abusham_09_optimization_798007.pdf.txt

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml">
<head>
<title>1475-2859-8-20.fm</title>
<meta name="Author" content="sufi01"/>
<meta name="Creator" content="FrameMaker 8.0"/>
<meta name="Producer" content="Acrobat Distiller 8.1.0 (Windows)"/>
<meta name="CreationDate" content=""/>
</head>
<body>
<pre>
Microbial Cell Factories

BioMed Central

Open Access

Research

Optimization of physical factors affecting the production of
thermo-stable organic solvent-tolerant protease from a newly
isolated halo tolerant Bacillus subtilis strain Rand
Randa A Abusham†1, Raja Noor Zaliha RA Rahman*†1, Abu Bakar Salleh†1
and Mahiran Basri†2
Address: 1Enzyme and Microbial Technology Research Group, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia and 2Enzyme and Microbial Technology Research Group, Faculty of Science, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
Email: Randa A Abusham - noia25@hotmail.com; Raja Noor Zaliha RA Rahman* - rnzaliha@biotech.upm.edu.my;
Abu Bakar Salleh - abubakar@biotech.upm.edu.my; Mahiran Basri - mahiran@science.upm.edu.my
* Corresponding author †Equal contributors

Published: 9 April 2009
Microbial Cell Factories 2009, 8:20

doi:10.1186/1475-2859-8-20

Received: 26 December 2008
Accepted: 9 April 2009

This article is available from: http://www.microbialcellfactories.com/content/8/1/20
© 2009 Abusham et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Many researchers have reported on the optimization of protease production;
nevertheless, only a few have reported on the optimization of the production of organic solventtolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant
protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant
protease and identify the culture conditions which support its production. The bacteria of genus
Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme
production by Bacillus species has been well-studied by a number of researchers. In the present
study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson,
Malaysia.
Results: A thermostable organic solvent-tolerant protease producer had been identified as Bacillus
subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological
characteristics and biochemical properties. The production of the thermostable organic solventtolerant protease was optimized by varying various physical culture conditions. Inoculation with
5.0% (v/v) of (AB600 = 0.5) inoculum size, in a culture medium (pH 7.0) and incubated for 24 h at
37°C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth
and production of protease (444.7 U/ml; 4042.4 U/mg). The Rand protease was not only stable in
the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic
solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and
80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60°C, respectively.
Conclusion: Strain Rand has been found to be able to secrete extra-cellular thermostable organic
solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability
towards temperature and organic solvent. This unique property makes it attractive and useful to
be used in industrial applications.

Page 1 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

Background
Currently, enzymes have attracted the world attention due
to their wide range of industrial applications in many
fields, including organic synthesis, clinical analysis, pharmaceuticals, detergents, food production and fermentation. Enzymes are gradually replacing the use of harsh
chemicals in various industrial processes [1]. Proteases are
one of the most important groups of industrial enzymes
and they account for nearly 60% of the total enzyme sale
[2-4].

http://www.microbialcellfactories.com/content/8/1/20

However, cultivation conditions are essential in a successful production of an enzyme, while optimization parameters, such as pH and temperature, are important in
developing this cultivation process [15].
In this study, the effects of physical factors on the production of a thermostable organic solvent-tolerant protease,
from Bacillus subtilis strain Rand, were identified and
investigated.

Results and discussion
In industrial applications, with thermopiles and thermostable enzymes, the isolation of enzymes is dominating over micro-organisms [5]. Bacterial proteases,
especially from Bacillus sp., are the most widely exploited
industrial enzymes and among the bacteria, Bacillus sp.,
are producers of extra-cellular proteases [6].
The industrial use of proteases, in detergents and in
leather processing, requires that the enzymes be stable at
higher temperatures. Thermostable proteases are advantageous in some applications because of the higher processing temperatures which can be employed, resulting in
much faster reaction rates, increasing the solubility of
non-gaseous reactants and products, and reducing the
incidence of microbial contamination by mesophilic
organisms [7].
Thermophilic enzymes are potentially applicable in a
wide range of industrial processes, particularly and mainly
due to their denaturant tolerance and extraordinary operational stability at high temperatures. Such enzymes are
used in chemical, food, pharmaceutical, paper, textile and
other industries [5,8,9].
Enzymatic conversions in non-aqueous media have been
shown to possess many potential industrial applications.
The areas of application vary widely from food additives,
flavours and fragrances to pharmaceuticals, pesticides and
specialty polymers [10]. Enzymes, which are stable and
active in non-aqueous media, are in large demand for
their increasing application in organic synthesis [11]. The
use of proteases in peptide synthesis is limited by the specificity and the instability of the enzymes in the presence
of organic solvents, since reactions occurred in organic
media. However, little attention has been given to the
study of organic solvent-stable protease [12].
Each organism or strain has its own special conditions for
the maximum enzyme production [13]. The general rules
for the optimization of microbial protease production are
affected by various physical factors which include pH, cultivation temperature, shaking condition and aeration.
These factors are important to promote, stimulate,
enhance and optimize the production of proteases [14].

