Purification and characterization of an alkaline protease from Bacillus licheniformis UV-9 for detergent formulations

Abstract

Alkaline protease produced by mutant strain B. licheniformis UV-9 was purified and characterized for its exploitation in detergent formulation. The enzyme was purified to homogeneity by employing ammonium sulphate precipitation and sephadex G-100 gel filtration chromatography with a 36.83 fold increase in specific activity and 11% recovery. The molecular weight of the protease was found to be 36.12 kDa by SDS-PAGE. The Km and Vmax values exhibited by purified protease were 5 mg/ml and 61.58ìM/ml/min, respectively, using casein as substrate. The enzyme exhibited highest activity at pH 11 and temperature 60°C. Stability studies showed that the enzyme retained higher than 80% residual activity in the pH and temperature ranges of 8 to 11 and 30 to 50°C, respectively. However, in the presence of 10 mM Ca2+ ions the enzyme tained more than 90% of its residual activity at pH 11 and temperature 60°C. Phenyl methyl sulphonyl fluoride (PMSF) completely inhibited the enzyme activity suggesting that it was serine protease. Among metal ions, the Mg2+ and Ca2+ ions enhanced activity up to 128% and 145%, respectively. The purified enzyme showed extreme stability towards various surfactants such as Tween-20, Tween-45, Tween-65 and Triton X-45. In addition, the enzyme also exhibited more than 100% residual activity in the presence of oxidizing agents, H2O2 and sodium perborate. These biochemical properties indicate the potential use of B. licheniformis UV-9 enzyme in laundry detergents.

Full text

Original Article
Purification and characterization of an alkaline protease from
Bacillus licheniformis UV-9 for detergent formulations
Muhammad Nadeem1*, Javed Iqbal Qazi2, Quratulain Syed1, and Muhammad Gulsher1
1 Biotechnology & Food Research Center, PCSIR Laboratories Complex, Lahore-54600, Pakistan.
2 Department of Zoology, University of the Punjab, New Campus, Lahore-54590, Pakistan.
Received 22 October 2012; Accepted 6 February 2013
Abstract
Alkaline protease produced by mutant strain B. licheniformis UV-9 was purified and characterized for its exploitation
in detergent formulation. The enzyme was purified to homogeneity by employing ammonium sulphate precipitation and
sephadex G-100 gel filtration chromatography with a 36.83 fold increase in specific activity and 11% recovery. The molecular
weight of the protease was found to be 36.12 kDa by SDS-PAGE. The Km and Vmax values exhibited by purified protease
were 5 mg/ml and 61.58ìM/ml/min, respectively, using casein as substrate. The enzyme exhibited highest activity at pH 11 and
temperature 60°C. Stability studies showed that the enzyme retained higher than 80% residual activity in the pH and temper-
ature ranges of 8 to 11 and 30 to 50°C, respectively. However, in the presence of 10 mM Ca2+ ions the enzyme tained more
than 90% of its residual activity at pH 11 and temperature 60°C. Phenyl methyl sulphonyl fluoride (PMSF) completely
inhibited the enzyme activity suggesting that it was serine protease. Among metal ions, the Mg2+ and Ca2+ ions enhanced
activity up to 128% and 145%, respectively. The purified enzyme showed extreme stability towards various surfactants
such as Tween-20, Tween- 45, Tween-65 and Triton X-45. In addition, the enzyme also exhibited more than 100% residual
activity in the presence of oxidizing agents, H2O2 and sodium perborate. These biochemical properties indicate the potential
use of B. licheniformis UV-9 enzyme in laundry detergents.
Keywords: alkaline protease, B. licheniformis, purification, characterization, oxidant stability
Songklanakarin J. Sci. Technol.
