WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Bole et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 4, 1150-1161.

Research Article

ISSN 2278 4357

ISOLATION, PURIFICATION AND CHARACTERIZATION OF

PROTEASE
Shivaji Bole*1., Prity Lata1., Madhu Singh R1
1

Dept. of Biotechnology, The Oxford College of Science, 19th Main, 17th B cross, HSR
Layout, Bangalore.

Article Received on
25 February 2014,
Revised on 16 March
2014,
Accepted on 04 April 2014

ABSTRACT
Among different types of enzymes obtained from microbial sources,
protease are the most widely used in industries. In the present study,
bacteria were isolated from air exposure and screened for the
production of protease. The bacterial isolate was identified as Bacillus

*Correspondence for Author

Shivaji Bole
Dept. of Biotechnology, The

sp. and was later used for further characterization. Maximum yield of
Protease was obtained after 48hrs of incubation. Enzyme activity of

Oxford College of Science, 19th

Protease was found to be 0.7917mol/ ml/min and specific Activity

Main, 17th B cross, HSR

was found to be 0.425 mol/ mg/min. The optimum temperature for

Layout, Bangalore.

enzyme activity was found to be at 300C and The optimum pH for

enzyme activity was found to be at pH 7.6.

Keywords: Protease, Casein, Cultural characterization, protease activity, pH, Temperature.

INTRODUCTION
Microorganisms are the most important sources for production of several commercial
enzymes. So selection of the right organism plays a key role in high yield of desirable
enzymes. For production of enzymes for industrial use, isolation and characterization of new
promising strains using cheap carbon and nitrogen source is a continuous process.
Microorganisms have become increasingly important as producer of industrial enzymes. Due
to their biochemical diversity and the ease with which enzyme concentrations may be
increased by environmental and genetic manipulation, attempts are now being made to
replace enzymes, which traditionally have been isolated from complex eukaryotes. [1, 2]
Proteolytic enzymes from microorganisms may be located within the cell (intracellular), cell
wall associated (periplasmic), or excreted into the media. Extracellular enzymes are usually
capable of digesting insoluble nutrient materials such as cellulose, protein and starch, and the

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digested products are transported into the cell where they are used as nutrients for growth.
Some extracellular enzymes are used in the food, dairy, pharmaceutical, and textile industries
than intracellular enzymes as their isolation and purification procedure are simple and cheap.
Hence they are produced in large amounts by microbial synthesis [3, 6].
Proteases are one of the most important groups of industrial enzymes and account for nearly
60% of the total enzyme sale. The major uses of free proteases occur in dry cleaning,
detergents, meat processing, cheese making, silver recovery from photographic film,
production of digestive and certain medical treatments of inflammation and virulent wounds.
[3,]
Certain proteases are used in food industry for proteolysis of inexpensive materials such as
soya protein present during the production of soy sauce and increase the value of their usage
and also improve the emulsifying capacity. Proteases are also used in leather and wool
industry for improving the quality of leather and reduction of waste material formation.
Alkaline proteases such as subtilisin from Bacilus subtilis are used to clean stock and
facilitate water uptake of hide or skin. [4]
In the present study, we report the isolation and characterization of Protease producing
bacteria from air-borne sample.
MATERIALS AND METHODS
ISOLATION OF PROTEASE PRODUCING MICROORGANISMS:
Samples were collected from different environmental sources by air exposure method and
plated on nutrient agar medium. The plates were incubated at 37C for 24 hours. Pure culture
were isolated by further subculturing.
SCREENING OF PROTEASE PRODUCING BACTERIAL ISOLATES
The bacterial isolates were checked for protease activity by streaking each isolates on plates
with nutrient agar containing casein. The plates were incubated at 37C for 24 hours. The
bacterial isolates showing growth were selected as protease producers and further
characterized.
MORPHOLOGICAL AND BIOCHEMICAL CHARACTERISTICS
Gram staining, motility, indole production, methyl red, Vogues Proskauer's, citrate

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utilization, triple sugar iron, nitrate reduction, catalase, oxidase, gelatin liquefaction, urease,
hydrolysis of casein, hydrolysis of starch were carried out.
Table 1: Biochemical characterization

Biochemical test

Result

Gram staining
Indole production
Methyl red
Vogues proskauer
Citrate utilization
Triple sugar ion
Nitrate reduction
Catalase test
Oxidase test
Gelatin liquefication
Urease test
Hydrolysis of casein
Hydrolysis of starch

