MATERIAL AND METHOD

3.1 Protease from biological (Bacteria and Plant) sources-Sample collection

3.1.1 Soil Collection

Soil was collected in sterilized sampling bags of Ghazipur poultry waste site, India. Soil was

selected from areas where naturally degraded feather were seen. The samples were brought to

the laboratory and processed for the analysis the same day. Soil samples (10%) were

suspended in basal media (PeptoneBroth - Beef extract 3g/l Peptone 5g/l, NaCl 75g/l,

Distilled water 1liter) and kept for growth at 37°C for 6 days. After 24 hrs of incubation at

regular intervals of 6 hrs the activity of protease was measured and sample showing

maximum activity was screened for protease producing strains.

3.1.2 Plant Material Collection

Green and Senesced leaves of different plants growing in and around the University area in

Noida, Uttar Pradesh, India were collected in forenoon in plastic bags and brought to the

laboratory and processed for the analysis the same day.

49

3.2 Isolation and Screening of protease-producing strains and plants

3.2.1 Soil Isolate

Soil isolate showing maximum protease activity were plated on skim milk agar (skim milk

powder 10 %, peptone 0.1%, NaCl 0.5% and 2 % agar). Plates were incubated at 37°C for 24

hours and the colony that showed clear zone were selected and was sub cultured in LB broth

The bacterial isolate was further incubated in cultivation media for 48 hrs and checked for

protease production.

3.2.2 Plant Source

Green and Senesced leaves of Asparagus officinalis (Asparagus), Ocimum sanctum (Tulsi),

Murraya koenigii (Karipatta), Tagetes erecta (Marigold), Hibiscus rosasinensis (China rose),

Carica papaya (Papaya), Vinca Rosea (Periwinkle), Azadirachta indica (Neem), Musa

paradisiaca (Banana), Lantana camara (Lantana) growing in and around the University area

in Noida Uttar Pradesh India were collected in forenoon in plastic bags and brought to the

laboratory and processed for the analysis the same day. Leaves of each plant were washed

with distilled water and blotted dry were weighed (15 gm), homogenized with mortar and

pestle in three volumes w/v 10 mM Tris-chloride buffer (pH 8.0) followed by filteration with

three layered cheese cloth. Then the filterate was centrifuged at 10,000 rpm for 10 min at

4°C. The supernatant of leaves of each plant was collected separately and used as crude

preparation. The crude extract was checked for protease activity.

3.3 Identification of soil isolates and plant species

3.3.1 The isolate GW1 was identified originally as a strain of pseudomonas based on

Morphological, Biochemical and 16S rDNA sequencing characteristics.

3.3.2 Lantana camara was selected as a biological source for protease isolation and it is

classified under family verbanaceae genus Lantana species camara according to Linnaeus

classification system.

50

3.4 Identification and classification of soil isolate

3.4.1 Microscopic Examination

3.4.1.1 Gram Staining

A drop of sterile saline water was placed on the microscope slide. A light suspension of the

test culture was made with normal saline and heat fixed. The slide was allowed to cool and

flooded with crystal violet for 60 seconds and then washed with distilled water. It was then

flooded with Gram’s iodine for 60 seconds, washed with water and excess water was drained

off. The stain was then decolorized with acetone which was washed off immediately.

Counterstaining was done with safranin for 2 minutes. The slide was then washed, air dried

and observed under a microscope.

3.4.1.2 Endospore Staining

A smear of the culture was prepared on a clean slide and heat fixed. The smear was flooded

and kept saturated with 5% malachite green stain while the slide was heated continuously for

5 minutes. The smear was then rinsed with distilled water and counter stained with safranin

for 2 minutes. The slide was once again washed with water, air dried and investigated under a

microscope for endospore formation.

3.4.2 Biochemical Test

3.4.2.1 Oxygen Requirement

The bacterium was inoculated into nutrient broth (peptone-5g/l, NaCl-3g/l, Beef extract-3g/l,

distilled water-1liter) to study the oxygen requirement of the pure isolate. Depending on the

growth of the bacteria are grouped as aerobic, micro-aerobic and anaerobic.

3.4.2.2 IMViC Test

IMViC is an acronym that stands for indole, methyl red, Voges-Proskauer, and citrate test.