35 (2), 187-195, Mar. - Apr. 2013
1. Introduction
Proteases represent one of the three largest groups of
industrial enzymes and account for about 60% of the total
worldwide sale of the enzymes (Rao et al., 1998). They have
diverse applications in various industries such as food,
pharmaceuticals, silk and diagnostic with predominant use in
detergent and leather industries (Kumar and Takagi, 1999;
Oberoi et al., 2001). Proteases in detergent formulations
facilitate washing efficiency of detergents by releasing pro-
teinaceous stains. The applications in detergent industries
require their improved stability at elevated temperatures and
pH and compatibility with various oxidants and metal ions
(Jaswal and Kocher, 2007; Haddar et al., 2009).
Several alkaline proteases have been purified and
characterized from many Bacillus strains (Bhaskar et al.,
2007; Doddapaneni et al., 2007, Padmapriya et al., 2012).
Subtilisin Carlsberg produced by Bacillus licheniformis
(Jacob et al., 1985) and Subtilisin Novo produced by Bacillus
amyloliquefaciems (Wells et al., 1983) have been the enzyme
of choice for detergent industries. Both enzymes have similar
molecular mass of 27.5 kDA but differ from each other in the
constitution of amino acids. These enzymes exhibit maximum
activity at alkaline pH values ranging from 8-10 (Horikoshi,
1999). Generally the alkaline proteases for detergent applica-
tions should be active at temperature higher than 40-50°C
and pH in the range of 9-12 (Sellami-Kamoun et al., 2008;
Haddar et al., 2009). Appropriate specificity and compatibi-
* Corresponding author.
Email address: mnadeempk@yahoo.com
http: //www.sjst.psu.ac. th

M. Nadeem et al. / Songklanakarin J. Sci. Technol. 35 (2), 187-195, 2013
188
lity with various detergent constituents continuously stimu-
late the thrust of new enzymes in the market. Purification and
characterization is the basic need to elucidate such precise
properties of a newly isolated enzyme for its applications in
the industry. The purification process also increases the
specific activities of enzymes, making them more specific for
industrial applications (Kumar, 2002; Adinarayana et al.,
2003). In the present study, we report purification and
characterization of alkaline protease produced by mutant
strain Bacillus licheniformis UV-9. Further studies regarding
stability and compatibility of the enzyme with various deter-
gent constituents were also conducted to examine its poten-
tial use as detergent additive.
2. Material and Methods
2.1 Enzyme production
Production of alkaline protease from mutant B.
licheniformis UV-9 was carried in a 2 L lab scale bioreactor
(Eyela, Japan) having 1.5 L growth medium comprising of
glucose, 1% (w/v); soybean meal, 1% (w/v); K2HPO4, 0.5%
(w/v); MgSO4.7H2O, 0.05% (w/v); NaCl, 0.05% (w/v) and
CaCl2.2H2O, 0.05% (w/v) at optimum conditions described
elsewhere (Nadeem et al., 2009). Cell-free supernatant was
used for subsequent studies.
2.2 Enzyme assay
Protease activity was determined by the method of
Yang and Haung (1994) with slight modifications in pH of
substrate and incubation temperature. The reaction mixture
containing 2 ml of 1% (w/v) casein solution in 0.05 M glycine-
NaOH buffer (pH 11) and 1 ml of enzyme solution was incu-
bated at 60°C for 15 min. The reaction was then stopped by
adding 3 ml of 10% (w/v) trichloroacetic acid. After that the
entire mixture was centrifuged at 9000 x g for 10 min at 4°C
and absorbance of the liberated tyrosine was measured at
280 nm against blank. One proteolytic unit (IU) was defined
as the amount of the enzyme that released 1 µg of tyrosine
per minute, under the assay conditions.
2.3 Protein assay
Total protein contents were determined according
to the method of Lowry et al. (1951), using bovine serum
albumin as a standard.
2.4 Protease purification
2.4.1 Ammonium sulphate precipitation
Cell free supernatant was precipitated by adding
ammonium sulphate at different saturation levels (40-80%).
