This presentation is a research work carried out by me in B.Tech 8 semester. and gives an idea about purification, optimization and characterization of protease from Bacillus Valismortis

1. 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

2. CONTENTS
 Introduction
 Aim and Objectives
 Materials and methods
 Result
 Conclusion
 References

3. 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 peptide
bonds that link amino acids together in
the polypeptide chain forming the protein.
 Proteases execute a large variety of functions,
extending from the cellular level to the organ and organism
level, to produce cascade systems such as homeostasis and
inflammation.

4.  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.

5.  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.

6.  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

7. 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.

8. 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,

9. METHODS
 Culture Media
 Autoclaving for sterilization
 Centrifugation
 Ammonium sulphate precipitation
 Dialysis
 Bradford method for Protein estimation (quantitative
analysis)
 Protease assay for enzyme activity (qualitative
analysis)
 SDS PAGE
 NATIVE PAGE

10. 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

11. 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 )

12. 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)

13. 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)

14. 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

15. 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

16. 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)

17. 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

18. 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

19. L – TYROSINE STANDARD GRAPH
0
0.2
0.4
0.6
1 2 3 4
Abs at
280nm
Conc.
(µg/µl)

20. 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

21. NATIVE PAGE (SILVER STAINING)
MARKER , CRUDE , A.SO4 SUPERNATANT , PELLET AFTER
DIALYSIS

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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)

28. ACTIVITY OF ENZYME AT VARIOUS
INCUBATION PERIOD AT 50 ͦC
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

29. GRAPH FOR ACTIVITY OF ENZYME AT
VARIOUS INCUBATION PERIOD AT 50 ͦC
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

30. ACTIVITY OF ENZYME AT VARIOUS
INCUBATION PERIOD AT 35 ͦC
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

31. GRAPH ACTIVITY OF ENZYME AT
VARIOUS INCUBATION PERIOD AT 35 ͦC
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

32. 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.

33. REFERENCES
 Isolation and partial characterization of a thermostable
extracellular protease of Bacillus polymyxa B-17
 Jens Waldeck, Gabriele Daum, Bernward Bisping, and Friedhelm
Meinhardt
 Laundry detergent compatibility of the alkaline protease
from Bacillus cereus
 Optimization of the production of an extracellular alkaline protease
from Bacillus horikoshii
 Purification and characterization of a protease from Thermophilic
bacillus strain HS08 HUANG Guangrong1,2, YING Tiejing1, HUO
Po2 and JIANG Jiaxing3
 Purification and characterization of a salt, solvent, detergent and
bleach tolerant protease from a new gamma-Proteo bacterium
isolated from the marine environment of the Sundarbans .

34. Thank You