51

3.4.2.2.1 Indole Production

The bacterial isolate was inoculated into Tryptone broth (tryptone-10g/l, NaCl-5g/l, distilled

water-1 liter) and incubated at 37°C for 48 hours. Then 0.5 ml of Kovac’s reagent was added

and shook the culture gently. The test tube was observed for colour reaction.

3.4.2.2.2 Methyl Red Test

Bacteria produce acid by fermentation of glucose, which changes the pH of the medium. It

falls and maintained below 4.5. This test detects the production of acid. The isolated test

sample was inoculated in glucose phosphate broth (Protease peptone-5g/l, D-glucose-5g/l,

Na2HPO4-5g/l, distilled water-1liter) and incubated at 37°C for 2 days. Five drops of 0.04%

solution of alcoholic methyl red solution were added to the culture, mixed well and the results

were read immediately.

3.4.2.2.3 Voges-Proskauer Test

The isolated test sample was inoculated into 5ml glucose phosphate broth (Protease peptone-

5g/l, D-glucose-5g/l, Na2HPO4-5g/l, distilled water-1liter) and incubated at 37°C for 48

hours. Then 1ml of 40% potassium hydroxide (KOH) containing 0.3% creatine and 3ml of

5% solution of α-napthol in absolute alcohol were added. The results were read immediately.

3.4.2.2.4 Citrate Utilization Test

This test is used to study the ability of an organism to utilize citrate present in Simmon’s

media (MgSO4-0.2g/l, NH4H2PO4-1g/l, K2HPO4-1g/l, sodium citrate-2g/l, NaCl-5g/l,

Bromothymol blue-0.08ml, agar-15g/l, distilled water-1liter) as a sole source of carbon for

growth. The Simmon’s media was prepared, sterilized and poured into the tubes. The media

was allowed to cool and solidify in the form of slants. The slant was then stabbed with the

isolated bacterial sample using a needle and incubated at 37°C for 36 hours.

3.4.2.3 Nitrate Reduction Test

The enzyme nitrate reductase reduces nitrate to nitrite, ammonia, nitrous oxide, nitrogen, etc.

This test is used to detect the production of nitrate reductase. The isolated test organism was

inoculated in 5ml of nitrate broth (beef extract-3g/l, peptone-5g/l, NaCl-5g/l, potassium

nitrate-1g/l, distilled water-1 liter) and incubated at 37°C for 96 hours. Then we added 1ml of

52

α-naphthylamine reagent and 1ml of sulphanilamide reagent and the results were immediately

read.

3.4.2.4 Motility Test

This test used to check for the ability of bacteria to migrate away from a line of inoculation

due to physical features like flagella. The motility media for the motility test was prepared,

sterilized and poured into tubes. The media was allowed to cool and solidify in the form of

slants. The slants were then stabbed with the isolated bacterial sample using an inoculation

needle and incubated at 37°C for 24 hours.

3.4.2.5 Catalase test

One drop of 30% hydrogen peroxide was placed on a slide. One loopful of the fresh bacterial

culture was taken by a sterile needle and placed on the drop of hydrogen peroxide. Then

observed for bubble formation.

3.4.2.6 Oxidase Test

Fresh growth of bacterial isolate was scraped from the plate with the help of disposable stick

and rubbed on to the Impregnated oxidase test strip then the strip was observed for colour

reaction.

3.4.2.7 Amylase test

This test detects the ability of an organism to produce amylase enzyme for starch hydrolysis.

The bacterial isolate was inoculated on the starch agar plates (Beef Extract -3g/l, Peptone

-5g/l, NacL-5g/l, Agar-15g/l, 10% starch solution) and incubated at 37°C for 24 hours. After

incubation period the plates were then flooded with Gram’s iodine.

3.4.2.8 Urease Test

This test detects the ability of an organism to produce urease enzyme. The test organism was

inoculated on the slant of Urea agar and incubated at 37°C for 24 hours.

53

3.4.2.9 Lactose Test

This test detects the ability of an organism to undergo lactose fermentation. The test

organism was inoculated into lactose broth (beef extract-3g/l, peptone-5g/l, lactose-5g/l) and

incubated at 37°C for 24 hours. The change in color was observed after adding phenol red

indicator.