After each addition, the enzyme solution was stirred for 1 h
at 4°C. The protein precipitated was collected by centrifuga-
tion at 12,000 x g for 20 min at 4°C and resuspended in
minimum volume of 0.05M Tris-HCl buffer, pH 8.0 to get
the concentrated enzyme suspension. After that the enzyme
suspension was dialyzed against the same buffer with 4-6
changes.
2.4.2 Sephadex G-100 gel filtration chromatography
The concentrated enzyme sample was purified on
sephadex G-100 (Pharmacia) column (1.5 cm x 30 cm) by suing
FPLC system (Biologic LP, Bio-Rad, USA). The Sephadex
column was equilibrated with 0.05 M Tris-HCl buffer of
pH 8.0. The dialyzed enzyme sample was loaded onto a
Sephadex G-100 column and then eluted with the same
buffer. Fractions each of 4 ml were collected at a flow rate of
30 ml/h by fraction collector (Model 2110, Bio-Rad). The
fractions showing absorbance at 280 nm were analyzed for
protease activities and the active fractions were pooled,
dialyzed and then freeze dried by freeze dryer (Eyela, Japan).
The freeze dried preparation was stored at -20°C for further
studies.
2.4.3 SDS-polyacrylamide gel electrophoresis
SDS-PAGE (12%) was performed according to the
method described by Laemmli (1970) using a mini slab gel
apparatus (8x8 cm glass plate). The molecular weight was
determined by interpolation from linear semi-logarithmic plot
of relative molecular weight versus the Rf value (relative
mobility) using standard molecular weight markers
(Fermentas).
2.5 Characterization of purified protease
2.5.1 Effect of pH on enzyme activity and stability
The activity of purified protease was measured at
different pH values (6-12) by using 1% (w/v) solution of
casein as a substrate dissolved in different buffers (0.05 M):
phosphate (pH 6-7) tris–HCl (pH 8-9) and glycine-NaOH
(pH 10-12). To determine pH stability, the enzyme was incu-
bated in different buffers of pH values ranging from 6 to 12
for 12 h at 40°C in the absence and presence of 5 and 10 mM
CaCl2.2H2O. The residual activities were then measured
according to the standard assay procedure.
2.5.2 Effect of temperature on protease activity and stability
Influence of temperature on purified protease activity
was studied by incubating reaction mixtures at different
temperatures ranging from 30 to 80°C by using 1% casein
solution. Thermal inactivation was examined by incubating
purified enzyme at different temperatures (30-80°C) for 1 h in
a water bath (Eyela, Japan) in the absence and presence of
Ca2+ ions at 5 mM and 10 mM of CaCl2.2H2O. The residual
activities were then measured as described above while

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M. Nadeem et al. / Songklanakarin J. Sci. Technol. 35 (2), 187-195, 2013
taking the activity of non-heated enzyme as 100%.
2.5.3 Effect of inhibitors and metal ions on protease activity
Effects on protease activity of various inhibitors (each
at 5mM) such as phenyl methyl sulphonyl fluoride (PMSF),
1,10-phenanthroline, pepstatin, di-isopropyl fluorophosphate
(DFP), cysteine inhibitors p-chloromercuric benzoate
(pCMB), ethylene diamine tetra acetic acid (EDTA) and metal
ions (Ca+2, Zn+2, Mg+ 2, Na+2, Hg+2, Cu+2, Al+3, Ni+2, Cd+2 and
Co+2), each at 5mM concentration on protease activity were
studied. The purified enzyme was pre-incubated with the
above inhibitors and metal ions for 30 min at 40°C. Then the
remaining activity was measured routinely, taking activity in
the absence of inhibitors and metal ions as 100%.
