Chapter 4

Materials and methods

  

 

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44

4.1 Materials and instruments

All chemicals used in this study were of analytical grade except for specific chemical. Almost all

the chemicals used for the preparation of various selective, differential and biochemical media

were of good quality mainly from the Sigma Aldrich Co., Merck (Germany), Sisco Research

Laboratories (SRL) Pvt. Ltd., Mumbai, Loba chemie Pvt. Ltd., Hi-media Laboratories Pvt. Ltd.,

(India), Spectrochem Pvt. Ltd., Mumbai, ACS Chemicals, S.D.Fine Chemicals (India),

Qualigens (India), The media and reagents were accurately prepared according to the

compositions and preparations mentioned in the appendix.

The glassware used were of Borosil, thoroughly cleaned, decontaminated and then sterilized (If

required) as per the standard method before use.

Various equipments, accessories and instruments used during the study are Plastiwares (Tarsons

Products Pvt. Ltd., Kolkata), Micropipettes (Span diagnostics Pvt. Ltd., India), Microcentrifuge

tubes (Tarsons Products Pvt. Ltd., Kolkata), UV-vis spectrophotomenter (Systronics).

Microscope (Olympus), Microscope eyepiece digital camera (Catalyst biotech, India), pH meter

(Systronics), Laboratory fermenter (Sartorius stedim, Germany) Vertical gel electrophoresis

system( Mini protean tetracell) and Gel Doc XR System (Bio-Rad Laboratories,

Inc.USA).Laminar air flow (Sun Instruments Pvt. Ltd. Ahmedabad), Orbital Shaking Incubator

(Remi Laboratory Instruments), Chromatography column with Teflon screw cap stopcock

(Durasil, India),

4.2 Sample collection

Samples were collected from waste of regional oil mill, soil and agricultural compost in a sterile

petridish with a spatula for isolation of cholesterol oxidase producing microorganisms.

4.3 Isolation of microorganisms

Cholesterol oxidase producing microorganisms were isolated by following modified procedure

of Nagasawa et al., (1969). 1 gm. of samples was suspended in 100 ml of distilled water. The

suspension was vigorously shaken for 30 min. A volume of 100 μl of supernatant was inoculated

in a triplicate of solid medium A containing cholesterol as the sole carbon source. Medium A

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contained (gm. / lit.): agar, 3.0 % (In case of solid medium); K2HPO4, 0.25; NH4NO3, 17;

MgSO4.H2O 0.25%; FeSO4, 0.001; NaCl, 0.005; cholesterol, 0.1% and Tween 80, 0.5 ml. The

pH of medium was adjusted to 7.0. For suspending cholesterol and avoiding its coagulation, it

was first dissolved in 10 ml solution of 20% isopropanol plus 10% of Tween 20 and then added

to the medium. After incubation period was completed, abscission colonies were appeared on the

plate surface. In order to fast growing and generation of larger colonies, they were subculture in

secondary medium (medium B) containing cholesterol as the only source of carbon as well as

yeast extract (Yazdi et al., 2001, Lashkarian et al. 2010). This medium contained yeast extract,

0.3 gm; (NH4)2HPO4, 0.1 gm; cholesterol, 0.15gm; Tween 80, 0.05 ml; pH – 7; agar, 3.0 % and

distilled water, 100 ml. Each colony on medium A was cultured in medium B and incubated at

30oC for 24 h. Then, larger colonies generated on medium B were used for further study.

4.4 Screening of CHO producing organisms

CHO producing organisms were screened by detecting the products produced after oxidation of

cholesterol by using indicator plate. CHO is able to convert Cholesterol into Cholest-4-en-3-

one and hydrogen peroxide. CHO producing colonies were selected on these plates were

prepared by adding 1.0 g Cholesterol, 1.0 g Triton X-100, 0.1g o-dianisidine and 1000 Units of

peroxidase to 1 liter of agar medium. Selected isolated colonies from medium B were cultured

on these plates and incubated at 30°C for 2 to 3 days. Cholesterol penetrates into bacterial cells

where it can be converted into hydrogen peroxide by Cholesterol oxidase. Reagents that exist

in the medium react with hydrogen peroxide (H2O2) to form azo compound which turns the

color of medium into intense brown color (Nishiya et al. 1997, Drzyzga et al. 2011, Ferna´ndez

de las Heras et al. 2011).