Screening and isolation of bacteria
Contamination and hot surrounding area may provide a
good environment for the growth of micro-organisms
producing thermostable, organic solvent-tolerant proteases. Several samples were obtained from a car service
workshop located in Port Dickson, and hot spring water
from Batang Kali and Selayang, Malaysia. The temperatures were between 45 to 90°C when the sample was collected. From a comprehensive screening on the Skim Milk
Agar (SMA) plate, ten isolates (L1, L2, BK, BK1, BK2, PD,
PD1, PD2, PD and Rand) showed positive results by forming zone of lyses around the colonies on the SMA. All
these isolates were found to be able to produce protease
(data not shown). Among the ten isolates, Rand was
detected to have the highest protease activity (34.9 U/ml),
and was selected for further study.
Identification of isolate Rand
16S rDNA analysis
The primers are highly conserved among prokaryotes and
found to amplify the whole region of the rRNA gene,
which is 1500 bp. The PCR product sequencing was done
by the First BASE Laboratories Sdn Bhd (Shah Alam,
Selangor, Malaysia). The DNA homology search on the
GenBank database http://www.ncbi.nih.gov was performed. A phylogenetic tree was constructed based on the
comparison of the 16S rDNA sequence of this isolate and
other strain of Bacillus. All the sequences were aligned
with CLUSTALW from Biology Workbench database at
http://workbench.sdsc.edu[16]. 16S rDNA sequences of
other Bacillus were obtained from the GenBank database
http://www.ncbi.nih.gov. The results gathered from the
16S rDNA analyses show that Bacillus subtilis strain Rand
is very close to Brevibacterium halotolerans, Bacillus malacintesis strain LMG 22477, Bacillus malacitensis strain CECT
5687 and Bacillus axarquiensis strain LMG 22476 (Figure
1). The partial sequencing of the 16S rDNA shows a
99.6% similarity to different strains of Bacillus subtilis. In
addition, the analysis of the cellular fatty acids also shows
a good correspondence to the profile of the Bacillus subtilis
group.

Strain Rand is an aerobic, rod-shaped, with 0.7–0.8 μm in
width and 2.5–3.0 μm in length gram positive bacteria.

Page 2 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

http://www.microbialcellfactories.com/content/8/1/20

DQ993672.1 Bacillus malacitensis stra...
DQ993671.1 Bacillus axarquiensis stra...
DQ993673.1 Bacillus malacitensis stra...
AM747812.1 Brevibacterium halotolerans
EU233271.1 Bacillus subtilis strain Rand
EF433403.1 Bacillus subtilis subsp. s...
DQ207730.2 Bacillus subtilis strain C...
AB018486.1 Bacillus subtilis strain:A...
AJ830709.1 Bacillus subtilis isolate ...
EU257449.1 Bacillus subtilis isolate ...
DQ451099.1 Bacillus sp. JB5
DQ451100.1 Bacillus sp. JB7

0.0015

0.0010

0.0005

0.0000

Figure 1
Phylogenetic position of strain Rand with other bacteria
Phylogenetic position of strain Rand with other bacteria. The members of bacteria used include Bacillus malacitensis
CECT 5687; Bacillus axarquiensis LMG 22476; Bacillus malacintesis LMG 22477; Brevibacterium halotolerans;Bacillus subtilis subsp.
spizizenii BCRC 10447;Bacillus subtilis CCM 1999; Bacillus subtilis AU30; Bacillus subtilis isolate KCM-RG5; Bacillus subtilis isolate
C10-1. Phylogenetic tree was inferred by using the neighbour-joining methods. The software package MEGA 4 was used for
analysis.

The biochemical, morphological and physiological properties of strain Rand are listed in Table 1.
According to the 16S rDNA analysis, the biochemical
results and morphological properties of the bacterium
were identified as Bacillus subtilis strain Rand.
Organic solvent-stability of crude enzyme
Enzymes are usually inactivated or denaturated in the
presence of organic solvents [12]. The effects of different
organic solvents on protease stability were studied. The
relative activity, which remained after 30 min of incubation in 25% (v/v) of organic solvent, is shown in Table 2.
The activity of the enzyme, without any solvent (control),
was taken as 100%. Rand protease showed a remarkable
stability in the presence of all of the solvents, except pyridine (log P 0.71) as shown in Table 2. The remaining
activity of the Rand protease was found to be 104, 197,
130, 134, 146, 209, 151, 152 and 151% in the presence of
butanol, benzene, toluene, p-xylene, n-hexane, n-decane,
n-dodacane, n-tetradecane and n-hexadecane, respectively
(data not shown). This level of stability, towards hydrophobic and hydrophilic solvents, is unique. The remaining activities of alkaliphilic protease, from Bacillus subtilis
TKU07, were 65% and 90% in the presence of only 20%