2.5.4 Effects of surfactants and oxidants on stability of
alkaline protease
Effects of different surfactants like Tween-20 (0.5%,
1.0%); Tween-45 (0.5%, 1.0%); Tween-65 (0.5 %, 1.0 %);
Triton-X-405 (0.5 %, 1.0 %); SDS (0.5 %, 1.0 %, 5.0 %) and
oxidizing agents like H2O2 (0.5%, 1.0%, 5.0%) and sodium per
borate (0.5%, 1.0%, 5.0%) on the alkaline protease stability
were studied by incubating the mixtures for 1 h at 40°C. The
residual activities were then measured and the activity of
enzyme without any additive was taken as 100%.
2.5.5 Substrate specificity
Effects of various substrates such as casein, bovine
serum albumin, gelatin, hemoglobin and keratin on purified
alkaline protease activity were determined according to the
method of Yang and Huang (1994) as described earlier. The
protease activity towards casein was taken as control.
2.5.6 Enzyme kinetics
The Km and Vmax values for alkaline protease were
calculated by linear regression analysis by Lineweaver-Burk
plot (double reciprocal plot) using various concentration of
casein (5, 10, 15, 20, 25 and 30 mg/ml). The experiments were
carried out in triplicate and then the activity measured
according to the standard assay conditions.
3. Results and Discussion
3.1 Purification of alkaline protease
Purification of alkaline protease of mutant B.
licheniformis UV-9 is summarized in Table 1. Initially the
enzyme solution was precipitated with ammonium sulphate
and 60-70% saturation level increased the protease activity
6.41 fold with 62% recovery, showing specific activity of
2106.02 U/mg. The dialyzed enzyme suspension of 60-70%
saturation level was then subjected to gel filtration chroma-
tography on a Sephadex G-100 column for further purifica-
tion. The elution profile yielded a well-resolved single peak
showing protease enzyme after activity measurement (Figure
1). The active fraction of this peak gave a 36.83 fold increase
in protease activity with a recovery of 11% and specific
activity of 12102.96 U/mg of protein. The results indicate that
gel filtration chromatography yielded a pure enzyme. Adi-
narayana et al. (2003) and Alam et al. (2005) have reported
purification of protease enzymes by gel filtration chromato-
graphy with Sephadex G-200 and Sephadex G-100 columns,
respectively. Wide-ranging results of protease purification
(4.25-200-folds) with various specific activities (13.33-
159381 U/mg of protein) and % recovery (2-21%) have been
described for different microbial species (Tunga et al., 2003;
Tang et al., 2004; Kim and Kim, 2005; Guangrong et al., 2006;
Yossan et al., 2006).
The enzyme purity was confirmed by SDS-PAGE
which demonstrated a single band, indicating homogeneous
preparation (Figure 2a). Molecular weight of the protease was
determined by interpolation from a linear logarithmic plot of
relative molecular mass versus the Rf value. The molecular
weight of the protease band was accordingly calculated and
found to be as 36.12 kDa (Figure 2b). Beg and Gupta (2003)
reported a 30 kDa molecular weight serine alkaline protease
produced by B. mojavensis. Generally, the molecular masses
of alkaline proteases from various Bacillus species range
between 17-40 kDa (Tang et al., 2004; Kim and Kim, 2005;
Guangrong et al., 2006; Yossan et al., 2006, Haddar et al.,
2009; Padmapriya et al., 2012), with few exception of high
molecular mass, such as up to 90 kDa from Bacillus subtilis
(Kato et al., 1992). The molecular masses of most popular
Subtilisin Carlsberg and Subtilisin BPN are 27.3 and 27.5
kDa, respectively (Wells et al., 1983; Jacobs et al., 1985).