3.4.2.10 Sucrose Test

This test detects the ability of an organism to undergo sucrose fermentation. The test

organism was inoculated into sucrose broth (beef extract-3g/l, peptone-5g/l, sucrose-5g/l) and

incubated at 37°C for 24 hours. The change in color was observed after adding phenol red

indicator.

3.4.2.11 Triple Sugar Iron Agar (TSI)

To determine the ability of an organism to attack a specific carbohydrate incorporated into a

basal growth medium, with or without the production of gas, along with the determination of

possible hydrogen sulphide (H2S) production.

TSI medium (Beef Extract – 3g/l, Yeast extract – 3g/l , Peptone - 15.0 g/l , Proteose peptone

-5.0 g/l, D-glucose monohydrate - 1.0 g/l , Lactose - 10.0 g/l, Sucrose - 10.0 g/l Ferrous

Sulphate (FeSO4.7H2O) - 0.2 g/l , Sodium Chloride - 5.0 g/l , Sodium thiosulphate

pentahydrate - 0.5 g/l, Phenol Red, 0.5 g/l aqueous solution- 48.0 ml , Agar - 12.0 g /l ) was

inoculated with an inoculating needle by stabbing the butt and streaking the slant. The tubes

were then incubated at 37°C for 24 hours.

3.4.2.12 Pseudomonas Agar P

Plates of Pseudomonas Agar P were prepared (Peptone-2%, potassium sulphate-1%, MgCl2 -

0.14%, Agar-1.8%) and the Bacterial isolate was streaked on Plates of Pseudomonas Agar P.

Then the plates were incubated at 37°C for 24 hours. After incubation period plates were

checked for pigment production for identification of isolate.

3.4.2.13 Pseudomonas Agar F

Plates of Pseudomonas Agar F were prepared (Peptone-1%, Proteose peptone- 1%, K2HPO4 –

0.15%, Agar- 1.5%, MgSO4). The bacterial isolate was streaked on Plates of Pseudomonas

54

Agar F. Then the plates were incubated at 37°C for 24 hours. After incubation period plates

were checked for pigment production for identification of isolate.

3.4.2.14 Cetrimide Agar

Bacterial isolate was streaked on cetrimide agar plates (Peptone 20gm/l, MgCl2- 1.4gm/l,

potassium sulphate- 10gm/l, Cetrimethyl diammonium bromide- 0.3gm/l, Agar- 15gm/l,

Distilled water- 1 liter) and incubated at 37°C for 24 hours. After incubation period plates were

checked for pigment production for identification of isolate.

3.4.3 16S rDna Sequencing

The sample was sent to Bangalore GeneI, Bangalore, India for identification. Genomic DNA

was isolated from the pure culture pellet. Using consensus primers, the ~1.5 kb 16S rDNA

fragment was amplified using Taq DNA Polymerase. The PCR product was bi-directionally

sequenced using the forward, reverse and an internal primer. Sequence data was aligned and

analyzed for finding the closest homologs for the microbe.

3.5 Protease production

3.5.1 Protease Production from Bacteria

The cultivation media for protease production composed of (g L-1): Peptone, 5; Glucose, 10;

NaCl, 0.5; CaCl2.2H2O, 0.1; K2HPO4, 0.3; KH2PO4, 0.4; MgSO4 · 7H2O, 0.1; and yeast extract,

5. The pH was maintained at 7.5. Microbes were allowed to grow in 500 ml conical flask

containing 50 ml of the culture media that was maintained at 37°C at 140 rpm. 5% (v/v) of the

20 hrs old culture was inoculated in cultivation media. [213].

i. Tryptic soy broth (TSB)

ii. Gelatin (1%) + cultivation medium for protease production

iii. Casein (1%)+ cultivation medium for protease production

iv. Skim milk powder (1%)+ cultivation medium for protease production

v. Pigeon feathers (1%)+ cultivation medium for protease production

The above cultivation media were checked for enzyme activity by the modified method of

Tsuchida et al. [214].