Positive
Negative
Negative
Positive
Positive
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive

PURIFICATION OF ENZYME
The enzyme was isolated by spinning the culture at low rpm for 10 mins to remove the cells.
The extract was then purified using acetone and ammonium sulphate precipitation method.
ACETONE METHOD
The crude extract is treated with different concentration of acetone. In this 30% and 50%
acetone was used for purification. Acetone is slowly added to the extract to precipitate out
the enzyme. This is done on an ice bath and kept for 1hr under continuous stirring. The
mixture is the centrifuged at 3000 rpm for 10mins. The supernatant and pellet are separated
and checked for enzyme activity.
AMMONIUM SULPHATE PRECIPITATION
The crude extract is treated with different concentration of Ammonium sulphate. In this 30%
and 50% Ammonium sulphate was used for purification. Ammonium sulphate is slowly
added to the extract to precipitate out the enzyme. This is done on an ice bath and kept for 1hr
under continuous stirring. The mixture is the centrifuged at 3000 rpm for 10mins. The
supernatant and pellet are separated and checked for enzyme activity.

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CHARACTERIZATION OF ENZYME PROTEASE

STANDARD CURVE FOR CASEIN
A standard casein curve is prepared using standard as 200 g/ml. Different aliquots of
standard casein solution is taken and made up to mark by adding distill water and then
incubated with 5ml of alkaline copper reagent for 10 minutes and followed with 0.6ml of FC
reagent for 30 minutes. This solution was then read for absorbance at 660 nM .
Observation
Table 2: Standard curve for casein

Sl.No

Standard
Alkaline
Water Concenteration
casein
copper
(ml)
of casein(g)
(ml)
reagent(ml)

1
2
3
4
5

0.0
0.2
0.4
0.6
0.8

1.0
0.8
0.6
0.4
0.2

0.0
40
80
120
160

1.0

0.0

200

5.0

FC
reagent
(ml)
Incubate
for 10mins
at room
temperature

0.6ml

OD
at
660
Nm
0.00
0.09
incubate
for 10mins 0.18
at room
0.27
temperature 0.36
0.45

0.35

OD @ 660nm

0.3
0.25
0.2
0.15
0.1
0.05
0
o

40

80

120

160

200

Concentration g/ml

Fig 1: Standard curve for casein

ENZYME ACTIVITY
Enzyme activity is determined by incubating the 0.5ml of isolated enzyme with phosphate
buffer (pH. 6.5) for 30 minutes. This is followed by addition of 1ml trichloroacetic acid (5%)
to stop the reaction. The solution is centrifuged @ 5000 rpm for 10 minutes. The supernatant
collected and 1ml of the supernatant is then incubated with 5ml alkaline copper reagent for

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10 minutes. Then 0.6ml of Folin ciocalteus reagent was added and incubated for 30
minutes. The solution is then read for absorbance at 660 nm.
Table 3: Enzyme activity

Sl.No

Phosp
hate
Buffer
(ml)

Substrate
Casein
(ml)

Enz

Ec

Et
Direct

0.5

Dilute
(1:10)
30%
Acetone
direct
30%
Acetone
dilute
(1:10)
50%
Acetone
direct
50%
Acetone
Dilute
(1:10)
40%
(NH4)2SO4
direct
40%
(NH4)2SO4
Dilute
(1:10)
50%
(NH4)2SO4
direct
50%
(NH4)2SO4
Dilute
(1:10)

0.5

1ml
0.5

I
N
C
U
B
A
T
I
O
N
30

Enz
(ml)

TCA
(ml)

Supern
atant

Alkaline
copper
reagent(
ml)

1ml

1ml

5ml

0.5
ml
----

----

FC
I reage
N
nt
C
U
B
A
T
I
0.6ml
O
N

OD
at
660
nm

I
N
C
0.0
U
B
0.57
A
T
I
O 0.26
N

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.36

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.08

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.17

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.05

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.7

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.1

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.4

0.5

1ml

0.5

1ml

1ml

5ml

0.6ml

0.1

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SPECIFIC ACTIVITY OF ENZYME

The amount of protein content in the culture filtrate was assayed by Lowry method. The
enzyme is incubated along with buffer, with 5ml of freshly prepared alkaline solution and
incubated for 10min at room temperature.0.6ml of FC reagent and incubated for 30minutes
and the absorbance is read at 660nm. The protein content is estimated using calibration curve
of standard BSA protein.