Table 1. Summary of purification of alkaline protease produced by B. licheniformis UV-9 in submerged fermentation
process
Purification Steps Total activity Total protein Specific activity Purification Yield
(U) (mg) (U/mg) fold (%)
Crude enzyme 2530620 7700 328.65 1 100
Ammonium sulphate precipitation (60-70%) 1568984 745 2106.02 6.41 62
Sephadex G-100 278368 23 12102.96 36.83 11

M. Nadeem et al. / Songklanakarin J. Sci. Technol. 35 (2), 187-195, 2013
190
3.2 Characterization of purified protease
3.2.1 Effect of pH and temperature on enzyme activity and
stability
The present protease was found to be active over a
broad range of pH 8-12 with optimal activity at pH 11 indicat-
ing alkaline nature of the enzyme (Figure 3a). Generally,
commercial proteases from microorganisms have maximum
activity in the alkaline pH range of 8-12 (Rao et al., 1998;
Kumar et al., 1999; Gupta et al., 2002). Optimum pH of 10 of
alkaline proteases from various Bacillus species has been
described by some workers (Adinarayana et al., 2003; Uchida
et al., 2004; Gupta et al., 2005).
pH of the laundry detergent is usually in the range of
9-12. The results of pH stability study indicated that the
enzyme was stable over a wide range of pH (8-11) and
retained its 80% activity at pH 11 (Figure 3b). However, 40%
remaining activity was observed at pH 12 in the absence of
Ca2+ ions. The presence of 5 and 10 mM Ca2+ ions increased
the stability against various pH. About 90 and 93% activities
are noted at pH 11 in the presence of 5 and 10 mM Ca2+ ions,
respectively. However, the enzyme retained its 60 and 72%
activity in the presence of Ca2+ ions at pH 12. Proteases
produced from Bacillus subtilis PE-11 and Bacillus subtilis
CN2 have been described as remaining stable in the ranges of
8-11 and 7-11, with relative activities of more than 90% and
70%, respectively (Adinarayana et al., 2003; Uchida et al.,
2004). Comparable results have been reported by Sookkheo
et al. (2000). These workers found 60% proteolytic retention
at pH 10 in the presence of 5 mM Ca2+ ions. All these investi-
gations indicate that pH stabilities of enzymes depend on
the available concentration of Ca2+ ions in the enzyme
solution.
Optimum temperature for protease of B. licheniformis
UV-9 was found to be 60oC when tested at pH 11 using casein
as substrate (Figure 4a). Maximum proteolytic activity of
Bacillus strains HR-08 and KR-8102 isolated from soil of
western and northern parts of Iran have been recorded at
65°C and 50°C, respectively (Moradian et al., 2006). Proteases
from P. aeruginosa MN1 and some other Bacillus species
have been described with optimum temperature of 60°C
(Banerjee et al., 1999; Beg and Gupta, 2003; Nascimento and
Martins, 2004: Khosravi-Darani et al., 2008; Olajuyigbe and
Ajele, 2008). These findings indicate that variation in the
characteristics of the extracellular proteolytic enzymes could
Figure 1. Elution profile of alkaline protease of B. licheniformis UV-9 from Sephadex G-100 column by FPLC
Figure 2. SDS-PAGE of the purified protease. Molecular mass markers and the purified protease were applied to lanes 1 and 2,
respectively. a: SDS-PAGE; b: Molecular weight determination

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M. Nadeem et al. / Songklanakarin J. Sci. Technol. 35 (2), 187-195, 2013
exist among various Bacillus species.
The investigations regarding thermostability of alka-
line protease showed that the enzyme was stable up to 50°C
and above this temperature its activity decreased. However,
presence of Ca2+ ions improved thermostability of the
alkaline protease produced by B. licheniformis UV-9. The
enzyme retained 72 and 98% residual activities at 60°C in the
presence of 5mM and 10 mM Ca2+ ions, respectively (Figure
4b). Ca2+ ions have been described to keep 78% of residual
activity of thermostable bacterial enzymes after incubation at
80 oC for 1 h in the presence of 10 mM Ca2+ ions (Johnvesly
and Naik, 2001). Most alkaline proteases have been reported
to remain stable at high temperature in the presence Ca2+ ions
(Rahman et al., 1994; Kumar et al., 1999). The Ca2+ ions may
stabilize the structure of the present enzyme which conse-
quently increased its thermal stability at elevated tempera-
tures.