55

3.5.2 Protease Production from Plant

Senesced and green leaves

Green and Senesced leaves of Lantana camara (Lantana) growing in and around the

University area in Noida Uttar Pradesh India were collected in forenoon in plastic bags and

brought to the laboratory and processed for the analysis the same day. The leaves were weighed

after washing and rinsing with distilled water, homogenized with mortar and pestle in three

volumes wt/vol 10 mM Tris-chloride buffer (pH 8.0) followed by filteration with three layered

cheese cloth. Then the filtrate was centrifuged at 10,000 rpm for 10 min at 4°C. The

supernatant was collected and used as crude preparation. The crude extract was checked for

protease activity.

3.6 Protein EstimationProtein concentration was determined by the method of Bradford [215]. BSA (Bovine Serum

Albumin) was used as a standard protein for the estimation. A stock of BSA was prepared with

1mg / ml concentration Different dilutions of BSA was prepared (10µg/ml – 100µg/ml) and

added in each tube (total volume made upto 1 ml with distilled water). 5 ml of Bradford reagent

was added in each tube. After 10 min, OD was measured at 595 nm.

The absorbance against protein concentration was plotted to get a standard calibration curve.

The absorbance of unknown sample was checked and the concentration of the unknown sample

was determined by using the standard curve.

3.7 Determination of Protease Activity (plant and microbial source)Protease activity was assayed by a modified method of Tsuchida et al. [214] by using casein as

substrate. 100 μl of enzyme solution was added to 900 μl of substrate solution (2 mg/ml (w/v)

casein in 10 mM Tris–HCl buffer, pH 8.0).The mixture was incubated at 45°C for 30 min.

Reaction was terminated by the addition of an equal volume of 10% (w/v) chilled

trichloroacetic acid then the reaction mixture was allowed to stand in ice for 15 min to

precipitate the insoluble proteins. The supernatant was separated by centrifugation at 10,000

rpm for 10 min at 4°C. The acid soluble product in the supernatant was neutralized with 5 ml of

0.5 M Na2CO3 solution. The colour developed after adding 0.5 ml of 3-fold-diluted Folin–

Ciocalteau reagent was measured at 660 nm. All assays were done in triplicate. One protease

56

unit is defined as the amount of enzyme that releases 1 µg of tyrosine per ml per minute under

the above assay conditions. The specific activity is expressed in the units of enzyme activity

per milligram of protein.

3.8 Protease Purification

3.8.1 Crude Extract Preparation

3.8.1.1 Bacterial Source

The bacterial strain was grown for 48 hrs at 37°C in the selected cultivation media. The

culture medium was centrifuged at 10,000 rpm for 10 min at 4°C and the cell-free

supernatant was collected.

3.8.1.2 Plant Source

Senesced leaves of Lantana camara was washed with distilled water and blotted dry were

weighed, homogenized with mortar and pestle in three volumes w/v 10 mM Tris-chloride

buffer (pH 8.0) followed by filteration with three layered cheese cloth. Then the filterate was

centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected and used as crude

preparation.

3.8.2 Ammonium Sulphate Precipitation

3.8.2.1 Bacterial Source

The supernatant was subjected for Ammonium sulphate precipitation. Supernatant was

precipitated with 0-60% ammonium sulphate. Ammonium sulphate was poured slowly into

the supernatant over a period of ten minutes, allowing the salt to slowly dissolve. The

supernatant was continually stirred at 4°C for an additional 1 hour. The precipitate was

collected by centrifugation (10,000 rpm for 10 minutes at 4ºC), and dissolved in a small

volume (1/50) of 10 mM Tris-HCl buffer (pH 8.0), and dialyzed against 4 liters of same

buffer for 12 hrs at 4°C. This step was repeated twice. The protease activity and protein

concentration was measured and specific activity calculated.

57

3.8.2.2 Plant Source

The crude enzyme preparation was precipitated with 0-30% and 30-60% ammonium sulphate

at 4°C, chilled for 1 hour at 4°C then centrifuged for 10 minutes (4°C) at 10,000 rpm. The

pellet was suspended in minimum volume of 10 mM Tris-Cl pH 8 and dialyzed against 6

liters Tris–chloride buffer (10mM, pH 8.0) at 4°C.

