Materials and Methods

MATERIALS AND METHODS

The present investigation was carried out in the Department of

Sericulture/ Life Science Bangalore University, Bangalore during the year

2010-2015. The material used and the methods followed in this study are

presented below.

3.1 COLLECTION AND CULTIVATION OF HYPSIZYGUS ULMARIUS

The edible mushroom Hypsizygus ulmarius (DMRP-253) spawn was

collected from Directorate of Mushroom Research, Chambaghat, Solan,

Himachal Pradesh, India. The mushrooms were cultivated by hanging bag

method (Peter oei, 2005) in association with Vinayaka mushroom cultivators,

Hebbal, Bangalore, Karnataka.

The fresh and well dried paddy straw was used as substrate. The paddy

straw were chopped into 3-5 cm pieces and soaked in fresh water for 8-16

hours, and then excess water was drained off and spread on mesh. The soaked

substrate was boiled in water for 30 minutes at 80-850 C to minimize

contamination and give higher yield. The excess hot water was drained and

spread on mesh. When the pasteurized substrate was cooled at room

temperature, filling and spawning was carried out. At this stage, substrate

moisture content was maintained at 70%. Polythene bags of 150 gauge was

used for layer spawning, wherein substrate was filled and pressed to a depth of

Page 54
 Materials and Methods

8 cm and a handful of spawn was spread above it. Similarly, second and third

layers were done and finally the bag was closed and hanged from the ceiling in

cropping room. After 18 days, white mycelium was observed in the polythene

bags. At this stage polythene cover was removed maintaining the temperature

range of 24-29 0C with relative humidity of 70%. About 5 days was required

for the appearance of pinhead mushroom stage, after which fruiting bodies was

seen within 5 days. They were plucked before it sheds spores and this was

referred as first crop. The total cropping cycle of this variety from spawning to

harvesting was found to be 35 days. After first crop harvest, 1 cm outer layer

of the bag was scrapped off. This helps to initiate second crop which appears

after 8 days. The fruiting bodies obtained was cleaned and used for further

experimental analysis (Figure 2 & 2.1).

3.2 CHEMICALS AND REAGENTS

The chemicals such as alpha napthyl acetate, diazo fast blue B salt,

sephacryl-1000, ammonium per sulphate, acrylamide and bis acrylamide and

reagents procured were of analytical grade from Sigma Chemicals,

St. Louis, MO, USA and Hi Media, Mumbai, India. The glassware were from

Borosil Glass Works Ltd.

Page 55
 Materials and Methods

3.3 MYCOCHEMICAL SCREENING OF HYPSIZYGUS ULMARIUS BY

QUALITATIVE AND QUANTITATIVE METHODS

3.3.1 Qualitative mycochemical screening

3.3.1.1 Methanol extraction

Fresh Hypsizygus ulmarius mushroom fruiting bodies were randomly

selected, air dried under sun for 5 days and powdered using blender. 50 gm of

mushroom powder was extracted with 400 ml of methanol (40-50 0C) using

soxhlet apparatus for 25 hrs and filtered through muslin cloth. The filtered

extract was evaporated under reduced pressure and vacuum dried to get

viscous residue (Figure 3A &B). The sample obtained was subjected to various

mycochemical analysis. The preliminary mycochemical analysis was carried

out to detect the chemical constituents such as alkaloids, saponins, phenolics,

flavonoids, tannins and glycosides present in Hypsizygus ulmarius according to

the methods described by Kokate (1994) and Khandelwal (2000).

3.3.1.2 Detection of alkaloids

Methanolic extract was dissolved in dilute hydrochloric acid and

filtered. The filtrate was tested for the presence or absence of alkaloids by

using the following tests.

Page 56
 Materials and Methods

Mayer’s test: The formation of a yellow cream precipitate indicates the

presence of alkaloids when the filtrate was treated with Mayer’s reagent

(potassium mercuric iodide).

Wagner’s test: Filtrate was treated with Wagner’s reagent (iodine in

potassium iodide) and observed. Formation of brown or reddish brown

precipitate indicates the presence of alkaloids.

Dragendorff’s test: The filtrate was treated with Dragendorff’s reagent

(solution of potassium bismuth iodide). Formation of red precipitate indicates

the presence of alkaloids.

Hager’s test: Filtrate was treated with Hager’s reagent (saturated picric

acid solution). Formation of yellow colored precipitate indicates the presence

of alkaloids.

