Purification Optimization And

Characterization Of Protease

From Bacillus vallismortis

SUBMITTED BY:

VAIBHAV KUMAR MAURYA

(07BBT254)

School of Bio Sciences and Technology (SBST)

Guide:

Mr. S. KARTHIKEYAN

ASST. PROFESSOR

CONTENTS

Introduction

Aim and Objectives

Materials and methods

Result

Conclusion

References

INTRODUCTION

A protease is proteolytic enzyme breaks down proteins.A protease is any enzyme that conducts proteolysis, that is,begins protein catabolism by hydrolysis of the peptidebonds that link amino acids together inthe polypeptide chain forming the protein.

Proteases execute a large variety of functions,extending from the cellular level to the organ and organismlevel, to produce cascade systems such as homeostasis andinflammation.

The current estimated value of the worldwide

sales of industrial enzymes is $1 billion. Of the

industrial enzymes, 75% are hydrolytic.

Proteases represent one of the three largest

groups of industrial enzymes and account for

about 60% of the total worldwide sale of

enzymes.

A combination of lipase, amylase, and

cellulase is expected to enhance the

performance of protease in laundry detergents.

They have been routinely used for various

purposes such as cheese making, baking,

preparation of soya hydrolysates, and meat

tenderization.

Proteases in the dairy industry is in the

manufacture of cheese.

Proteases have been used from ancient times to

prepare soy sauce and other soy product

AIM AND OBJECTIVES

Aim

To purify and characterize of protease from

Bacillus vallismortis.

Objectives

Sequential extraction and optimization of the

bacterial protease enzyme for the industrial

purpose.

MATERIALS

Chemicals:

Nutrient broth, sodium chloride, casein, Bradford reagent,

Ammonium sulphate, L – tyrosine, Folin – Ciocalteu reagent, Acryl

amide/Bis SDS, APS ,TEMED, AgNO3, BSA( Bovine serum

albumin).mono/di basic sodium phosphate , Sodium carbonate ,

Trichloro acetic acid,

Glass wares:Test tube ,centrifuge tubes ,conical flasks , micropipette ,beaker ,

Instruments:Centrifuge , autoclave, flasks, PAGE Apparatus, spectrophotometer,

pH meter,

METHODS

Culture Media

Autoclaving for sterilization

Centrifugation

Ammonium sulphate precipitation

Dialysis

Bradford method for Protein estimation (quantitativeanalysis)

Protease assay for enzyme activity (qualitativeanalysis)

SDS PAGE

NATIVE PAGE

METHODOLOGY

O

P

T

I

M

I

Z

A

T

I

O

N

SDS

PAGE,

NATIV

E

PAGE,

ZYMO

GRAM

P

R

O

T

E

A

S

E

A

S

S

A

Y

B

R

A

D

F

O

R

D

A

S

S

A

Y

Culture media

Inoculation with strain

Centrifugation affter 24 hours

Supernatent

(NH4 )2SO4 prcipitation

Centrifugation

pellet

Dissolve in PO4 Buffer

Dialysis

Dialyzed sample

CULTURE MEDIA

Composition:

1. Nutrient broth(1.3 gm/100 ml)

2. NaCl (2% e.g 2gm/100ml)

3. Casein(2% e.g 2gm/100ml )

PROTEIN ESTIMATION

Bradford method is used for protein estimation of

sample.

Reagents:

a) BSA ( Bovine serum albumin).

b) Sample

c) Phosphate buffer(Na2HPO4, NaH2PO4)

d) Coomassie brilliant blue G250(Bradford

reagent)

PROTEASE ASSAY

To check enzyme activity of protease enzyme

present in sample.