3.8.3 Chromatographic Techniques

3.8.3.1 Ion-Exchange Chromatography (for bacterial sources)

The dialyzed ammonium sulphate preparation was applied on a DEAE-cellulose column (2 X

24 cm) pre-equilibrated with 10 mM Tris-HCl (pH 8.0). The unadsorbed protein fraction was

eluted with the same buffer (150 ml). The enzyme was eluted with 2mM and 4 mM NaCl in

the same buffer at a flow rate of 1ml/min. The protease activity and protein concentration of

fractions was measured and specific activity calculated. Active fractions that contained

(80%) of the enzyme activity were pooled, and subsequently used for characterization. All

steps were conducted at 4°C.

3.8.3.2 Gel filtration Chromatography (for plant sources)

The protein pellet obtained after saturation with 0-30% ammonium sulphate was dissolved in

10mM Tris-HCl buffer and loaded onto a column of Sephadex G-250 (2 × 24 cm)

equilibrated with Tris-HCl buffer, pH 8.0. The one ml fractions were collected and checked

for protein and protease activity. From the activity profile, it was observed that the protease

was eluted as a well-resolved single peak of caseinase activity (11-20 ml fraction of one ml

each) coinciding with a single protein peak(11-20 ml fraction of one ml each) All the

fractions with high protease activities were pooled, dialyzed, and concentrated by

lyophilization and used for further studies.

58

3.9 Electophoretic Techniques

3.9.1 Polyacrylamide Gel Electrophoresis (Bacterial and Plant Sources)

SDS-PAGE was performed on a slab gel containing 10% (w/v) polyacrylamide by the

method of Laemmli [216].

Resolving Gel (10%) Stacking Gel (4%)

Distilled Water- 4.1 ml Distilled water- 3.075ml

1.5M Tris (pH: 8.8)-2.5ml 1.5M Tris (pH:6.8)-1.25ml

10%SDS-50µl 10%SDS-25µl

Acrylamide-3.3 ml Acrylamide-670µl

10%ammonium persulphate-50µl 10%ammonium persulphate-25µl

Temed-5µl Temed-5µl

All the above reagent of resolving gel was mixed and after polymerization of the resolving

gel all the component of the stacking gel was mixed and poured inside the glass plate, wells

were made with the help of comb. After that protease sample was loaded in each well and

electrophoresed at 100V for 1:30 hour after electrophoresis completed, gel was removed from

in between the glass plates placed in the staining trough containing the staining solution,

stained for overnight on rocker. The gel was destained in destaining solution.

3.9.2 Zymography

Casein zymography was performed in 10% polyacrylamide slab gels containing SDS and

casein (0.12% w/v) as co-polymerized substrate, as described by Choi etal, [217]. After

electrophoresis, the gel was incubated for 30 minutes at room temperature on a gel rocker in

50 mM Tris-Cl (pH 7.4), which contained 2.5% Triton X-100 to remove SDS. The gel was

then incubated in a zymogram reaction buffer (30 mM Tris-HCl, pH 7.4, 200 mM NaCl and

10 mM CaCl2) left at 37°C for 12 hrs on rocker shaker. The gel was stained with Coomassie

brilliant blue (0.5% w/v) for 30 min. The activity band was observed as a clear colourless

area depleted of casein in the gel against the blue background when destained in 10%

methanol and 5% acetic acid for a limited period of time.

59

3.10 Characterization of Proteases (bacterial and plant source)

3.10.1 Hydrolysis of Protein Substrates

Protease activity with various protein substrates including casein, egg albumin, and gelatin

(2mg/ml) was assayed by mixing 2µg of the enzyme and 200 μl of assay buffer containing

the protein substrates. After incubation at 45°C for 30 minutes, the reaction was stopped by

adding 200 μl of 10% of chilled trichloroacetic acid (TCA) (w/v) and allowed to stand in ice

for 15 minutes. The undigested proteins were removed by filtration or centrifugation at

10,000 rpm for 5 min. and activity of the enzyme was measured.

3.10.2 Effect of pH on Enzyme Activity

The protease activity of the purified enzyme was measured at different pH values (3.0-12).

The pH was adjusted using the following buffers (0.2M) of Acetate (pH 2.0-4.0), phosphate

(pH 5.0-7.0), Tris-HCl (pH 8.0), and glycine-NaOH (pH 9.0-12.0). Reaction mixtures were

incubated at 45°C for 30 minutes and the activity of the enzyme was measured.