4.5 Identification of isolates

Primary characterization of isolates was carried out by studying their morphological, cultural and

biochemical characteristics by standard method. Molecular characterizations of isolates were

carried out by studying their partial 16S or ITS (Internal Transcribed Spacer) ribosomal DNA

(rDNA) sequences.

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The ITS regions of fungal rDNA are highly variable sequences of great importance in

distinguishing fungal species by PCR analysis (Figure 4.1). The amplification of internal

transcribed spacer (ITS1-5.8S-ITS2) of rDNA by the polymerase chain reaction (PCR) combined

with sequencing of the amplicon and analysis of similarity between the sequences obtained and

those already deposited in the gene bank, has been frequently employed for identification of

fungal species.

Figure 4.1. Structure of the rDNA gene cluster and positions of fungal PCR primers. The

cluster is split into coding (18S, 5.8S and 28S genes) and non-coding ITS regions.

The positions of the PCR primers and their direction of synthesis are indicated by

arrows.

16S / ITS rDNA sequencing of bacterial isolates were carried out at Microbial culture

collection, National center for cell science, Pune university campus, Pune 400011, India. The

16S rDNA of bacterial isolates were amplified using 8F (5’-AGAGTTTGATCCTGGCTCAG-

3’) and 907R (5’- CGTCAATTCMTTTRAGTTT-3’) as forward and reverse primer

respectively.

The amplification of ITS region in fungal isolates were carried out by universal primers ITS1

(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCC GCTTATTGATATGG-3’)

(White et al. 1990).

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The outline for 16S / ITS rDNA sequencing of the isolates is given below.

Sequencing pure isolate(s) using multiple PCR primers in ABI 3730XL sequencing machine.

DNA isolation (PCR Template preparation) by Phenol-Chloroform method

PCR amplification by using 16S / ITS rDNA region primers

Check the amplification on agarose gel

PCR purification by PEG-NaCl method

Cycle sequencing using primer

Cycle sequencing clean up

Loaded samples on a machine 3730 XL

Identification of isolates was carried out by comparing the partial 16S/ ITS rDNA sequence with

known sequences contained within large database using BLAST

(Basic Local Alignment Search Tool) program of NCBI.

The results of primary characterization and partial sequences of 16S or ITS rDNA were used to

identify the isolates. Bergey’s manual of systematic bacteriology, 2nd

edition and Illustrated

genera of imperfect fungi, 4th

edition by Barnet & Hunter were used to identify bacterial and

fungal isolates respectively.

The phylogenetic analyses of isolates were carried out on the basis of partial 16S/ ITS rDNA

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sequences. The nucleotide sequence of related organisms was obtained from the NCBI database

and used for alignment and calculating the homology level. ClustalW2 programme was used to

align the sequences. The Phylogenetic trees were constructed by the neighbor-joining method

using the MEGA 5.1 software (Molecular Evolutionary Genetic Analysis, Version 5.1)

(http://www.megasoftware.net) (Tamura et al., 2011). . Bootstrap analysis of the neighbor-

joining data, using 500 replicons, was carried out to evaluate the validity and reliability of the

tree topology by the same software. Partial sequence of 16S / ITS of some isolates were

deposited in NCBI database using BankIt submission tool of Gene bank.

4.6 Isolation and identification of cholesterol oxidation product

In order to know the cholesterol degradation products accumulated in the culture medium due to

the action of microbial cells, 50 ml of medium A in 250 ml Erlenmeyer flasks were inoculated

with a single isolated colony and incubated for 7 days at 30°C at 150 rpm. After incubation time,

Cells were harvested by centrifugation and 5 ml supernatant extracted by mixing with 2 ml.

chloroform and centrifuged at 7000 rpm for 10 min., After centrifugation 5 µl of chloroform

extract was spotted on pre-coated TLC silica gel 60 F plate (0.25 mm thick, 20 cm x 20 cm)

(Merck, Germany) at room temperature. Plates were activated at 110°C for 1 hour. Benzene:

ethyl acetate 9:1 (v:v) was used as solvent system and the development was carried out by

spraying H2SO4: methanol 5:95 (v:v) solution followed by heating at 90°C until visualization of

the spots. 5 µl of 2.0 mg/ml solutions of cholesterol and 4-cholesten-3-one was spotted as

standard along with extract (Salva et al. 1999).