(v/v) of butanol and toluene [17]. Gupta and Khare
reported that crude P. aeruginosa PseA protease showed a
remarkable stability in the presence of most solvents, having the logarithm of the partition coefficient (log P) above
2.0, but was less stable in the presence of hydrophilic solvents [11]. The stability of the Pseudomonas aeruginosa protease, in the presence of organic solvents (of which the
values of the log P were equal to or more than 3.2), was
almost the same as the one found in the absence of
organic solvents [18]. Purified protease, from Pseudomonas
aeruginosa PseA strain, was found to be stable in the presence of a range of organic solvents, but was detected to be
less stable in benzene and isooctane [19]. In another
study, the protease from Pseudomonas aeruginosa stain K
was activated when compared to the control, in the presence of 25% organic solvents, with the Log P values
exceeding 4.0; however, in the presence of 25% organic
solvents with the Log P values below 4.0, the stability of
the protease was lesser after 30 min of incubation [20].
Slightly over 20% of the activity remained [21] in the
crude protease derived from Pseudomonas aeruginosa sanai, in the presence of butanol, chloroform, and hexane.
However, the results in this study showed that the protease, from the Rand strain, was not only stable in the
presence of various organic solvents (with the Log P val-

Page 3 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

Table 1: Morphological and biochemical characteristics of
Bacillus subtilis strain Rand

http://www.microbialcellfactories.com/content/8/1/20

Table 2: Effect of organic solvents on Rand protease stability

Organic solvents

Rods
Width μm
Length μm
Aminopeptidase test
KOH test
Oxidase
Catalase

+
+

Gram stain

+

Spores
Sporangium swollen

+
-

Anaerobic growth
VP reaction
pH in VP broth

+
5.5

Growth positive at
Growth negative at

50°C
55°C

Growth in medium pH5.7
NaCl 2%
5%
7%
10%

+
+
+
+
+

Acid form
D-glucose
L-arabinose
D-xylose
D-mannitol
D-frucrose

+
+
+
+
+

Use of citrate
propionate

+
-

NO2 from NO3
Indol reaction
Phenylalanine deaminase
Arginine dihydrolase

+
-

Hydrolysis of Starch
Gelatin
Casein
Tween 80

+
+
+
+

pyridine
butanol
benzene
toluene
p-xylene
n-hexane
n-decane
n-dodacane
n-tetradecane
n-hexadecane
None

+
0.7–0.8
2.5–3.0

Log P *

Protease activity (U/ml)
0 min
30 min

0.71
0.80
2.0
2.5
3.1
3.5
5.6
6.0
7.6
8.8

Isolate Rand results

22.66
23
61.9
45.7
54.4
47.22
53.4
65
44.7
61.9
37

0.0
38.3
72.7
47.9
49.6
53.9
77.4
55.7
56.4
56
37

* Adopted from Laane et al. [22]
Note: 25% (v/v) of organic solvents were added to the cell-free
supernatant and incubated at 50°C with shaking 150 rpm for 30 min.

incubated at different temperatures ranging from 37 to
70°C for 30 min, rapidly cooled, and the protease activities were measured by the standard assay procedure. The
protease appeared to be stable and was found to be able
to retain its full activity after 30 min of incubation in the
temperature ranging from 37 to 55°C (Figure 2). The
crude enzyme retained 80% activity (0.086 mg/ml), even
after the heat treatment at 60°C for 30 min. A reduction
in the enzyme activity was observed at the temperature
values above 60°C.
The Rand protease is more thermostable than other
organic solvent tolerant proteases. Ghorbel et al. isolated
a protease from Bacillus cereus BG1 which retained 89.5 of
its original activity, after 15-min incubation at 55°C, in
the presence of 2 mM Ca2+; meanwhile, no activity was
detected in the absence of Ca2+ [12]. An organic solvent-

120
Relative Activity (%)

Characteristics

100
80
60
40
20
0

ues equalled to or more than 2.0), but also in the presence
of some organic solvents with the Log P values below 2.0.
These results indicated that this protease might be a novel
solvent-stable protease.
Thermostability of crude enzyme
Another remarkable feature of the Rand protease is its stability in high temperatures. To study the stability of
enzyme at different temperatures, crude enzyme was pre-

30

35

40

45

50

55

60

65

70

75

o

Temperature ( C)

Figure Temperature on Protease Stability
Effect of2
Effect of Temperature on Protease Stability. The
crude enzyme was incubated at different temperatures (37–
70°C) for 30 min with shaking 150 rpm. Protease activity at
37°C was considered as 100%.

