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Iraqi J. Biotech.,Vol.6, No.

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

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IV
Pseudomonas aeruginosa

/ /
2005/3/15

2004 /12 /6

Pseudomonas aeruginosa No.3 .


. 191
) %80(
) (Batch-wise CMC Sephadex
.G-75
,
9.5 ) (10 -8 30 .
35
45 . 30 %90
60 40 80 . )(Protease IV
(%20) phenylmethylsulfonyl fluoride .(%75 ) Diisopropyl fluorophosphate
50) EDTA ( %22 .
10 ) dithiothreitol ( 150 ) 2- mercaptoethanol (

.

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INTRODUCTION
Pseudomonas aeruginosa is an opportunistic human pathogen that causes
severe morbidity and mortality in-patients with burns, cystic fibrosis, pneumonia,
urinary tract infections, skin infections, cancer, acquired immunodeficiency
syndrome, ocular disease and others (1,2,3). At least part of the pathogenic potential
of this organism stems from its ability to produce a myriad of extracellular virulence
factors, including toxins, siderophores, and proteases. P. aeruginosa produces
several proteases, including alkaline protease (aeruginolysin), elastase (LasB,
pseudolysin). LasA (staphlolysin) and Las D (staphylolysin) is considered to be
important in its pathogencity and in tissue damage during infection [1]. One of the
functions of proteases is to hydrolyze peptides for nutrient acquisition either by
degrading host enzyme or even by causing tissue damage to further the survival of
the bacterium. These proteases are often under complex regulation. For example,
expression of lasB depends on an intact lasR gene and the autoinducer PAI (4,5).
Often, as in the case of the metalloprotease LasB, efficient production and processing
of certain proteases require zinc and calcium ion (6). Invasiveness of P. aeruginosa
in burn patients is correlated with elastase production (7). Pseudomonas alkaline
protease and elastase are produced in the lungs of patients with cystic fibrosis and
have been show to damage respiratory epithelium (8). Protease IV (lysyl
enopeptidase; EC.3.4.24.26) has been demonstrated to correlate with corneal
virulence. Protease IV has also recently been identified as the iron- regulated protein
PrpL [9]. Protease IV is a lysine- specific protease with molecular mass of 26 KDa, an
isoelectric point 8.7, and optimum enzymatic activity at pH 10 and 45C that has
been identified in culture supernatants of P. aeruginosa (3,9,10). The purified
enzyme demonstrates activity for the carboxyl side of lysine-containing peptides and
can digest a number of biologically important proteins, including complement
component, immunoglobulin G, fibrinogen and plasminogen. Protease IV not inhibit
by thiol-, carboxyl- and metalloproteinase inhibitors and partial inhibited by PMSF
and DFP that protease is a serine protease (3). The enzyme can be completely
inhibited by protease inhibitor, such as tosyl lysyl chloromethyl ketone (9).
Protease IV has been implicated as a virulence factor that contributes to the
pathogencity of Pseudomonas keratitis (9,10). Purified protease IV induced corneal
epithelial damage within three hours after injection into the coreneal stroma and
increased the virulence of protease IV deficient bacteria (11). Protease IV may be
involved in the processing mechanism of elastase A (Las A) because lysine- specific
protease of P. aeruginosa converts the Las A proenzyme to active enzyme (12).
The current study focuses on the partial purification of protease IV and some
characteristics of the enzyme.