For identification of cholesterol oxidation product, selected strain was cultivated in medium A,

the total culture broth (100 ml) was extracted with chloroform (10 ml). 20µL of the chloroform

layer extract was spotted onto a preparative silica gel plate (0.5mm, 10cm×20 cm). The plate was

then developed in the above solvent system for an initial separation of residual cholesterol from

its catabolic product. The spot of this product was scraped from the plate and re dissolved in

ethyl acetate (40 mL). The ethyl acetate phase containing the products was first centrifuged, in

order to discard insoluble residue, and then evaporated to dryness in a rotary evaporator. Steroid

residue was dissolved in chloroform (5 mL) and again chromatographed. This TLC was repeated

three times for the initial product spot. The structure of final product, purified by the TLC, was

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elucidated by NMR and MS analyses. λmax of the product was detected in UV and visible

spectrum. (Salva et al. 1999, Lin et al. 2010)

4.7 Determination of CHO activity

Intracellular and extra cellular CHO activity was measured by harvesting the cells by

centrifugation at 10,000 rpm for 20 min. at 4oC (Kreit et al. 1992). The supernatant (extracellular

CHO) was kept at 4oC and the pellet was resuspended in sodium phosphate buffer (20 mM, pH

7.0) containing 5% (v/v) isopropanol and 1% (v/v) Triton X-100. Intracellular CHO fraction was

obtained by magnetic stirring of the cellular suspension for 30 min at room temperature. The

enzymatic solution was collected by centrifugation (15,000 rpm, 30 min, and 4 ◦C) and stored at

4o

C until use. The enzymatic activity in both fractions (Intracellular and extracellular) were

measured by modified method based on the study of Allain et.al (1974) (Lin et al., 2010). In this

reaction, hydrogen peroxide generated during Cholesterol oxidation process was coupled with 4-

aminoantipyrine and phenol by peroxidase to produce quinoneimine dye with maximum

absorption in 500nm. The reaction mixture was consisted of 1mM 4-aminoantipyrine , 5 mM

phenol, 5 U/ml of horseradish peroxidase and sodium phosphate buffer (20 mM, pH 7.0). 50 μL

of 6 gm. / lit Cholesterol dissolved in dimethyl formamide containing 5% (v/v) Triton X-100 was

added to 1ml of reaction mixture. 3 ml. of reaction mixtures were then pre incubated for 3 min.

at 30°C. The reaction was initiated by adding 50 μl of enzyme sample and was continued for 5

min at 30°C. The assay mixture was boiled in a water bath for 2 min. to stop the reaction, and

then place in an ice bath for 2 min. Absorbance of the reaction solution was monitored at 500

nm. (Systronic 2203, India). The assay mixture containing heat inactivated enzyme was used as

the blank.

One unit of CHO activity was defined as the amount of enzyme that converts 1μmol of

cholesterol in to 4-cholesten-3-one per minute at 30°C.

Cholesterol + O2 CHO

4- cholesten-3-one + H2O2

Peroxidase

2H2O2 + 4 - aminoantipyrene + Phenol Quinoneimine dye + 4H2O

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The units of CHO activity were calculated by following equation.

( O.D.500nm/minTest - O.D.500nm/min Blank) (3.05) (D.F.)

Units/ml = --------------------------------------------------------------------------- (0.5) (13.78) (Volume of enzyme)

Where, 3.05 = Total volume (in milliliters) of assay

D.F. = Dilution factor

13.78 = Millimolar extinction coefficient of quinoneimine dye at 500 nm under the assay

conditions

0.5 = Conversion factor based on one mole of H2O2 produces half a mole of

quinoneimine Dye

4.8 Optimization of fermentation medium

4.8.1 One–factor-at-a-time method

The one factor-at-a-time method i.e., varying one factor while keeping all others constant was

use to determine the effect of fermentation time, inoculums age and concentration, medium

components (carbon and nitrogen source) and pH on biomass and CHO production (Chauhan

et al., 2009). The study was carried out in 250 ml. Erlenmeyer flasks containing 100 ml. liquid

medium B, on rotary shaker at 150 rpm and 30oC for 72 hrs. The medium was inoculated with

10% (v/v) of 48 hrs. old culture grown in the same medium. Dry cell weight (DCW) was

determined by centrifugation of fermentation broth at 10,000 rpm for 15 min. and washed

twice with distilled water. The recovered biomass was dried to a constant weight at 80oC for 24

hrs.

4.8.1.1 Effect of fermentation time

In order to investigate the optimum fermentation time for CHO production, a series of flasks

were inoculated and harvested for 24 to 120 hrs. at time interval of 12 hrs. The parameters

monitored were pH, biomass and CHO activity.