3.3.1.3 Detection of saponins

Froth’s test: The methanolic extracts was diluted with 20 ml of distilled

water individually and shaken for 15 mins in a graduated cylinder. A layer of

foam measuring about 1 cm indicates the presence of saponins.

Page 57
 Materials and Methods

3.3.1.4 Detection of phenolics

Ferric chloride test: The extract was treated with few drops of neutral

ferric chloride solution (5 %). The formation of bluish black color indicates the

presence of phenolic compound.

3.3.1.5 Test for flavonoids

Lead acetate test: The extract was treated with few drops of 10 % lead

acetate solution. The formation of yellow precipitate confirms the presence of

flavonoid.

Alkaline reagent test: The extract was treated with few drops of sodium

hydroxide separately. Formation of intense yellow color, which turned

colorless on addition of few drops of dilute acid, indicates the presence of

flavonoid.

Shinoda test: The mushroom extract was treated with few fragments of

magnesium metal separately, followed by drop wise addition of concentrated

hydrochloric acid. The formation of magenta color indicates the presence of

flavonoid.

Page 58
 Materials and Methods

3.3.1.6 Detection of proteins

Millons test: The extract was treated with 2 ml of millon’s reagent. The

formation of white precipitate, which turned to red upon heating, indicates the

presence of proteins and amino acids.

Biuret test: The extract was treated with 1ml of 10 % sodium hydroxide

solution and heated. A drop of 0.7 % copper sulphate solution was added to the

above mixture. The formation of purplish violet color indicates the presence of

proteins.

Ninhydrin test: To the mushroom extract, 0.25 % ninhydrin reagent was

added and boiled for few minutes. Formation of blue color shows the presence

of amino acid.

3.3.1.7 Detection of carbohydrates

Extract was dissolved individually in 5 ml of distilled water and filtered.

The filtrate was used to test the presence of carbohydrates.

Molisch’s test: To the filtrate, 2 drops of alco -napthol solution

and 2 ml concentrated sulphuric acid was added carefully by the sides of the

test tube. Formation of violet ring at the junction indicates the presence of

carbohydrates.

Page 59
 Materials and Methods

Benedict’s test: The filtrate was treated with Benedict’s reagent and

heated on water bath; formation of an orange red precipitate indicates the

presence of reducing sugars.

Fehling’s test: To 1 ml of the filtrate, 1 ml each Fehling’s A and B

solutions was added and boiled in water bath. Formation of red precipitate

indicates the presence of carbohydrates.

3.3.1.8 Detection of tannins

Vanillin hydrochloric test: The extract was treated with few drops of

vanillin hydrochloride reagent. The formation of pinkish red color indicates the

presence of tannins.

Gelatin test: To the extract, 1 % gelatin solution containing sodium

chloride was added. The formation of white precipitate indicates the presence

of tannins.

3.3.1.9 Detection of glycosides

Mushroom extract was hydrolyzed with dilute hydrochloric acid and the

hydrolysate was subjected to glycoside tests.

Page 60
 Materials and Methods

Borntrager’s test: 2 ml of the filtrate was treated with 3 ml of

chloroform and shaked vigorously. Chloroform layer was separated and to this

10 % NH3 solution was added. Appearance of pink color confirms the presence

of glycosides.

Legal’s test: The extract was treated with sodium nitroprusside in

pyridine and methanolic alkali. The formation of pink to red color indicates the

presence of cardiac glycosides.

3.3.2 Quantitative mycochemical analysis of Hypsizygus ulmarius

3.3.2.1 Determination of total phenolic content (TPC)

Total soluble phenol in the methanol extract of Hypsizygus ulmarius

(DMRP-253) was determined with Folin–Ciocalteu (FC) reagent according to

the method followed by Slinkard (1977) using pyrocatechol as a standard

(100 / ml). To 1ml of extract, 46 ml of distilled water and 1 ml of FC

reagent was added and mixed thoroughly. After 3 mins, 3 ml of 2 % sodium

carbonate (Na2CO3) was added and incubated for 2 hrs with intermittent

shaking. The absorbance was measured at 760 nm using UV-Visible

Spectrophotometer (Shimadzu UV-1800 UV-VIS Spectrophotometer). The

total phenolic content was expressed as pyrocatechol equivalent in micrograms

per gram of sample.