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MATERIAL AND METHODS
Bacterial Strain and Culture
P.aeruginosa No.3 strain used in this study was isolated from clinical specimen
of patient with corneal ulceration and it has ability to produce extracellular protease.
Enzyme Assay
Casinolytic activity of protease IV was measured as follows: 0.1 ml of the
enzyme was mixed with 0.9 ml of Tris- HCl buffer (0.1M, pH 8.5) which contained 5
mg Hammerstein casein (Merck). After incubation at 40C for 10 min, 2ml 0f 5%
trichloroacetic acid (TCA) was added to terminate the reaction. The mixture was
passed through Watman No.2 filter paper to remove denatured proteins. The
absorbancy of the supernatant fraction was read at 275 nm. Blanks were stopped with
the addition of TCA before the addition of the enzyme. One unit of activity was
defined as the amount of enzyme required to produce an increase 0.001 in the
absorbancy at 275 nm per min (13). Specific activity was expressed as enzyme units
per mg protein. The protein content was estimated by the method of Lowry et al.(14),
with bovine serum albumin (sigma) as the protein standard.
Partial purification of the enzyme
The purification of protease IV was performed as described by Engel et al. with
some modification (3). Briefly, culture supernatant of P. aeruginosa was prepared by
growing bacterial cells in with shaking (150 rpm) for 48 hours at 37C in 50ml
Mu`eller-Hinton broth in 500ml flasks at pH 8.5 supplemented with calcium (50 g /
ml) and magnesium (20 g / ml) to increase protease production. The culture was
then centrifuged, and the supernatant was sterilized by filtration through a filter with
0.45 m pores. The protein concentration of culture supernatant was determined.
Proteins from culture supernatant (one liter) were fractionated with ammonium sulfate
60, 65, 70, 75 and 80% saturation at 4 C respectively (15). The precipitation was
removed from culture supernatant by centrifugation at 10000 xg for 20 min at 4 C for
each step. The precipitates was dissolved in 3 ml of ml of 5mM Tris-buffered pH 6.5
separately, then the enzyme activity detected in each fraction and supernatant. The
fraction (20 ml) with protease activity (80%) was then dialyzed overnight against 5
mM Tris buffered saline (pH 6.5). The dialyzed supernatant (25ml) was applied to
Carboxymethyl Cellulose Cation exchange (Bio-Rad). The cation exchange matrix
was washed with 5mM Tris-buffer (pH 6.5) and eluted by the addition of 5 ml of 10
mM ammonium acetate buffer at pH 9. Then two ml of the concentrate was
fractionated on a Sephadex G-75 column (1.6 100 cm; Pharmacia Biotech, Uppsala,
Sweden) at 4C that had a 65ml void volume. The column was equilibrated with
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10mM Tris-buffer, pH8, containing 0.02% sodium azide. The column was eluted with
the same buffer at flow rate of 15ml/h, and 5ml fractions were collected. Protease
activity was detected in all of the fractions. Fractions with protease IV activity were
pooled, and lyophilized. Total protein and the enzyme activity from each step of the
purification were determined using Lowry method and the casienolytic assay
respectively.
Effect of pH on the Activity and Stability of Protease IV
The reaction between partially purified protease IV (2.5 g) and 0.5% casein
substrate was assayed in triplicates for 10 min in solutions ranging from pH 6 to 12.
The effect of acidic pH (< 6) could not be determined, since the casein precipitates at
acid pH conditions. Controls consisted of the casein substrate at pH values tested
without protease IV and the enzyme was added after the addition of trichloroacetic
acid solution. The absorbance of the supernatant fraction was read at 275 nm.
To determine the effect of pH on stability of protease IV, protease IV (2.5 g)
was preincubated in triplicates at 37C for 30 min in various pH values ranging from 3
to 12. By using the following buffers (20 mM); citrate-phosphate (pH 4.0- 7.0);
phosphate (pH 6.0- 8.0); glycine-NaOH (pH 8-10.5) and carbonate (pH 11-12.3). Then
the residual activity was measured with casein at 37C for 10 min.
Effect of Temperature on the Activity and Stability of Protease IV
Effect of temperature on enzymatic activity was determined by the reactions
between partially purified protease IV (2.5 g) and 0.5% casein substrate assayed in
triplicates for 10 min in 20mM glycine-NaOH buffer (pH9) at various temperatures
ranging from 5 to 70 C , each reaction was stopped by addition of trichloroacetic acid
solution. . Blanks consisted of the casein substrate in the same buffer without protease
IV and the enzyme was added after the addition of trichloroacetic acid solution. The
absorbance of the supernatant fraction was read at 275 nm.
To determine the effect of temperature on the stability of the enzyme, protease
IV (2.5 g) had been preincubated in triplicate for 30 min at various temperature
(0-70C) in 20mM glycine-NaOH buffer (pH9). Cooling in ice bath stopped heating of
the enzyme, and then the residual activity was measured at 37C for 10 min. Controls
consisted of the casein substrate in the same buffer without protease IV and the
enzyme was added after the addition of trichloroacetic acid solution. The absorbance
of the supernatant fraction was read at 275 nm.
Inhibition Studies
The inhibition studies were performed as described by Yan et al. (16) with some
modification. Preincubation of protease (50 g in 10l of 50 mM Tris, pH 8.5) and
inhibitor (80 l in 50 mM Tris-HCl, pH8.2, and 10 l of NaCl, 150 mM) was carried
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out at room temperature for 30 min. before adding the substrate (Hammerstein casein).
Assays were performed in triplicate. An appropriate control, in which the
inhibitor was replaced with solvent, were assayed in parallel .The effect of each
inhibitor used was confirmed by demonstrating its activity against susceptible
enzymes Table (1).
Table (1): Inhibitor Reactivity with Protease IV
Inhibitor

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

Inhibitor class refer to those proteases known to be susceptible to each compound


tested.
b
Enzyme reactivity refer to the specific enzymes used to demonstrate the activity of
the inhibitor preparation.
c
These values represent the change in protease activity measured as units of activity
relative to inhibitor- free protease assayed using casein as substrate.