Page 4 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

http://www.microbialcellfactories.com/content/8/1/20

stable protease from Pseudomonas aeruginosa PST-01 was
reported to be stable at the temperature below 50°C [23].
A solvent stable protease, from Pseudomonas aeruginosa
PseA retained 80% of its initial activity after heating, for
30 min at 55°C [11]. In particular, TKU004 metalloprotease had 10% of its activity retained at 60°C, but was
completely inactivated at 70°C [24]. The Rand protease
displayed a greater stability at higher temperatures, and
thus was suitable to be used in industrial and biotechnological applications.
The effects of temperature on the production of protease
Temperature is a critical parameter which needs to be controlled and this is usually varied from organism to another
[13]. The optimum temperature for the production of protease and bacterial growth was investigated from 30°C to
65°C. In shaken cultures, 37°C was found to the optimum temperature for both protease production and bacterial growth (Figure 3). The incubation at 30, 40, 45 and
50°C was found to decrease the production of protease,
and no protease activity was detected at 55, 60 and 65°C.
The optimum temperature for crude protease, produced
from B. subtilis strain 38, was 47°C [25]. The optimum
temperature for the protease produced by Bacillus sp. MIG
was found to be 30°C [26]. Meanwhile, the optimum
temperature for the production of protease by Bacillus sp
SMIA-2 was 60°C [7]. The optimum temperature for the
protease produced by Bacillus licheniformis was 50°C [27].
The studies by Frankena et al. [28] showed that there was
a link between enzyme synthesis and energy metabolism
in bacteria, and this was controlled by the temperature
and oxygen uptake. As for the extra-cellular enzymes, temperature was found to influence their secretion, possibly
by changing the physical properties of the cell membrane

[14]. On the other hand, a lower growth of Rand strain at
high temperatures could be due to the lack of dissolved
oxygen in the medium, which resulted to a low protease
activity. It is a well-known fact that protein conformation
changes or degraded at higher temperatures, and hence,
causes a decrease in the protease activity [29].
The effects of pH on the production of protease
The pH of the medium started to decrease after 4 h to 4.5
after 8 h of growth (Figure 4). The increased acidity was
due to the production of acids during the bacterial
growth. After that, the pH was slightly increased to 5.2 at
12 h, and to 6.4 at 16 h. It turned neutral at 20 h and
remained constant up to 48 h. The rise in the pH after 8 h
of incubation could be due to utilization of organic acids
or the production of alkaline compounds.

Extra-cellular protease was detected over a broad pH range
(pH 4.0 to 9.0), with the optimum production of protease
and bacterial growth exhibited at pH 7.0 (Figure 5). The
bacterial growth and production of protease in an acidic
medium at pH 6.0 were higher as compared to that in
alkaline at pH 8.0. However, at an extreme acidity of pH
4.0, the production of protease was found to be greatly
reduced. The optimum pH for the production of protease
determined in this study is in agreement with the optimum pH for the protease from Bacillus sp. MIG [26]. The
crude protease enzyme, produced from B. subtilis strain
38, had the optimal pH at 6.5 [25]. Malathu et al. reported
an extra-cellular protease from a novel bacterial isolate
showing the maximum activity at pH 7.5 [1]. Meanwhile,
Nascimento and Martins reported an optimum pH of 8.0
for a protease derived from therophilic Bacillus sp strains
SMIA-2 [7].

2.5

8

120

80
1.5
60
1
40
0.5

20

Relative Activity (%)

2

Log CFU/ml

Relative Activity (%)

100

7

100

6
80

5

60

4
3

40

2
20

1

0
0

0
30

37

40

45
50 o
55
60
Temperature ( C)
Protease activity
CFU

65

Figure temperature on protease production
Effect of3
Effect of temperature on protease production. Culture media were incubated at 30, 37, 40, 45, 50, 55, 60 and
65°C with shaking at 150 rpm for 24 h. Protease activity at
37°C was considered as 100%.

pH

120

0
0

4

8

12

16

20 24 28
Time (h)
Protease activity

32

36

40

44

48

pH

Figure 4
Time course of protease activity and pH of the medium
Time course of protease activity and pH of the
medium. Culture media were incubated at 37°C with shaking at 150 rpm for 48 h. Samples were taken at 4 h intervals
to determine the protease activity and the pH level.

Page 5 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

http://www.microbialcellfactories.com/content/8/1/20

120

2.5

100

100

2

1.5
60
1

80
1.5
60
1

Log CFU/ml

80

Relative Activity (%)

2

Log CFU/ml

Relative Activity (%)

120

2.5

40

40

0.5

20

0.5

20

0

0
0

0

0
4

5

6

7
8
9
pH Growth
Protease activity

10

11

12

13

CFU

Figure pH
Effect of5 on protease production
Effect of pH on protease production. Bacterial cultures
were adjusted to pH 4, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0,
13.0 and incubated at 37°C with 150 rpm for 24 h. Protease
activity at pH 7 was considered as 100%.