Then the specificity of enzyme is calculated by the amount of

protein in mg present per ml of sample.

OBSERVATION
Table 4: Standard curve for BSA

Sl.no

0.35
0.3
OD @ 660nm

1
2
3
4
5

Standard
Concenteration
Alkaline
FC
OD at
Water
BSA
of BSA
copper
reagent
660
(ml)
(ml)
(g)
reagent(ml)
(ml)
nm
0.0
1.0
0.0
0.00
0.2
0.8
40
0.06
Incubate
incubate
0.4
0.6
80
0.12
for 10mins
for 10mins
0.6
0.4
120
0.18
at room
at room
0.8
0.2
160
temperature 0.6ml temperature 0.24
5.0
1.0
0.0
200
0.30

0.25
0.2
0.15
0.1
0.05
0
o

40

80

120

160

200

Concentration g/ml

Fig 2: Standard curve for BSA

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Specific activity of enzyme

Table 5: Specific activity of enzyme

Enzyme Water
(ml)
(ml)

Sl.no
Control
Test
(direct)
Dilute
(1:10)

0.5

Alkaline
copper
reagent(ml)

Enzyme
(ml)
0.5ml

0.5

0.5

----

0.5

0.5

----

5ml

FC
reagent
Incubate for
10mins at
room
temperature

0.6ml

OD at
660nm
Incubate for
10mins at
room
temperature

0.0
1.397
1.3290

Effect of substrate concentration

Different concentration of substrate (standard 1% casein) is incubated with 0.5ml of enzyme
along with buffer for 30mins. Then 1ml of TCA is added and mixture is incubated for
10mins. The solution is then centrifuged @ 5000 rpm for 10 minutes. The supernatant
collected and 1ml of the supernatant was then incubated with 5ml alkaline copper reagent for
10 minutes followed by 0.6ml of Folin ciocalteus reagent for 30 minutes. The solution is
then read for absorbance at 660 nm.
Table 6: Effect of substrate concentration
Sl.No

Sub

o.o

Enzyme

1/v

1/s

.0

o.05

.5735

1.74

. 002

0.1

.0925

10.81

. 001

0.15

.1535

6.52

. 0006667

0.20

.2486

4.02

. 0005

0.25

.4294

2.33

. 0004

0.30

.4082

2.45

. 0003333

0.35

.3986

2.51

. 00028571

0.40

.4378

2.28

. 00025

10

0.45

.495

2.02

0000222

11

0.50

.6036

1.66

.0002

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activity(v)

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Table 7: Effect of pH

pH
5.8
6.2

6.5

7.2
7.6
8.0

Substrate
(ml)

Enzyme (ml)

Control
0.5ml
Dilute
Control
0.5ml
Dilute
Control
Dilute

0.5ml

Control
0.5ml
Dilute
Control
0.5ml
Dilute
Control

0.5ml

Incubate
At
Room
temper
ature
For
30mins

1ml

C
E
N
T
R
I
F
U
G
E
At
5000
Rpm
10mins

Alkaline
copper
reagent(ml)

FC
reagent
(ml)
In
cu
ba
tI
on

In
cu
ba
tI
on

1ml

5ml

For
10
min

OD at
660nm
0.0
0.06
0.00
0.15
0.00
0.18

0.6ml

For
30
min

0.0
0.20
0.0
0.27
0.00

0.5ml

Dilute

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Supernatant
(ml)

TCA

0.24

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Effect of pH
0.5ml of substrate (casein) is incubated with 0.5ml enzyme along with buffer for 30mins at
various pH strength. Then 1ml of TCA is added and mixture is incubated for 10mins. The
solution is centrifuged at 5000 rpm for 10 minutes. The supernatant collected is then
incubated with 5ml of alkaline copper reagent for 10 minutes followed by 0.6ml Folin
ciocalteus reagent for 30 minutes. The solution is then read for absorbance at 660 nm.