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4.8.1.2 Effect of inoculums volume

The effect of inoculums volume were monitored by inoculating medium with different

inoculums concentration like 3, 5, 7,10,12,15 % v/v.

4.8.1.3 Effect of inoculums age

The effect of inoculums age were monitored by inoculating medium with different hours old

culture (12, 24, 36,48,60,72 hrs.).

4.8.1.4 Effect of carbon source

1 gm% of Glucose, Lactose, Sucrose, Maltose, Glycerol and Starch, was studied as an

alternative source of carbon. Cholesterol (0.002%) as an inducer of CHO was added, in

suspended form in 1ml of 5% Tween 80 solution, to the medium B.

4.8.1.5 Effect of nitrogen source

Cells were cultivated in the medium B containing various organic and inorganic nitrogen

sources, including meat extract, yeast extract, malt extract, peptone, ammonium sulfate,

ammonium phosphate and ammonium nitrate. Medium B contains yeast extract and

ammonium phosphate a concentration of 0.3% (w/v) and 0.1% (w/v) respectively, were

replaced with different nitrogen source at a concentration of 0.5% (w/v).

4.8.1.6 Effect of initial pH

To investigate the effect of initial pH of medium on CHO production, fermentation runs were

carried out by adjusting initial pH of the medium B in the pH range of 5 , 5.5,6,6.5,7,7.5,8 to

8.5.

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4.8.2 Orthogonal array method

Orthogonal array method was used for screening of the most significant fermentation parameters

influencing CHO production. The design for the orthogonal array was developed and analyzed

using “MINITAB 15” software (Pennsylvania State University, University Park, Pennsylvania).

The experimental results were analyzed to extract independently the main effects of the factors.

The controlling factors were identified, with the magnitude of effects qualified and the

statistically significant effects determined. Accordingly, the optimal conditions were determined

by combining the levels of factors that had the highest main effect value. All experiments were

performed in triplicates.

4.8.3 Validation of experiments at bioreactor level

The validation of data was done by using optimized process parameters and fermentation media

components in shake flask and laboratory level bioreactor. A laboratory fermenter of Sartorius

stedim, Germany (Biostat A plus, 5 lit. capacity) with 2 lit. working volume were used for

fermentation. The fermenter was autoclaved at 121°C, 15 lb/in2 pressure for 20 min and allowed

to cool at room temperature. During fermentation, the parameters like pH, Temperature and

dissolved oxygen concentration were monitored.

4.9 Purification of cholesterol oxidase

4.9.1 Ammonium sulfate precipitation

The culture broth was centrifuged (10,000 rpm) for 20 min at 4oC, and the supernatant was

collected. It was subjected to ammonium sulfate fractionation (at 60% saturation) to precipitate

the produced CHO. Ammonium sulfate was slowly added to the supernatant fluid with stir (on

magnetic stirrer) and allowed to stand for 3 hrs. at 4oC and kept at 4

oC overnight. The precipitate

thus formed was obtained by centrifugation (10,000 rpm, 15 min and 4oC) and dissolved in 20

mM sodium phosphate buffer (pH 7.0). Precipitate solution was checked for CHO activity and

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the protein content was measured by the method of Lowry et al., (1951) using bovine serum

albumin (BSA) as the standard.

.

4.9.2 Dialysis

Precipitate solution was dialyzed against the same buffer for removal of ammonium sulfate and

impurities by dialysis Membrane-50Av. flat width - 22.54 mm, Av. diameter - 14.3 mm, capacity

approx - 1.61 ml/cm (Hi-media, Mumbai). Dialysis tube was prepared by boiling in 2% sodium

bi carbonate and 0.05% EDTA at 80oC for 15 min. Rinse the tube in distilled water then boil in

warm water twice for 15 min. Dialysis was performed on a magnetic stirrer at 4oC for 4 hrs.

Follow buffer change for 3 times and then kept at 4oC overnight. CHO activity and the protein

content were tested after dialysis by following the same method as mention above.

4.9.3 Column chromatography

The resulting dialyzed mixture was filled in a column (1.6cm×20 cm) and equilibrated with 20

mM sodium phosphate buffer (pH 7.0) to separate CHO from the polysaccharides. These

polysaccharides, containing negatively charged glucuronic acid, were bound to the DEAE-

cellulose column, while CHO was eluted with the buffer. (Lin et al. 2010).

4.10 Determination of protein concentration

Protein concentration of crude enzyme and at each step of purification was determined by the

method of Lowry et al., (1951) using bovine serum albumin (BSA) as the standard.

