Professional Documents
Culture Documents
1:94-106 , 2007
PARTIAL PURIFICATION AND CHARACTERIZATION OF
PROTEASE IV FROM PSEUDOMONAS AERUGINOSA
Rabab Omran
Biology Department/ College of Science/ Babylon University
Received 6/12/2004
Accepted 15/3/2005
ABSTRACT
Clinical strain Pseudomonas aeruginosa No.3 was isolated from human
corneal ulceration. The bacterial cells secreted the extracellular protease in
liquid culture. The enzyme was partial purified 191 fold from culture filtrate by
sequential steps such as salting out with ammonium sulfate precipitation (80%
saturation), ion exchange CM- Cellulose Chromatography, and by Sephadex
G-75 Gel filtration.
Characterization study of the partially purified enzyme revealed that the
enzyme had an optimum activity at pH 9.5 and the activity was stable in the
alkaline pH range (8- 10 )for 30 min. Enzyme activity toward casein increased
with temperature raise up to 35C and maximal activity was attained at 45 C.
The enzyme was stable at temperature under 30C and approximately 90% of
the activity was abolished by incubation of the enzyme at 60 C for 40 min or at
80 C for one min. Protease IV activity was partially inhibited by
phenylmethylsulfonyl fluoride (20%) and Diisopropyl fluorophosphate (75%).
EDTA at 50mM caused a 22% inhibition of protease activity, which suggested
that the enzyme is a serine protease. The reducing agents dithiothreitol (1.0 mM)
and 2- mercaptoethanol (150-mM) also demonstrated complete inhibition of the
enzyme, which suggests that the enzyme protein containing disulfide bonds could
be important in maintaining the molecular conformation required for activity.
__________________________________________________________
Keywords: Pseudomonas aeruginosa, corneal ulceration, Protease IV, purification,
characterization
94
2004 /12 /6
95
96
Inhibitor
class a
Enzyme
reactivity
Solvent
Inhibitor
conc.
Serine
Trypsin
H2O
1- propanol
1.0 mM
Percent.
activity of
remain c %
100
80
Serine
Trypsin
2-propanol
5.0 mM
25
Metallo
Alkaline
protease
Alkaline
protease
H2O
1.0 mM
100
H2O
50 mM
78
H2O
H2O
1.0 mM
150mM
0
0
None
Phenylmethylsulfony
l fluoride
Diisopropyl
fluorophosphate
EDTA
EDTA
Metallo
Dithiothreitol
2- Mercaptoethanol
a
RESULTS
The active extracellular protease IV was secreted into the culture medium during
late logarithmic phase of bacterial growth. The enzyme was fractionated by 60-80%
saturation of ammonium sulfate to remove impurity protein. The enzyme precipitated
at 80% saturation and dialyzed. Protease IV was purified from concentrated
supernatant fraction (25ml) by ion exchange chromatography. The protein of enzyme
adhered to the cation exchange resin (Carboxymethyl Cellulose Cation exchange). It
returned by elution from the resin at pH 9. The batch-wise purification of protease IV
showed more than 97.5 fold increase in specific activity which was 1102 U/mg protein.
The enzyme was further purified using size exclusion column (Sephadex G-75). The
99
Total
vol.
Protein
conc.
mg/ml
Enzyme
activity
U/ml
Total
activity
U
1.9
Total
protein
conc.
mg
1900
Crude filtrate
1000
Ammonium
sulfate
precipitation
(80% saturation)
Batch wise
CM-Cellulose
Chromatography
Gel filtration
(Sephadex G-75)
Purification
Fold
Recovery
(%)
21500
Specific
activity
U/mg
protein
11.3
21.5
100
50
7.85
392.5
401
20050
51
4.5
93.26
2.55
12.75
2810
14050
1102
97.5
65.3
36
0.16
5.76
347
12492
2168.7
191.9
58
100
0.4
200
0.35
0.3
150
0.25
0.2
100
0.15
0.1
50
0.05
0
0
20
40
60
80
100
120 140
160 180
Absorbance 280 nm
0.45
250
0
200 220
102
140
100
90
80
100
70
60
80
50
60
40
30
40
120
20
20
10
0
0
0
10
12
14
pH
A. Effect of pH on enzyme activity (). The caseinolytic activity was measured in total
volume of 1.0 ml that contained 2.5 g of protease IV in various buffers (20mM)
[citrate-phosphate (pH 4.0 - 7.0); phosphate (6.0 - 8.0); glycine NaOH (8-10.5) and
carbonate (pH11.0- 12.3)] at 37C for 10 min. B. Effect of pH on the stability of the
enzyme (). After protease IV (2.5 g) had been preincubated at 37C for 30 min in the
same buffers, the residual activity was measured with casein under assayed conditions.
103
100
140
90
120
100
70
60
80
50
60
40
30
40
80
20
20
10
0
0
0
10
20
30
40
50
60
70
80
Temperature (C)
A. Effect of temperature on enzyme activity (). Protease (2.5 g) has been assayed
with casein substrate (0.5%) in 20mM glycine buffer (pH9) at various temperature
(5,10,15,20,25,30,35,40,45,50,55,60,65, and 70) for 10 min. B. Effect of temperature
on the stability of enzyme (). Protease (2.5 g) had been preincubated for 30 min at
various temperature (0-70C) in 20mM glycine buffer (pH9). Cooling in an ice bath
stopped heating of the enzyme, and then the residual activity was measured with
casein substrate (0.5%) at 37C for 10 min.
104
106