Moon and Parulekar [30] reported that the pH of culture
has been shown to strongly affect many enzymatic processes and transportation of various components across the
cell membrane.
The effect of agitation rate on the production of protease
Micro-organisms vary in their oxygen requirements. In
particular, oxygen acts as a terminal electron acceptor for
oxidative reactions to provide energy for cellular activities.
The variation in the agitation speed has been found to
influence the extent of mixing in the shake flasks or the
bioreactor, and also affect the nutrient availability [7].

The effects of agitation rate on the production of protease
and bacterial growth were investigated. Figure 6 shows
that the highest protease production and bacteria growth
were obtained when agitated at 200 rpm. At this speed,
the aeration of the culture medium was increased, and
this further led to a sufficient supply of dissolved oxygen
in the media [13]. Although the production of protease
was found to decrease when shaken at 250 rpm, the static
condition almost inhibited its production.
As a conclusion, higher agitation rates could increase the
oxygen pressure of the system but did not bring about the
increase in production, probably because at a high agitation rate, the structure of enzyme would be altered [31].
However, lowering the aeration rate could cause a drastic
reduction in the protease yields [24]. This indicates that a

50

100
150
200
Agitation Rate (rpm)
Protease activity
CFU

250

Figure agitation rate on protease production
Effect of6
Effect of agitation rate on protease production. Culture media were incubated at 37°C with different shaking
rates (0, 50, 100, 150, 200 and 250 rpm) for 24 h. Protease
activity at 200 rpm was considered as 100%.

reduction in oxygen supply is an important limiting factor
for growth as well as protease synthesis [7].
The effects of inoculum size on the production of protease
The finite volume of a culture medium means that it can
only contain limited nutrients for the micro-organism.
Furthermore, the consumption of the nutrients is largely
dependent on the population of bacteria. To ensure a high
production of enzyme in the limited volume of medium,
the bacterial inoculum size should therefore be controlled.

Inoculating several production media, with various inoculum sizes (from 1% to 11%) of the isolate Rand, could
affect the production of protease and bacterial growth.
The maximum production of protease and bacterial
growth were achieved with an inoculum size of 5% (v/v)
(Figure 7). Similar result was also reported by Mabrouk et
al. [32] who found the maximum production of protease
by Bacillus licheniformis ATCC21415 with an inoculum
size of 5% (v/v). A higher inoculum of 11% (v/v) was
found to reduce the production of protease more than if
the lower inoculum size of 1% (v/v) was used. Therefore,
high inoculum sizes do not necessarily give higher protease yield. The increase in the production of protease
using small inoculum sizes was suggested to be due to the
higher surface area to volume ratio, which resulted in the
increased production of protease [14]. In addition, an
improved distribution of dissolve oxygen and more effective uptake of nutrient also contributed to a higher protease production. If the inoculum sizes are too small,
insufficient number of bacteria would then lead to a
reduced amount of secreted protease [33].

Page 6 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

http://www.microbialcellfactories.com/content/8/1/20

100

Methods

3

2.5

80

2

60

1.5

40

1

20

0.5

0

Log CFU/ml

Relative Activity (%)

120

0
1

3

5
7
Inoculum Sizes % (v/v)
Prorease activity

9

11

CFU

Figure inoculum size on protease production
Effect of7
Effect of inoculum size on protease production. Culture media were incubated with 1.0, 3.0, 5.0, 7.0, 9.0 and
11.0% (v/v) of inoculum and incubated at 37°C with shaking
at 200 rpm for 24 h. Protease activity at with an inoculum
size of 5% (v/v) was considered as 100%.

However, higher inoculum sizes could lead to or cause a
lack of oxygen and depletion of nutrient in the culture
media. Different optimum inoculum sizes have been
reported by other researchers for different bacteria: 1.0%
(v/v) for Aspergillus flavus [34] and 4.0% (v/v) for Pseudomonas aeruginosa strain K [14].
Under optimized conditions (growth temperature of
37°C; bacterial inoculum size at AB600 = 0.5, 5% (v/v); initial pH of 7.0; 24 h of incubated time; agitation rate of 200
rpm), the highest protease activity of 444.7 U/ml (4042.4
U/mg) was obtained.

Conclusion
The aim of this study was to optimize the physical factors
affecting the productions of thermostable and organic solvent-tolerant protease. For this purpose, the organic solvent-tolerant and thermostabe protease were isolated
from a newly isolated bacterium (Bacillus subtilis strain
Rand). The bacterium was identified, based on the 16S
rDNA analysis, biochemical tests and morphological
study conducted.
It can be concluded that the maximum bacterial growth
and production of protease were achieved under optimized conditions.
The extra-cellular protease was found to exhibit a remarkable stability towards several organic solvents. In more
specific, it was found to retain 100% and 80% activity at
55 and 60°C, respectively, after 30 min of incubation.