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
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enzyme eluted from Sephadex G-75 column with two peaks one of them had
enzymatic
activity and the other had none Fig. (1). The stepwise purification of the enzyme in
greater than a 190 fold increase in specific activity (2168.7 U/mg protein) as
summarized in Table (2).
Table (2): Partial Purification of Protease IV from P. aeruginosa
Purification step

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

Enzyme assays conducted at seven different pH values indicated that the


maximal protease IV activity occurred at pH 9.5 Fig. (2). Protease activity increased
from pH 6 to 9; pH 11 demonstrated approximately 85% of maximal activity, but there
was no activity at pH 12. Studies of the pH stability of protease IV demonstrated that
approximately 90% of the activity was abolished by incubation of the enzyme at pH 4
and 11 but the enzyme retained its activity at pH 9 to 10 for 30 min Fig. (2).
Enzyme activity of protease IV increased with temperature from 10 to 45 C
with maximal activity occurring at 45C Fig. (3). Studies of the thermal liability of
protease IV demonstrated that approximately 90% of the activity was abolished by
incubation of the enzyme at 65 C for 30 min Fig. (3).
Five enzyme inhibitors were tested for their ability to block the hydrolysis of
casein. The Serine protease inhibitor Phenylmethylsulfonyl fluoride demonstrated
(20%) and Diisopropyl fluorophosphate (75%) inhibition of protease activity Table (1).
EDTA at 50mM caused a 22% inhibition of protease activity. The reducing agents
dithiothreitol (1.0 mM) and 2- mercaptoethanol (150-mM) also demonstrated
complete inhibition of the enzyme.

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DISCUSSION
Protease IV activity is correlated with virulence and is known to contribute to the
corneal damage that often results in a loss of visual a cutie and even blindness
following Pseudomonas keratitis (11,17). Pseudomonas mutant deficient in protease
IV activity has significally reduced virulence in experimental keratitis thus the
protease IV has important role in the corneal virulence by directed toxicity and its
ability to significantly augment the virulence of proteaseIV-deficient Pseudomonas
(18,19). This study focuses on the initial characterization of extracellular protease IV
as has been reported previously is active only as an extracellular enzyme (3,19) .
Ammonium sulfate fractionation followed by batch wise ion exchange by
CM-cellulose and molecular sieve chromatography by Sephadex G-75 were effective
in increasing the specific activity of protease IV by more than 190-fold.
Characteristic of the extracellular protease from P. aeruginosa are similar to
those previously reported in the literature (3, 20). The protease is heat stable and active
over wide range of temperatures (10-50). The protease is also stable and active over
wide range of neutral and alkaline pH values ( 7-10) with maximal activity at pH 9-10
as has been reported previously (3,19) .
Inhibition studies suggest that protease IV (alkaline protease) is a serine protease.
Phenylmethylsulfonyl fluoride and Diisopropyl fluorophosphate, which irreversibly
and specifically react with active site Serine residues (12,19), partially inhibited
extracellular protease activity as has been reported previously (3, 16,21). Engel et al.
(3) found that protease IV is a lysine-specific endoprotease produced by Pseudomonas
aeruginosa whose activity has been correlated with corneal virulence and the protease
IV amino acid sequence suggested that amino acid His-72, Asp-122 and Ser-198 could
form catalytic triad that is critical for protease IV activity. Furthermore they found that
mutation at any amino acid of the predicted catalytic triad or Ser-197 caused a loss of
enzymatic activity and absence of the mature form of protease IV. However, our
inhibitor data overall suggest that the extracellular protease of P.aeruginosa is a Serine
protease. EDTA did inhibit the enzyme activity but not at low concentrations
inhibitory for Pseudomonas metalloproteinases as has been reported previously (3, 22,
23). Furthermore, inhibition by dithiothreitol and 2- mercaptoethanol suggests that
disulfide bonds could be important in maintaining the molecular conformation
required for activity as has been reported previously (3, 20). Engel et al (3) found
similarities of protease IV to the lysine specific endoprotease of Achromobacter
lyticus which have three possible disulfide bonds in protease IV. These results identify
the catalytic triad of the enzyme, which demonstrate that autodigestion is essential for
the processing of protease IV into amateur protease, and predict sites essential enzyme
conformation.
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Protease IV is a distinct and unique form of other Pseudomonas proteases
associated with virulence (3,9,21). Research is currently in progress to determine the
therapeutic value of protease inhibitors that are reactive with protease IV in reducing
tissue damage that occurs during Pseudomonas infection.

Protease Activity (U/ml)

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

Elution Volume (ml)

Fig. (1): Chromatochraghy of protease IV from P. aeruginosa on


Sephadex G-75
Protease IV was fractionated at 4C on a Sephadex G-75 column (1.6 100 cm;
Pharmacia Biotech, Uppsala, Sweden). The column was eluted with 10mM Trisbuffer, pH8, containing 0.02% sodium azide, at flow rate of 15 ml/h, and 5ml
fractions were collected.( : Absorbance 280 nm, protease activity).

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140

100

90
80

100

70
60

80

50
60

40
30

40

Residual activity (%)

ProteaseIV activity (U/ml)

120

20
20
10
0

0
0

10

12

14

pH

Fig. (2): Effect of pH on the activity and stability of protease IV

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.

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100

140

90
120

100

70
60

80

50

60

40
30

40

Protease activity (U/ml)

Residual activity (%)

80

20
20
10
0

0
0

10

20

30

40

50

60

70

80

Temperature (C)

Fig. (3): Effect of temperature on the activity and stability of protease IV

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.

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