Chapter 4
Materials and methods
Materials and methods
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.