Page 61
 Materials and Methods

3.3.2.2 Determination of total flavonoid content (TFC)

The total flavonoid content was measured according to the method of

Atanassova (2011). To 1 ml of extract 4 ml of distilled water, 0.3 ml of 5 %

sodium nitrite (NaNO2) and 0.3 % aluminum chloride (AlCl3) was added and

incubated for 5 mins. The reaction was arrested by adding 2 ml of 1 M sodium

hydroxide (NaOH) and absorbance was measured against blank at 410 nm. The

total flavonoid content was expressed as quercetin (100 / ml) equivalent in

micrograms per gram of the sample.

Page 62
 Materials and Methods

3.4 ANTIOXIDANT POTENTIAL OF HYPSIZYGUS ULMARIUS

Methanol extraction was carried out as earlier explained in section

3.3.1.1. The samples obtained were used to analyze antioxidant activity.

3.4.1 DPPH radical scavenging assay

Hydrogen donating radical scavenging ability of Hypsizygus ulmarius

methanolic extract was determined according to the method of Sharmila

(2011). 1 ml of 100 2, 2-diphenyl-1-picrylhydrazyl (DPPH) solution in

methanol was mixed with 1 ml of extract and incubated in dark for 30 min. The

absorbance was measured at 517 nm using methanol as control and ascorbic

acid (20-100 µg / ml) as reference compound.

3.4.2 Nitricoxide radical scavenging assay

The interaction of Hypsizygus ulmarius methanolic extract with nitric

oxide (NO) was assessed by using spectrophotometer according to the method

of Govindharajan et al., 2003. To 1 ml of sample, 1 ml of 5 mM Sodium

nitroprusside was added and incubated at 250C for 2hrs. The reaction was

stopped by adding Greiss reagent (1% sulphanilamide, 2% O-phosphoric acid

and 0.1% of (N-(1-naphthyl) ethylenediaminedihydrochloride) and absorbance

was measured at 546 nm by using ascorbic acid (100-500 µg/ml) as standard.

Page 63
 Materials and Methods

3.4.3 Hydroxyl radical scavenging assay

Hydroxyl radical scavenging activity of Hypsizygus ulmarius

methanolic extract was measured according to the method of Halliwell (1992).

To 1 ml of the extract, 0.1 ml 1 mM ethylenediaminetetra acetic acid (EDTA),

0.01 ml 10 mM of ferric chloride (FeCl3), 0.1 ml 10 mM hydrogen peroxide

(H2O2), 0.36 ml of 10 mM deoxyribose, 0.33 ml of 50 mM sodium phosphate

buffer (pH 7.4) and 0.1 ml of 1 mM ascorbic acid was added and incubated at

37 0C for 60 mins. To 1 ml reaction mixture, 1 ml of 10 % trichloroacetic acid

(TCA) and 1 ml of 0.5 % Thiobarbituric acid (TBA) (0.025 M NaOH

containing 0.025 % butylated hydroxyl anisole) was added and absorbance

was recorded at 532 nm using ascorbic (200-1000 µg / ml) as standard.

Page 64
 Materials and Methods

3.5 ANTIMICROBIAL ACTIVITY OF HYPSIZYGUS ULMARIUS

Methanol extraction was carried out as earlier explained in section

3.3.1.1. The samples obtained were used to analyze antioxidant activity.

3.5.1 Antimicrobial assay

Antimicrobial susceptibility was performed by using six

microorganisms such as Bacillus cereus (NICM 2106), Bacillus subtilis

(NICM 2063), Escherichia coli (NICM 2065), Pseudomonas aeroginosa

(NICM 2036), Aspergillus flavus (NICM 524) and Candida albicans (NICM

3102). The bacterial strains were maintained in nutrient agar (NA) and fungal

strains on sabouraud dextrose agar (SDA) slants at 4 0C. Zone of inhibition of

Hypsizygus ulmarius extract for the above mentioned microbes were carried

out by agar well diffusion method (Mukherjee, 1988). Bacterial cultures were

grown at 37 0C for 24 hrs in nutrient broth, whereas fungal culture on

sabouraud dextrose broth at 37 0C for 48 hrs. The culture suspension was

adjusted to 0.3 OD (bacteria) and 0.11 OD (fungi) at 650 nm respectively. To

sterile nutrient agar and sabouraud dextrose agar plates, 0.1 ml of various

inoculum was spread uniformly and wells were made using a sterile cork borer

(5 mm). 100 µl of the sample (1000 and 500 µg / well), 100 µl of dimethyl

sulfoxide (DMSO) as control and 100 µl of 100 µg / ml standards ciprofloxacin

(bacteria) and ketoconazol (fungi) were added. The plates were placed at 4 0C

Page 65
 Materials and Methods

for 1hr to allow the diffusion and incubated at 37 0C for 24 hrs and 48 hrs

respectively. The zone of inhibition around the well was measured in mm

(Hugo and Russell., 1987).