Bacterial isolation
The bacteria used in this study were isolated from contaminated soil mixed with engine oil collected from a car service workshop in Port Dickson, Malaysia. A soil sample (3
g) was suspended in a sterilized Tryptic Soy Broth (TSB)
(50 ml). The sample was incubation at 50°C (the temperature during sampling) with an agitation rate at 150 rpm
for 24 hr.
Identification of the bacteria
In this study, Bacillus subtilis strain Rand was identified
based on the 16S rDNA analysis, morphological properties and biochemical characteristics. The 16S rDNA
sequence was amplified via the polymerase chain reaction
(PCR), using two universal primers, known as forward (5'GAGTTTGATCCTGGCTCAG-3')
and
reverse
(5'CGGCTACCTTGTTACGACTT-3').
The
16S
rDNA
sequence of Bacillus subtilis strain Rand was analyzed using
the software package MEGA 4 [35]. Prior to gram staining,
pure bacterial strain was streaked on the nutrient agar
plate and incubated for 24 h at 50°C for the morphological study. An observation of this was then done under a
light microscope. The morphological and physiological
characteristics were further determined at Deutsche
Sammlung Von Mikroorganismen (DSMZ), Germany.
The physiological characteristics study included catalase
and oxidase test, anaerobic growth, Voges-Proskauer test,
growth at 30, 50 and 55°C, growth in medium at pH 5.7,
2%, 5%, 7% and 10% NaCl, fermentation of D-glucose, Larabinose, D- xylose, D-mannitol, D-fructose, hydrolysis
starch, gelatine, casein and Tween 80, use of citrate and
propionate, nitrate reduction, indole production, phenylalanine deaminase and arginine dihydrolase test.
Production media and growth condition
The culture was grown in standard 500 ml blue cap bottle
containing 50 ml of production media. The medium consisted of (g/l); CaCl2.2H2O 0.5, KH2PO4 0.2,
MgSO4.7H2O 0.5, NaCl 0.1 and 1% peptone [36]. The pH
of the media was adjusted to 7.0 before being autoclaved
at 121°C for 15 min. The bacterium was grown for 18 h at
37°C on a shaker at 150 rpm. The culture was centrifuged
at 10,000 × g for 10 min and the supernatant was used as
crude enzyme for further study.
Protease assay
The protease activity was determined by a slight modification method proposed by Rahman et al. [37]. Azocasein
(0.5%, 1 ml) was dissolved in 0.1 M Tris-HCl-2 mM CaCl2
pH7.0. The reaction was initiated by adding 100 μl of
enzyme solution into the azocasein solution and incubated at 50°C for 30 min. An equal volume of 10% (w/v)
TCA was added to terminate the reaction, and the mixture
was then allowed at room temperature for 30 min, before

Page 7 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

centrifugation in eppendoft micro-centrifuge at 13,000 ×
g for 10 min. The supernatant was removed and mixed
with an equal volume of 1 N NaOH. The absorbance was
read at 450 nm. One unit of protease activity is defined in
the assay conditions, giving an increase of 0.001 absorbance unit at 450 nm per minute [38]. As a control, the
enzyme was added at the end of the incubation period.
Protein assay
Protein was measured using the method suggested by
Bradford [39], with bovine serum albumin as the standard.
Organic solvent-stability of crude enzyme
Three millilitre of crude protease was incubated with 1.0
ml of organic solvent at 50°C with a constant shaking at
150 rpm for 30 min. The proteolytic activities were measured at zero time and the after incubation period using the
assay method described above. For control, the solvent
was replaced by distilled water. The organic solvents chosen in this study were toluene, n-tetradecane, n-hexadecane, n-dodacane, pyridine, p-xylene, n-hexane, benzene,
n-decane and butanol.

http://www.microbialcellfactories.com/content/8/1/20

protease production) for 24 h under 150 rpm of agitation
rate.
The effects of agitation rate on the production of protease
The effects of agitation rate on the growth and production
of protease were studied by cultivating the bacteria under
different agitation rates (0 to 250 rpm). These cultures
were incubated at 37°C for 24 h.
The effects of inoculum size on the production of protease
The effects of bacterial inoculum size (A600 = 0.5) on the
growth and production of protease were investigated
using different inoculum sizes ranging from 1 to 11% (v/
v). The cultures were incubated at 37°C for 24 h, under
the agitation rate of 200 rpm (i.e. the optimum agitation
rate for the production of protease). Each experiment was
carried out in triplicates and the results were taken in the
means of three independent determinations.
Statistical analysis
For statistical analysis, a standard deviation for each
experimental result was calculated using the Excel Spreadsheets available in the Microsoft Excel.

