD of RSM

Std Run Amount of pulp

Min Incubation time (min)

Wavelength (nm)

1.0 26.0 0.5 0.5 30 260

2.0 3.0 2.0 0.5 30 260

3.0 28.0 0.5 1.5 30 260

5.0 7.0 2.0 1.5 30 260

5.0 9.0 0.5 0.5 200 260 6.0 18.0 2.0 0.5 200 260

7.0 21.0 0.5 1.5 200 260

8.0 29.0 2.0 1.5 200 260

9.0 2.0 0.5 0.5 30 340

10.0 15.0 2.0 0.5 30 340

11.0 27.0 0.5 1.5 30 340

12.0 8.0 2.0 1.5 30 340

13.0 5.0 0.5 0.5 200 340

15.0 19.0 2.0 0.5 200 340

15.0 17.0 0.5 1.5 200 340 16.0 13.0 2.0 1.5 200 340

17.0 15.0 0.0 1.0 115 300

18.0 22.0 2.8 1.0 115 300

19.0 1.0 1.3 0.0 115 300

20.0 25.0 1.3 2.0 115 300

21.0 16.0 1.3 1.0 0 300

22.0 25.0 1.3 1.0 285 300

23.0 30.0 1.3 1.0 115 220

25.0 23.0 1.3 1.0 115 380

25.0 12.0 1.3 1.0 115 300 26.0 10.0 1.3 1.0 115 300

27.0 11.0 1.3 1.0 115 300

28.0 20.0 1.3 1.0 115 300

29.0 5.0 1.3 1.0 115 300

30.0 6.0 1.3 1.0 115 300