3.5.2 Minimum inhibitory concentration (MIC)

The resazurin solution was prepared by dissolving 270 mg resazurin

powder in 40 ml of sterile distilled water. MIC for bacteria was carried out

according to the method of Satyajit (2007). To a sterile 96 well plate, 100 µl of

sample (10 mg / ml) in DMSO and standard ciprofloxacin (1 mg/ml) was

pipetted individually. 50 µl of nutrient broth was added. A serial dilution in

descending concentrations was carried out so that each well contains 50 µl.

10 µl of resazurin indicator solution and 30µl broth was added. Finally 10 µl of

bacterial (0.3 OD adjusted) suspension was added to each well. Each plate was

wrapped loosely to ensure that bacteria do not become dehydrated. Each plate

had a set of positive control and negative controls. The plates were incubated at

37 0C for 18–24 hrs. The color change from purple to pink or colorless was

recorded as positive. The lowest concentration at which color change occurred

was taken as the MIC value.

Minimum inhibitory concentration for fungi was carried out according

to the method of Gibbons (2002). For the assay, 1ml of sterile SD broth and

1ml of known concentration (1 mg / ml) of sample was added and serial

Page 66
 Materials and Methods

dilution was carried out in two fold decreasing concentrations. Finally 1 ml of

inoculum (0.11 OD adjusted) and 20 ul of resazurin indicator was added and

incubated at 28 0C for 48 hrs. The tubes were inspected for color change from

purple to pink or colorless and recorded as positive. The lowest concentration

at which color change occurred was taken as the MIC value.

Page 67
 Materials and Methods

3.6 PURIFICATION AND CHARACTERIZATION OF CARBOXYLESTERASE

FROM HYPSIZYGUS ULMARIUS

3.6.1 Enzyme extraction

The fresh fruiting bodies of Hypsizygus ulmarius mushroom was

collected and stored at -20 0C until the sample was processed. The frozen

fruiting bodies of mushroom were ground to a fine powder in liquid nitrogen.

The powdered sample (80 gm) was further homogenized in 85 ml of 0.15 M

sodium chloride (NaCl) by using blender and left overnight with constant

stirring at 4 0C. The homogenate was centrifuged at 12,000 rpm for 30 mins at

4 0C. The crude enzyme extract was measured and enzyme assay was carried

out.

3.6.2 Determination of Protein

Protein concentration was determined according to Lowry et al., (1951)

method using bovine serum albumin (BSA) as standard.

3.6.3 Enzyme assay

Esterase activity was assayed according to the modified Gomori (1953)

method (Van asperen et al., 1962). The assay mixture consisting of 5 ml of

0.3 mM 1-naphthyl acetate and suitably diluted enzyme (1 ml) was incubated

at 27 0C for 15 mins. The reaction was stopped by addition of 1 ml of DBLS

Page 68
 Materials and Methods

reagent (2 parts of 1 % diazo blue B and 5 parts of 5 % sodium lauryl

sulphate). In the control, enzyme was inactivated by DBLS prior to incubation

with substrates. The absorbance was measured at 600 nm using -napthol

(20-100 ) as standard. One unit of enzyme activity was defined as the

amount of enzyme that released 1 µ mol of product (1-napthol) per min at pH

7.0 and 27 0C.

3.6.4 Purification of carboxylesterase enzyme

The carboxylesterase enzyme purification procedures were carried out

at 4 0C. The enzyme was purified from the crude supernatant by the following

steps.

3.6.4.1 Ammonium sulphate fractionation

The crude sample was subjected to 90 % saturation using ammonium

sulphate (80.76 gm / 122 ml), with constant stirring on an ice bath. The

proteins were allowed to precipitate for 5 hrs and then centrifuged at

10,000 rpm for 30 mins. The pellet was resuspended in minimum volume of

50 mM sodium phosphate buffer pH 6.0.