3.10.3 Effect of Temperature on Enzyme Activity and Stability

In bacterial source the activity of the enzyme was determined by incubating the reaction

mixture at different temperatures ranging from 20, 30, 40, 50, 60, 70 and 80°C were studied.

The activity of the enzyme was measured as described previously.

In plant source the activity of the enzyme was determined by incubating the reaction mixture

at different temperatures ranging from 30°C, 40°C, 50°C, 60°C, 70°C at different pH ranges

(pH 3.6, pH 5.0, pH 10.0, pH 11.0) were studied . The activity of the enzyme was measured.

3.10.4 Effect of Various Metal Ions on Protease Activity

In bacterial source the effects of metal ions on enzyme activity (e.g., Ca2+, Mg2+, Fe2+, Mn2+,

Zn2+, Hg2+, and Cu2+ [5 mM]) were investigated by adding them to the reaction mixture and

pre-incubated for 30 min at 45°C pH 10.0. The activity of the enzyme was measured as

described previously.

In plant source the effects of metal ions (e.g., Ca2+, Mg2+, Co+2, Fe+3, Mn2+ , Zn2+, Hg2+, and

Cu2+ [10mM]) were investigated by adding them to the reaction mixture and preincubated for

30 minutes at 45°C pH 10.0. The activity of the enzyme was measured.

60

3.10.5 Effect of Protease Inhibitors and Organic Solvents on Enzyme Activity

The effect of various protease inhibitors (10mM) such as serine inhibitors

phenylmethylsulphonyl fluoride [PMSF],3,4-dichloroisocoumarin(3,4-DCI) and 4-(2-

aminoethyl)benzene sulfonyl fluoride (AEBSF), cysteine inhibitors E-64 and iodoacetic acid ,

Aspartic protease Pepstatin and Metalloprotease 1,10-Phenanthroline were determined by

preincubation with the enzyme solution for 30minutes at 45°C. The relative protease activity

was measured

The organic solvents used were Methanol, Ethyl acetate, Benzene, Glycerol, Sucrose,

Toluene, Acetone, Hexane, DMSO, Isopropanol and Ethanol. In the stability test, 1.0 ml of

organic solvent (100% v/v) was added to 1 ml of the reaction mixture and pre incubated at

37°C for 30 min. The remaining proteolytic activity was measured as described previously.

Stability was expressed as the remaining proteolytic activity relative to the solvent-free

controls (0%, v/v).

3.11 Industrial Application

3.11.1 Compatibility with Detergents

The compatibility of protease with laundry detergents was studied in the presence of 10mM

CoCl2. Detergents used were Henko (Henkel Spic, India), Surf, Surf Excel, Tide, Rin

(Hindustan Lever Ltd, India) and Ariel (Procter and Gamble, India). The detergents were

diluted in distilled water (0.7% w/v) and incubated with protease for 30 min at 60°C, and the

specific activity was determined. This was then compared with the control samples without

detergents.

61

3.12 General methodology

3.12.1 Preparation of Dialysis Tubing

Dialysis tubing was cut into convenient length and boiled for 10 min in 500 ml of 2% sodium

bicarbonate and 1mM EDTA (pH 8.0) solution after that the tubing was rinsed thoroughly in

distilled water and boiled for 10 min in 1 mM EDTA then the tubing was allowed to cool and

stored at 4°C in 1mM EDTA buffer.

3.12.2 Preparation of Electrophoresis Reagents

Resolving Gel (10%) Stacking Gel (4%)

Distilled Water- 4.1 ml Distilled water- 3.075ml ml

1.5M Tris (pH: 8.8)-2.5ml 1.5M Tris (pH:6.8)-1.25ml

10%SDS-50µl 10%SDS-25µl

Acrylamide-3.3 ml Acrylamide-670µl

10%ammonium persulphate-50µl 10%ammonium persulphate-25µl

Temed-5µl Temed-5µl

Acrylamide- Bisacrylamide solution Staining Solution

Acrylamide – 30.0 gm Coomassie brilliant blue – 1.25 gm

Bisacrylamide – 0.8gm Glacial acetic acid – 35 ml

Distilled water – 100 ml Methanol - 200 ml

Final vol. - 500 ml (Distilled Water)

