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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