3.6.4.2 Dialysis

The 90 % ammonium sulphate precipitate (enzyme suspensions) were

dialyzed in dialyzing membrane-40 against 0.025 M sodium phosphate buffers,

Page 69
 Materials and Methods

pH 6.0 for 24 hrs and to remove ammonium sulfate ions from protein

molecules at 4 0C. Further, the buffer was replaced every 6 hrs to increase the

efficiency of desalting. Finally esterase activity and protein concentration of

diluted sample was measured. The specific activity of enzyme was calculated

based on the assay.

3.6.4.3 Gel filtration chromatography

The glass column measuring 60 x 1.5 cm was washed with hot water

before use. Sephacryl-1000 (5 gm) gel was equilibrated with 0.05 M sodium

phosphate buffer, pH 6.0 and allowed to swell overnight. 105 ml of equilibrated

Sephacryl 1000 matrix was packed into the column and allowed to settle. The

upper surface of the sephacryl was protected by filter paper disc. 50 ml of

equilibration buffer (0.05 M sodium phosphate buffer, pH 6.0) was passed

through the column. 7 ml of sample was loaded on top of the column bed. The

proteins were eluted with 0.05 M sodium phosphate buffer, pH 6.0 and fractions

of 1 ml were collected at a flow rate of 1 ml / 5 min. The fractions were

analyzed for presence of enzyme by measuring the esterase activity.

Additionally, the selected fractions were subjected to native page analysis

(zymogram) followed by in gel enzyme assay.

Page 70
 Materials and Methods

3.6.4.4 Determination of molecular mass of purified carboxylesterase and

zymogram

In order to determine the molecular mass of the carboxylesterase,

SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was performed with

10 % polyacrylamide gel (Laemmli, 1970). Approximately 15 crude,

dialysed ammonium sulphate fraction and purified protein was loaded on 10 %

separating gel, 5 % stacking gel along with standard molecular mass markers

(14.3-66 kD) and electrophoresis was carried out at a constant current of

50 mA. After electrophoresis the gel was incubated in 0.05 M sodium

phosphate buffer, pH 6.0 for 15 mins at 37 0C. The gel was stained with

Coomassie brilliant blue (CBB) R-250 and destained (5 % methanol and 7.5 %

acetic acid) to determine the molecular mass of the carboxylesterase. Native

PAGE (zymogram) was performed by using a non denaturing acrylamide gel

of 10%. After electrophoresis, in gel esterase activity was carried out according

to the method of Hunter and market (1957). The gel was stained for esterase

activity using 100 ml of 0.05 M phosphate buffer, pH 7.0 containing 40 mg of

fast blue RR and 20 mg of 1-napthyl acetate for 20 mins at room temperature.

The gels were stored in 7.5 % acetic acid at 4 0C.

Page 71
 Materials and Methods

3.6.5 Biochemical characterization of carboxylesterase

3.6.5.1 Determination of optimum pH and pH stability

The effect of pH on carboxylesterase activity was determined by using

the following buffers. 0.1 M citrate phosphate buffer (pH 3.0 & 4.0), 20 mM

acetate buffer (pH 5.0), 50 mM phosphate buffer (pH 6.0, 7.0 & 8.0) and

Tris–HCl buffer (pH 9.0). The optimum pH was determined by assaying the

enzyme with different buffers of pH 3.0-9.0 at room temperature using alpha

naphthyl acetate. Similarly pH stability was determined by preincubating the

enzyme with different buffers for 24 hrs at RT. The percentage relative

activities were calculated by comparison with un-incubated enzymes

(Faiz et al., 2007).

3.6.5.2 Determination of optimum temperature and thermal stability

To determine the optimum temperature of carboxylesterase activity, the

enzyme assay was carried out at various temperatures ranging from 10-80 0C

using alpha napthyl acetate as substrate. Similarly temperature stability was

determined by preincubating aliquots of enzyme at different temperature

(10-80 0C) for different time intervals (10, 20, 30 and 60 mins) followed by

rapid cooling. The enzyme was then assayed at room temperature. The

percentage relative activities were calculated by comparison with un-incubated

enzyme (Faiz et al., 200

Page 72
 Materials and Methods

3.6.5.3 Effect of metal ions on carboxylesterase activity

Various metal ions (Na+, Zn2+, Li+, Ni2+,Mg2+, Cu2+, Fe2+ and Ca2+) was

individually preincubated with enzyme at a concentration of 10 mM. The

enzyme activity was carried out. The percentage residual activities were

determined by comparison with the standard assay mixture with no metal ion

added (Lee et al., 1999).