Thermostability of crude enzyme
In this research, the effects of temperature on the crude
protease stability were studied. The crude enzyme (without any CaCl2) was incubated for 30 min at different temperatures (37, 40, 45, 50, 55, 60, 65, 70°C). The treated
enzyme was immediately put in ice-bath for 15 min
before measuring the activity. The proteolytic activities
were measured at zero time and after the incubation
period, using the assay method described in the earlier
section.

Competing interests

The effect of temperature on the production of protease
The ability of the Bacillus subtilis strain Rand to grow and
produce protease, at elevated temperatures (30 to 65°C),
was studied. For this purpose, separate cultures were incubated at 30, 37, 40, 45, 50, 55, 60 and 65°C for 24 h, with
agitation at 150 rpm.

References

The effect of pH on the production of protease
A loop-full of 24h-old single colony of Rand strain was
transferred from a fresh Nutrient agar plate into a 1 L blue
cap bottle of production medium (pH 7.0). The culture
was incubated at 37°C and 150 rpm on the shaker for 48
hours. Then, some samples were taken at 4 h intervals so
as to determine the protease activity and the pH level of
the culture medium. The effects of the initial medium pH
on the growth and production of protease were studied by
adjusting the media (with 1 M NaOH or HCl) to different
pH levels ranging from 4 to 13. The media were autoclave,
cooled and inoculated with an overnight culture of isolate
Rand, and incubated at 37°C (optimum temperature for

The authors declare that they have no competing interests.

Authors' contributions
All authors have read and approved the final version of
the manuscript.

Acknowledgements
This project was supported by the Ministry of Science, Technology and
Innovation (MOSTI), Malaysia 07-01-04-SS001.

1.

2.

3.
4.

5.
6.
7.
8.

Malathu R, Chowdhury S, Mishra M, Das S, Moharana P, Mitra J,
Mukhopadhyay UK, Thakur AR, Chaudhuri SR: Characterization
and wash performance analysis of microbial extracellular
enzymes from East Calcutta Wetland in India. Am J Appl Sci
2008, 5:1650-1661.
Adinarayana K, Ellaiah P, Prasad DS: Purification and partial characterization of thermostable serine alkaline protease from a
newly isolated Bacillus subtilis PE-11. AAPS PharmSciTech 2003,
4(4):56.
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV: Molecular and
biotechnological aspects of microbial proteases. Microbiol Mol
Biol Rev 1998, 62:597-635.
Thumar J, Singh SP: Two-step purification of a highly thermostable alkaline protease from salt-tolerant alkaliphilic
Streptomyces clavuligerus strain Mit-1. J Chromatogr B 2007,
854:198-203.
Turner P, Mamo G, Karlsson EN: Potential and utilization of
thermophiles and thermostable enzymes in biorefining.
Microb Cell Fact 2007, 6:1-23.
Priest FG: Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev 1977, 41:711-753.
Nascimento WCA, Martins MLL: Production and properties of
an extracellular protease from thermophilic Bacillus sp. Braz
J Microbiol 2004, 35:91-96.
Haki GD, Rakshit SK: Developments in industrially important
thermostable enzymes: a review. Bioresource Technol 2003,
89:17-34.

Page 8 of 9
(page number not for citation purposes)

Microbial Cell Factories 2009, 8:20

9.
10.
11.
12.
13.
14.

15.
16.

17.

18.

19.
20.
21.
22.
23.

24.

25.

26.

27.
28.

29.

30.