Reagents:a) 50 mM Potassium Phosphate buffer, pH 8.0 at 37ºC.

b) 1% (w/v) Casein Solution (Casein)

c) 10 % Trichloroacetic Acid Reagent (TCA)

d) 500 mM Sodium Carbonate Solution (Na2CO3)

PROTEASE ASSAY

S.No. Reagent Blank(ml) Test(ml) Control(ml)

1 casein 0 1 1

2 enzyme 0 1 2ml TCA

15 minutes incubation period At 37◦C

3 TCA 2ml 2ml 1ml enzyme

Incubate for

for

To

10 mins at 37◦C

12 mins

the

And centrifuge

supernatant

at 10,000 rpm

add

4 Na2CO3 1 1 1

Take the observation At 280 nm

BRADFORD ASSAY FOR STANDARD

GRAPH

S.No. Sample(µg/ml) O.D. AT 595 nm

1 10 0.103

2 20 0.120

3 30 0.156

4 40 0.200

5 50 0.280

6 60 0.360

7 70 0.430

8 80 0.460

9 90 0.470

10 100 0.530

BRADFORD ASSAY STANDARD GRAPH

0

0.2

0.4

0.6

0.8

1

1.2

10 20 30 40 50 60 70 80 90 100

O.D at

595 nm

Conc (µg/ml)

RESULTS PROTEIN ESTIMATION

S.No. sample O.D. Value at

595nm

Protien conc.

(µg/ml)

1 Blank 0.00 0.00

2 crude 1.126 602

3 A.SO4 Crude 1.652 1470

4 Pellet before

dialysis

1.228 670

5 Pellet after dialysis 1.652 220

TYROSINE STANDARD

S.No. Reagent Blank S1 S2 S3 S4 S5 S6

1 Tyrosine(µl) 0 15 30 60 75 90 120

2 Water(µl) 250 245 240 230 225 220 210

3 Na2CO3(µl) 625 625 625 625 625 625 625

4 Dil FC

Reagent

125 125 125 125 125 125 125

5 Abs at 280

nm

0 0.194 0.347 0.430 0.546 0.580 0.787

L – TYROSINE STANDARD GRAPH

0

0.2

0.4

0.6

1 2 3 4

Abs at

280nm

Conc.(µg/µl)

PROTEASE ASSAY

S.No. Sample Test(T) Control(C) T- C Enzyme activity

(U/ml)

1 Blank 0.00 0.00 0.00 0.00

2 Crude 0.301 0.198 0.103 6.86

3 A.So4 Crude 0.191 0.166 0.025 1.67

4 Pellet before

dialysis

0.261 0.192 0.069 4.67

NATIVE PAGE (SILVER STAINING)

MARKER , CRUDE , A.SO4 SUPERNATANT , PELLET AFTER

DIALYSIS

PROTEASE PRECIPITATION ACTIVITY AT

VARIOUS SUBSTRATE CONCENTRATION

S.No. % substrate Test

(T)

Control

(C)

T-C Enzyme activity

(U/ml)

1 0.5 2.120 2.055 0.0655 4.33

2 1 1.922 1.620 0.302 20.13

3 2 1.787 1.453 0.334 22.26

4 3 1.962 1.604 0.358 23.67

5 4 2.092 1.708 0.384 25.60

6 5 2.013 1.624 0.389 25.93

PROTEASE PRECIPITATION ACTIVITY AT

VARIOUS SUBSTRATE CONCENTRATION

0

5

10

15

20

25

30

0.5 1 2 3 4 5

Enzyme

activity

(U/ml)

Casein concentration(%) for Crude

AMMONIUM SULPAHTE PRECIPITATION ACTIVITY

AT VARIOUS SUBSTRATE CONCENTRATION

S.No % substrate Test(T) Control(C) T-C Enzyme

activity

(U/ml)

1 0.5 0.259 0.174 0.085 5.66

2 1 0.843 0.491 0.352 23.50

3 2 0.637 0.287 0.360 24.00

4 3 0.672 0.301 0.371 24.70

5 4 0.569 0.179 0.390 26.00

6 5 0.504 0.110 0.394 26.26

AMMONIUM SULPAHTE PRECIPITATION ACTIVITY

AT VARIOUS SUBSTRATE CONCENTRATION

0

5

10

15

20

25

30

0.5 1 2 3 4 5

Enzyme

activity

(U/ml)

Casein concentration(%) for A.So4 ppt

ACTIVITY OF ENZYME AT VARIOUS

TEMPERATURE

S.No. Temp

(Celsius)