3.6.5.4 Determination of Km and Vmax of carboxylesterase enzyme

Effect of alpha napthyl acetate at different concentration ranging from

0.1-3 mM was used and enzyme assay was carried out. The kinetic data were

plotted as reciprocals of activities versus substrate concentrations. The

Michaelis –Menten constant (Km) and maximum velocity (Vmax) values were

determined as the reciprocal absolute values of the intercepts on the X and Y

axis respectively. To obtain linear regression curve LB plot was drawn

(Lineweaver & Burk, 1934).

Page 73
 Materials and Methods

3.7 PURIFICATION AND CHARACTERIZATION OF ALKALINE PROTEASE

FROM HYPSIZYGUS ULMARIUS

3.7.1 Enzyme extraction

Enzyme extraction was carried out according as described in earlier

section 3.6.1 with only difference of centrifugation at 12,000 rpm for 30 min at

40C.

3.7.2 Protein determination

Protein concentration was determined according to Lowry et al., (1951)

method using bovine serum albumin (BSA) as standard.

3.7.3 Protease assay

Protease activity was assayed according to the method of Wu (2011)

with tyrosine (100 µ mole / ml) as standard. Crude enzyme and 1 % casein was

incubated at 37 0C for 30 mins 5 % trichloroaceticacid (TCA) was added at the

end of incubation period and centrifuged at 10,000 rpm for 10 mins.

Absorbance at 280 nm was recorded with control. One unit of protease activity

is defined as amount of enzyme required to release 1 µ mole of tyrosine per

minute under the experimental conditions.

Page 74
 Materials and Methods

3.7.4 Purification of alkaline protease

3.7.4.1 Ammonium sulphate fractionation

Ammonium sulphate fractionation was carried out as described in

earlier in section 3.6.4.1. The obtained pellet was resuspended in minimum

volume of 50 mM Tris-HCl buffer, pH 10.0.

3.7.4.2 Dialysis

The 90 % ammonium sulphate precipitate (enzyme suspensions) were

dialyzed in dialyzing membrane-30 against 50 mM Tris-HCl buffer, pH 10 for

24 hrs and to remove ammonium sulfate ions from protein molecules at 4 0C.

Further the buffer was replaced every 6 hrs to increase the efficiency of

desalting. Finally, protease activity and protein concentration of diluted sample

was measured. The specific activity of enzyme was calculated based on the

assay.

3.7.4.3 Gel filtration chromatography

The glass column measuring 60 x 1.5 cm was washed with hot water

before use. Sephacryl-1000 (5 gm) gel was equilibrated with 0.05 M Tris-HCl

buffer, pH 10 and allowed to swell overnight. 105 ml of equilibrated Sephacryl

1000 matrix was packed into the column and allowed to settle. The upper

surface of the sephacryl was protected by filter paper disc. 150 ml of

Page 75
 Materials and Methods

equilibration buffer (0.05 M Tris-HCl buffer, pH 10.0) was passed through the

column. 8 ml of sample was loaded on top of the column bed. The proteins

were eluted with 0.05 M Tris-HCl buffer, pH 10.0 and fractions of 1 ml were

collected at a flow rate of 1 ml / 5 min. The absorbance was carried out at

280 nm. The fractions were analyzed for presence of enzyme by measuring the

alkaline protease activity. Further, the selected fractions were subjected to

zymogram analysis followed by in gel enzyme assay.

3.7.4.4 Determination of molecular mass of purified alkaline protease and

zymogram

In order to determine the molecular mass of the alkaline protease,

SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was performed

(Laemmli, 1970) using 10 % separating gel and 5 % stacking gel.

Approximately 15

purified protein was loaded with standard medium molecular mass markers

(14.3-66 kD) and electrophoresis was carried out at a constant current of

50 mA. After electrophoresis, the gel was incubated in Tris-HCl buffer, pH 10.0

buffer for 15 mins at 37 0C. The gel was stained with Coomassie brilliant blue

(CBB) R-250 and destained (5 % methanol and 7.5 % acetic acid) to determine

the molecular mass of the protease. For zymography, 10% non denaturing

PAGE was performed and the gel was then transferred into 1 % casein solution

and incubated for 30 mins at 37 0C in the presence of 50 mM Tris buffer with

Page 76
 Materials and Methods

pH 10.0. Following incubation the gel was stained with Coomassie brilliant

blue (CBB) R-250 to visualize the hydrolysis zones (Garcia-Carreno et al.,

1993).