Destaining Solution Reservoir Buffer

Methanol- 45 ml Tris - 3.0 gm

Acetic Acid – 10 ml SDS – 1.0 gm

Distill Water – 45 ml Glycine – 14.4 gm

Distilled water – 1 liter

62

pH – 8.3

Sample Buffer (2X) Sample Buffer (2X)

1M Tris-Hcl (pH-6.8) – 12.5 ml for Zymography

SDS – 4.0 gm 0.5 M Tris-HCl, pH 6.8

β – mercaptoethanol – 10.0 ml SDS - 10

Glycerol – 20.0 ml Glycerol - 20%

1% Bromophenol blue – 4.0 ml Bromophenol blue - 0.5%

Final volume made up upto 500 ml with distilled water

Staining Solution for Zymography Destaining Solution for

Coomassie brilliant blue - 0.5% Zymography

Methanol - 10%

Acetic acid - 5%

Zymogram Reaction Buffer

30 mM Tris-HCl, pH 7.4,

200 mM NaCl,

10 mM CaCl2,

3.12.3 Preparation of Bradford Reagent

100mg of Coomassie brilliant Blue G250 dissolved in 50 ml of 95% ethanol then 100ml of

85% of Phosphoric acid was added. Final volume to 1 liter was made up with distilled water.

63

3.12.4 Source of Chemicals, Media and Reagents

S.nos Name of chemical Source1 Acrylamide Sigma2 acetic acid CDH,Qualigens3 Acetone CDH,Qualigens4 Agar Hi Media5 Ammonium persulphate Sigma6 Ammonium sulphate CDH,Qualigens

74-(2-aminoethyl)benzene sulfonyl

fluoride Sigma8 Beef extract Sigma9 Benzene Sigma10 Bisacrylamide Sigma11 Bromophenol blue Sigma12 BSA Sigma13 Casein Sigma14 Cetrimide agar Hi Media15 CoCl2 CDH,Qualigens16 Coomassie brilliant blue R-250 Sigma17 Coomassie brilliant blueG-250 Sigma18 DEAE-cellulose Pharmacia19 3,4-dichloroisocoumarin Sigma20 D-glucose monohydrate CDH,Qualigens21 DMSO, Sigma22 E-64 Sigma23 Ethanol CDH,Qualigens24 Ethyl acetate CDH,Qualigens25 Ferrous Sulphate pentahydrate Merck26 Folin–Ciocalteau reagent Merck27 Gelatin Merck28 Glacial acetic acid CDH,Qualigens29 Glycerol CDH,Qualigens30 Glycine CDH,Qualigens31 Hexane CDH,Qualigens32 Hydrogen peroxide Merck33 Iodoacetic acid Sigma34 Isopropanol Merck35 K2HPO4 CDH,Qualigens36 KH2PO4, CDH,Qualigens37 Lactose CDH,Qualigens

64

38 LB broth Hi Media39 Methanol Merck40 MgCl2 CDH,Qualigens41 MgSO4 CDH,Qualigens42 MgSO4 · 7H2O, CDH,Qualigens43 Motility Agar Hi Media44 Na2CO3 Merck45 NaOH CDH,Qualigens46 Pepstatin Sigma47 Peptone Hi Media48 1,10-Phenanthroline Sigma49 Phenol red CDH,Qualigens50 Phenylmethylsulphonyl fluoride Sigma51 Phosphoric acid CDH,Qualigens52 Potassium sulphate CDH,Qualigens53 Protein molecular weight marker Sigma54 Proteose peptone Hi Media55 SDS Sigma56 Sephadex G-250 Pharmacia57 Simmon’s media Hi Media58 Skim milk powder Hi Media59 Sodium Chloride CDH,Qualigens60 Sodium thiosulphate CDH,Qualigens61 Starch CDH,Qualigens62 Sucrose CDH,Qualigens63 Temed Sigma64 Toluene Merck65 Trichloroacetic acid CDH,Qualigens66 Tris Sigma67 Triton X-100 Sigma68 Tryptic soy broth Hi Media69 Urea agar Hi Media70 Yeast extract Hi Media71 β – mercaptoethanol Sigma

65