Test

(T)

Control

(c )

T-C Enzyme

Activity

(U/ml)

1 30 0.684 0.463 0.221 14.73

2 35 0.831 0.189 0.642 43.20

3 45 1.301 0.427 0.874 58.26

4 50 1.256 0.397 0.859 57.20

5 60 0.454 0.317 0.137 9.13

6 70 0.211 0.182 0.029 1.93

7 80 1.082 0.390 0.692 46.13

8 90 0.287 0.143 0.144 9.60

9 100 0.310 0.380 -0.07

ACTIVITY OF ENZYME AT VARIOUS

TEMPERATURE GRAPH

0

10

20

30

40

50

60

70

30 35 45 50 60 70 80 90 100

Enzyme

activity

(U/ml)

Temp (°C)

ACTIVITY OF ENZYME AT VARIOUS

INCUBATION PERIOD AT 50 Cͦ

S.No. Time (min) Test (T) Control (C) T-C Enzyme

Activity(U/ml)

1 10 0.408 0.181 0.227 22.70

2 20 0.490 0.108 0.382 19.1

3 30 0.292 0.082 0.210 7.00

4 40 0.350 0.158 0.192 4.80

5 50 0.370 0.200 0.170 3.40

6 60 0.349 0.192 0.157 2.61

7 70 0.406 0.205 0.141 2.01

8 80 0.388 0.297 0.090 1.12

9 90 0.498 0.312 0.086 0.95

10 100 0.269 0.200 0.069 0.69

11 110 0.240 0.212 0.028 0.25

12 120 0.298 0.281 0.017 0.14

GRAPH FOR ACTIVITY OF ENZYME AT

VARIOUS INCUBATION PERIOD AT 50 Cͦ

0

5

10

15

20

25

10 20 30 40 50 60 70 80 90 100 110 120

Enzyme

activity

(U/ml)

Time (min) at 50 °C

ACTIVITY OF ENZYME AT VARIOUS

INCUBATION PERIOD AT 35 Cͦ

S.No. Time (min) Test (T) Control (C) T-C Enzyme

Activity(U/ml)

1 10 0.229 0.112 0.117 11.70

2 20 0.502 0.246 0.256 12.80

3 30 0.339 0.104 0.235 7.830

4 40 0.362 0.171 0.189 4.725

5 50 0.618 0.438 0.180 3.600

6 60 0.288 0.109 0.179 2.980

7 70 0.357 0.213 0.144 2.050

8 80 0.332 0.212 0.120 1.500

9 90 0.283 0.182 0.101 1.120

10 100 0.266 0.169 0.097 0.970

11 110 0.323 0.293 0.030 0.272

12 120 0.243 0.192 0.051 0.425

GRAPH ACTIVITY OF ENZYME AT

VARIOUS INCUBATION PERIOD AT 35 Cͦ

0

2

4

6

8

10

12

14

10 20 30 40 50 60 70 80 90 100 110 120

Enzyme

activity

(U/ml)

Time (min) at 35°C

CONCLUSION

This strain had shown the maximum activity in the following optimal conditions:

an optimum substrate concentrations 5 %

optimum incubation period 20 minutes

optimum temperature and the optimum pH was 45ºC and 8 respectively.

Vmax =25.93; Km = 0.7 mg/ml

Data emphasized the possibility of the production and purification of microbial protease enzyme for application under industrial scale.

REFERENCES

Isolation and partial characterization of a thermostableextracellular protease of Bacillus polymyxa B-17

Jens Waldeck, Gabriele Daum, Bernward Bisping, and FriedhelmMeinhardt

Laundry detergent compatibility of the alkaline proteasefrom Bacillus cereus

Optimization of the production of an extracellular alkaline proteasefrom Bacillus horikoshii

Purification and characterization of a protease from Thermophilicbacillus strain HS08 HUANG Guangrong1,2, YING Tiejing1, HUOPo2 and JIANG Jiaxing3

Purification and characterization of a salt, solvent, detergent andbleach tolerant protease from a new gamma-Proteo bacteriumisolated from the marine environment of the Sundarbans .

Thank You