Zamost BL, Nielsen HK, Starnes RL: Thermostable enzymes for
industrial applications. J Ind Microbiol Biot 1991, 8:71-82.
Sheldon RA: Large scale enzymatic conversions in non-aqueous media. In Enzymatic Reactions in Organic Media Edited by: Koskinen AMP, Klibanov AM. London: Chapman and Hall; 1996:266-302.
Gupta A, Khare SK: Enhanced production and characterization
of a solvent stable protease from solvent tolerant Pseudomonas aeruginosa PseA. Enzyme Microb Tech 2007, 42:11-16.
Ghorbel B, Kamoun AS, Nasri M: Stability studies of protease
from Bacillus cereus BG1. Enzyme Microb Tech 2003, 32:513-518.
Kumar CG, Takagi H: Microbial alkaline proteases from a bioindustrial view point. Biotechnol Adv 1999, 17:561-594.
Rahman RNZR, Geok LP, Basri M, Salleh AB: Physical factors
affecting the production of organic solvent-tolerant protease
by Pseudomonas aeruginosa strain K. Bioresource Technol 2005,
96:429-436.
Wang JJ, Shih JCH: Fermentation production of keratinase
from Bacillus licheniformis PWD-1 and a recombinant B. subtilis FDB-29. J Ind Microbiol Biot 1999, 22:608-616.
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties
and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680.
Wang SL, Yeh PY: Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp shell
wastes fermented by Bacillus subtilis TKU07. Process biochem
2006, 41:1545-1552.
Ogino H, Yasui K, Shiotani T, Ishihara T, Ishikawa H: Organic solvent-tolerant bacterium which secretes an organic solventstable proteolytic enzyme.
Appl Environ Microbiol 1995,
61:4258-4262.
Gupta A, Roy I, Khare SK, Gupta MN: Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA. J Chromatogr A 2005, 1069:155-161.
Geok LP, Razak CNA, Rahman RNZA, Basri M, Salleh AB: Isolation
and screening of an extracellular organic solvent-tolerant
protease producer. Biochem Eng J 2003, 13:73-77.
Karadzic I, Masuib A, Fujiwara N: Purification and characterization of a protease from Pseudomonas aeruginosa grown in
cutting oil. J Biosci Bioeng 2004, 98:145-152.
Laane C, Boeren S, Vos K, Veeger C: Rules for the optimization
of biocatalysis in organic solvents. Biotechnol Bioeng 1987,
30:81-87.
Ogino H, Uchiho T, Doukyu N, Yasuda M, Ishimi K, Ishikawa H:
Effect of exchange of amino acid residues of the surface
region of the PST-01 protease on its organic solvent-stability. Biochem Biophys Res Commun 2007, 358:1028-1033.
Wang SL, Kao TY, Wang CL, Yen YH, Chern MK, Chen YH: A solvent stable metalloprotease produced by Bacillus sp.
TKU004 and its application in the deproteinization of squid
pen for β-chitin preparation. Enzyme Microb Tech 2006,
39:724-731.
Chantawannakula P, Oncharoena A, Klanbuta K, Chukeatiroteb E,
Lumyong S: Characterization of proteases of Bacillus subtilis
strain 38 isolated from traditionally fermented soybean in
Northern Thailand. Sci Asia 2002, 28:241-245.
Gouda MK: Optimization and purification of alkaline proteases produced by marine Bacillus sp. MIG newly isolated
from Eastern Harbour of Alexandria. Pol J Microbiol 2006,
55:119-126.
Abdulrahman AM, Yasser MS: Production and some properties
of protease produced by Bacillus licheniformis isolated from
Aseer, Saudi Arabia. Pakistan J Biol Sci 2004, 7:1631-1635.
Frankena J, Koningstein GM, Verseveld HW, Stouthamer AH: Effect
of different limitations in chemostat cultures on growth and
production of exocellular protease by Bacillus licheniformis.
Appl Microbiol Biotechnol 1986, 24:106-112.
Johnvesly B, Naik GR: Studies on production of thermostable
alkaline protease from thermophilic and alkaliphilic Bacillus
sp. JB-99 in a chemically defined medium. Process Biochem
2001, 37:139-144.
Moon SH, Parulekar SJ: Aparametric study of protease production in batch andfed-batch cultures of Bacillus firmus. Biotechnol Bioeng 1991, 37:467-483.

http://www.microbialcellfactories.com/content/8/1/20

31.
32.
33.

34.
35.
36.
37.

38.
39.

Roychoudhury S, Parulekar SJ, Weigand WA: Cell Growth and aAmylase Production Characteristics of Bacillus amyloliquefaciens. Biotechnol Bioeng 1988, 33:197-206.
Mabrouk SS, Hashem AM, El-Shayeb NMA, Ismail AMS, Abdel-Fattah
AF: Optimization of alkaline protease productivity by Bacillus
licheniformis ATCC21415. Bioresource Technol 1999, 69:155-159.
Shafee N, Aris SN, Rahman RNZA, Basri M, Salleh AB: Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium
Bacillus cereus strain 146. J Appl Sci Res 2005, 1:1-8.
Mehrotra S, Pandey PK, Gaur R, Dramwal NS: The production of
alkaline protease from Aspergillus flavus culture filtrates. Appl
Microbiol Biot 1999, 46:138-142.
Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol
Biol Evol 2007, 24:1596-1599.
Rahman RNZR, Basri M, Salleh AB: Thermostable alkaline protease from Bacillus stearothermophilus F1; nutritional factors
affecting protease production. Ann Microbiol 2003, 53:199-210.
Rahman RNZA, Razak CNAR, Ampon K, Basri M, Yunus WMZW,
Salleh AB: Purification and characterisation of heat-stable
alkaline protease from Bacillus stearothermophilus F1. Appl
Microbiol Biot 1994, 40:822-827.
Sarath G, De La Motte RS, Wagner FW: Protease assay methods.
In Proteolytic Enzymes: a practical approach Edited by: Beynon RJ, Bond
JS. Oxford: IRL Press; 1989:25-55.
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-54.

Publish with Bio Med Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK

Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright

BioMedcentral

Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp

Page 9 of 9
(page number not for citation purposes)

</pre>
</body>
</html>