3.7.5 Biochemical characterization of alkaline protease

3.7.5.1 Determination of optimum pH and pH stability

Effect of pH and pH stability on protease activity was determined by

using the following buffers 100 mM citrate phosphate buffer (pH 3.0 & 4.0),

20 mM acetate buffer (pH 5.0), 50 mM phosphate buffer (pH 6.0, 7.0 & 8.0)

50 mM Tris–HCl buffer (pH 9.0) and 50 mM sodium phosphate-sodium

hydroxide buffer (pH 10.0, 11.0 & 12.0). The optimum pH was determined by

assaying the enzyme with different buffers of pH 3.0-12.0 at RT using 1 %

casein. Similarly pH stability was determined by pre-incubating the enzyme

with different buffers for 24 hrs at room temperature. The percentage relative

activities were calculated by comparison with un-incubated enzymes

(Faiz et al., 2007).

3.7.5.2 Determination of optimum temperature and thermal stability

To determine the optimum temperature of alkaline protease, the enzyme assay

was carried out at various temperatures (20-80 0C). For thermal stability,

aliquots of enzyme was preincubated at different temperatures (20-80 0C) for

different intervals of time (10, 20, 30 & 60 mins) followed by rapid cooling
Page 77
 Materials and Methods

and assaying at room temperature. The percentage relative activities were

calculated by comparison with un-incubated enzyme (Faiz et al., 2007).

3.7.5.3 Effect of metal ions on alkaline protease activity

Various mono and divalent metal ions (Na+, Zn2+, Li+, Ni2+, Mg2+, Cu2+,

Fe2+ and Ca2+) was individually preincubated with enzyme at a concentration

of 10 mM. The enzyme activity was determined. The percentage residual

activities were determined by comparison with the standard assay mixture with

no metal ion added (Lee et al., 1999).

3.7.5.4 Effect of protease inhibitors on enzyme activity

To determine the effect of protease inhibitor such as

phenylmethylsulfonyl fluoride (PMSF) and Ethylene-diamine-tetraacetic acid

(EDTA), the enzyme assay was carried out in presence of varied concentration

of individual inhibitors (0.04, 0.2, 1.0, 10 mM) at room temperature. The

relative enzyme activity in presence of inhibitors was calculated and compared

with the unincubated contol assay (Lee et al., 1999).

3.7.5.5 Determination of Km and Vmax

Protease activity was carried out in presence of casein at different

concentration (0.1 %, 0.2 %, 0.5 %, 1 %, 2 %, and 5 %) as substrate. The

kinetic data were plotted as reciprocals of activities versus substrate

Page 78
 Materials and Methods

concentrations. The Michaelis–Menten constant (Km) and maximum velocity

(Vmax) values were determined as the reciprocal absolute values of the

intercepts on the X and Y axis respectively. The linear regression curve was

also plotted (Lineweaver and Burk, 1934).

3.8 STATISTICAL ANALYSIS

All the experimental results were pooled and expressed as mean ± SE of

three parallel measurements. Statistical analysis of data was processed using

analysis of variance (ANOVA) and means were compared by Turkeys test

(SPSS version 20.0). 0.05.

Page 79
 Materials and Methods

Figure 2 (A-F) Cultivation of Hypsizygus ulmarius hanging bag method

[

A B
A & B Seeds of Hypsizygus ulmarius and spreading of pasteurized paddy straw in
polytene bags.

C D
C & D Layer spawning of Hypsizygus ulmarius seeds.

E F

E & F Development of mycelia in polythene bag.

Page 80
 Materials and Methods

Figure 2 (G-L) Cultivation of Hypsizygus ulmarius hanging bag method

G H
G & H Pin head stage of Hypsizygus ulmarius.

I J

I & J Button stage of Hypsizygus ulmarius.

K L

K & L Fruiting bodies of Hypsizygus ulmarius.

Page 81
 Materials and Methods

Figure 3 Mycochemical screening of Hypsizygus ulmarius

A Powdered fruiting bodies of Hypsizygus ulmarius

B Methanolic extract of Hypsizygus ulmarius

Page 82