0.3

OD @ 660nm

0.25
0.2
0.15
0.1
0.05
0
0

10

pH

Fig 3: Effect of pH
Effect of temperature
0.5 ml Substrate (casein) is incubated with 0.5ml of enzyme along with 0.5ml of buffer for
30mins at various temperatures. Then 1ml of TCA is added and solution is incubated for
10mins. The solution is then centrifuged at 5000 rpm for 10 minutes. The supernatant
collected and 1ml of it is then incubated with 5ml of alkaline copper reagent for 10 minutes
followed by 0.6ml of Folin ciocalteus reagent for 30 minutes. The solution is then read for
absorbance at 660 nm.

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Table 8: Effect of Temperature.

Temper
ature

20

30

40

50

60

Enzyme
(ml)

Contr
0.5m
ol
l
Dilute
Contr
0.5m
ol
l
Dilute
Contr
ol
0.5m
l
Dilute
Contr
0.5m
ol
l
Dilute
Contr
0.5m
ol
l
Dilute
Contr
0.5m
ol
l
Dilute

0.5ml

Supe
rnata
nt
(ml)

TC
A

Inc
uba
te
At
Ro
om
tem
per
atu
re
For
30
min
s

1ml

C
E
N
T
R
I
F
U
G
E
At
5000
Rpm
10mi
ns

Alkaline
copper
reagent
(ml)
I
n
c
u
b
at
I
o
n

1ml

5ml

OD
at
660
nm

FC
reagent

Fo
r
10
mi
n

I
n
c
u
b
at
Io
n

0.6ml

Fo
r
30
M
in

0.03
0.00
0.14
0.00
0.35
0.0
0.32
0.0

0.07

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
10

20

30

40

50

60

Temperature 0C

Fig 4: Effect of temperature.

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0.0

0.08
0.00

0.4

OD @ 660nm

10

Substrate
(ml)

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RESULT AND DISCUSSION

Bacillus sp. Isolated from air was found as an effective producer of protaese evident from the
findings. The enzyme together helps in the stain removal having varied applications in
detergent industries. A bacterial isolates were obtained from air samples. They were initially
screened for protease production based utilization of casein present in nutrient media.
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and
are essential for cell growth and differentiation. The extracellular proteases are commercial
value and find multiple applications in various industrial sectors. Although there are many
microbial sources available for producing proteases, only a few are recognized as commercial
producers.
The enzyme obtained was characterized and from the observation it is found that the enzyme
activity is 0.7917mol/ ml/min. and specific Activity is 0.425 mol/ mg/min. The optimum
temperature was 300C and optimum p H was 7.6.
ACKNOWLEDGMENT
We are sincerely grateful to The Oxford College of Science, Bangalore, Karnataka, India, for
allowing us to use all facilities for our work, and their encouragement and support.
REFERENCES
1. Barindra Sana, Debashish Ghosh, Malay Saha, Joydeep Mukherjee . Purification and
characterization of a salt, solvent, detergent and bleach tolerant protease from a new
gamma-Proteobacterium isolated from the marine environment of the Sundarbans.
Process Biochemistry; 2006; 41(1). 208-215.
2. Remold H, Fasold H, Staib F. Purification and characterization of a proteolytic enzyme
from Candida albicans. Biochim Biophys Acta; 1968 ; 167(2):399-406.
3. Morgan

F,

Priest

F.

Characterization

of

Thermostable

-Amylase

from

Bacilluslicheniformis NCIB 6346). J. Appl. Bacterio; 1981; 50: 107114.

4. Ishtiaq Ahmed, Muhammad Anjum Zia and Hafiz Muhammad Nasir Iqbal.Purification
and Kinetic Parameters Characterization of an Alkaline Protease Produced from Bacillus
subtilis through Submerged Fermentation Technique. World Applied Sciences
Journal;2011; 12 (6): 751-757.
5. Patil M, Shastri N.V. Purification and properties of proteases produced by Alternaria
alternata (Fr.) Keissl, regulation of production by fructose. J. Biosc;1985; 9: pp. 111.

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6. Shanmugam S, Satishkumar T,. Shanmugaprakash M. Enzyme Technology 2nd Edition

2012 I.K. International Publishing House Pvt. Ltd. pp 154,185-186.
7. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B: (Microbial-amylases: A
Biotechnological perspective). Process Biochem 2003; 38, 1599-1616.
8. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. (Advances in microbial
amylases) Biotechnol. Appl. Biochem, 2000; 31, 135-152.

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