4.11 Determination of the properties of the purified cholesterol oxidase

4.11.1 Determination of molecular weight

The molecular weight of the purified enzymes was determined by sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE). Protein extracts were prepared by precipitating

either aliquot of culture broth supernatant or purified protein solution with 24% trichloroacetic

acid. The pellets were washed twice with cold acetone and allow pellets to air dry.

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Discontinuous polyacrylamide gels consist of a resolving or separating (lower) gel and stacking

(upper) gel was prepared in a Miniprotean tetra cell system (Bio-Rad). The stacking gel acts to

concentrate large sample volumes, resulting in a better band resolution than is possible using the

same volumes on a gel without a stack. 12% SDS-polyacrylamide slab gel (0.1 × 18.0 × 18.0 cm)

was prepared as described by Laemmli (1970).Pellets were dissolved in gel loading buffer

containing SDS and 2-mercaptoethanol, denatured by boiling at 100 °C for 5 min and subjected

to gel electrophoresis according to the method of Laemmi in 1 X Tris/glycerine-SDS-running

buffer. After the run was completed, the gel was stained in 50 ml of 0.1% coomassie blue R-250,

40% methanol, 10% glacial acetic acid. The gel was destained in destaining solution (10%

glacial acetic acid and 40% methanol). The molecular weight of the purified enzymes was

determined by comparison with the standard protein markers: Phosphorlase B (98 KDa) (Kilo

Dalton), Bovine serum albumin (68 KDa), Ovalbumin (44 K Da), Glutathione S transferase (29

K Da), Lysozyme (16 K Da). The Gel Doc XR system (Bio-Rad) was use to capture the image of

the gel.

4.11.2 Determination of substrate specificity

In order to examine the substrate specificity for CHO, different steroid compound were tested by

standard assay method. Cholesterol, Dihydrocholesterol, Stigmasterol, β-Sitosterol,

Ergosterol,Cholic acid, Deoxycholic acid, Pregnenolone, Dihydrocholesterol, and Stigmastanol

were used as a substrate.

4.11.3 Determination of Km and Vmax values of the cholesterol oxidase

Reaction kinetics of the CHO were examined with cholesterol as substrate under standard assay

conditions with the substrate concentration as only variable (0–0.2 mM). The Km (Michaelis

constant) value and Vmax for cholesterol were determined of protein from the data obtained by

the method of Lineweaver-Burk (Eisenthal et al. 1974), Eadie-Hofstee (Hofstee 1959) and

Hanes-Woolf plot (Hanes 1932).

The equation for the linear regression is y = kx + m where k is the slope of the line and m

represents the intercept of the y-axis. The R2 value is the square of the correlation coefficient

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which gives us a measure of the reliability of the linear relationship between the x and y values

(values close to 1 indicate excellent linear reliability). Km and Vmax in all the plots were

calculated by following equations.

Plot Km Vmax Lineweaver-Burk -1/(-m/k) 1/m

Eadie-Hofstee -k m Hanes-Woolf M

x Vmax 1/K

4.11.4 Effect of temperature and pH on CHO activity and stability

Effect of temperature on CHO activity was assayed under standard assay condition except for the

reaction temperature. The reaction temperature was varied between 4 to 70oC. Thermo stability

of the enzyme was assayed by incubating at different temperatures, ranging from 4o C to 70

o C,

in 20 mM sodium phosphate buffer pH 7.0 for 2 hrs. The percentage of residual activity was

obtained.

The CHO activity was determined at different pH and temperature values. Effect of pH on CHO

activity was assayed under standard assay conditions except that pH of reaction mixture varied

between 3.0 to 10. The buffers (20 mM each) used were: pH 4.0, citric acid–sodium phosphate;

pH 5.0–9.0, phosphate; pH 10.0, sodium carbonate–bicarbonate. Stability of CHO were

determined after incubating the enzyme in above mention various buffers having pH ranging

from 3.0 to 10.0 at 30oC for 2 hrs. and residual activity was measured.

4.11.5 Effect of metal ions and chemicals on CHO activity

Enzyme was incubated in 20mM sodium phosphate buffer (pH 7.0) containing a metal ion (1mM

final concentration) among the examined metals, for 2 hrs. at 30 o

C; relative enzyme activity

was measured.

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Effect of detergents and other chemicals were assayed under standard assay conditions with

0.1% chemicals which were incubated at (20mM sodium phosphate, pH 7.0) 30 o

C and 150

rpm for 2 hrs. Residual enzyme activities were then determined.