3.2.2 Effects of inhibitors and metal ions on enzyme activity
Effects of different inhibitors and metal ions on alka-
line protease of B. licheniformis UV-9 are given in Table 2.
The results showed that the enzyme was completely inhibited
by serine protease inhibitor phenylmethyl sulphonyl fluoride
(PMSF), suggesting its serine nature. PMSF is a well known
serine protease inhibitor which results in complete loss of the
enzyme activity after inhibition (Tsuchida et al., 1995; Jeong
(a)
(b)
Figure 3. (a) Effect of different pH levels on alkaline protease
activity produced by B. licheniformis UV-9. Bars re-
present ± S.D. (b) Effect of various pH on the stability
of protease produced by B. licheniformis UV-9 in the
presence and absence Ca2+ ions. The stability is
expressed in percentage of residual activity. Bars re-
present SD.
(a)
(b)
Figure 4. (a) Effect of different temperatures on protease activity
produced by B. licheniformis UV-9. Bars represent ±
S.D. (b) Effect of various temperatures on stability of
protease produced by B. licheniformis UV-9. The stabi-
lity is expressed in percentage of residual activity. Bars
represent SD.
Table 2. Effect of inhibitors and activators on the relative
acti vity of alk ali ne protease produced by B.
licheniformis UV-9
Inhibitors/ Activator Relative Activity (%)
Control 100
PMSF 0
1,10-phenanthroline 97
Pepstatin 103
DFP 03
PCMB 88
EDTA 90
Ca2+ (CaCl2) 145
Zn2+ (ZnCl2) 80
Mg+2 (MgCl2) 128
Na2+ (NaCl2) 102
Hg2+ (HgCl2) 70
Cu2+ (CuCl2) 106
Al3+ (AlCl3) 65
Ni2+ (NiCl2) 98
Cd2+ (CdCl2) 90
Co2+ (CoCl2) 93
PMSF = Phenylmethyl sulphonyl fluoride; DFP = di-Isopro-
pyl fluorophosphate; pCMB = p-Chloromercuric benzoate;
EDTA = Ethylene diamine tetra acetic acid. The concentra-
tion of all inhibitors and metal ions was adjusted at 5 mM.

M. Nadeem et al. / Songklanakarin J. Sci. Technol. 35 (2), 187-195, 2013
192
et al., 2000; Adinarayana et al., 2003). The inhibitor di-iso-
propyl fluorophospahte (DFP) reduced the protease activity
up to 97%. Similar results of inhibition by DFP have been
recorded for serine protease produced by B. licheniformis,
B. pumilus and B. inetermedius 3-19 (Aoyama et al., 2000;
Huang et al., 2003; Tang et al., 2004). The presence of suit-
able metal ions plays an important role in maintaining active
conformation of enzyme against thermal denaturation
(Donaghy and Mckay, 1993). Therefore, identification of
proper metal ions has significant impact on enzyme applica-
tions at commercial level. Results of some metal ions on the
protease activity indicated that Ca+ 2 and Mg+2 ions increased
relative enzyme activity up to 145 and 128%, respectively.
More than 100% relative activities also appeared in the
presence of Cu+2 and Na+1 ions. Siriporn et al. (2006) described
that Mn+2, Ca+2 and Mg+2 ions increased relative activity of
protease produced by Bacillus megatarium. These cations
have also been reported to increase activity and thermo-
stability of Bacillus alkaline proteases (Johnvesly and Naik,
2001).
3.2.3 Effect of surfactants and oxidants on stability of
alkaline proteases
In addition to activity and stability at high tempera-
ture and pH ranges, a good detergent protease must be
compatible and stable with all commonly used detergent
compounds such as surfactants, bleaches, oxidizing agents
and other additives which might be present in the formulation
(Gupta et al., 1999; Kumar and Takagi, 1999; Oberoi et al.,
2001). Therefore, the effects of various surfactants and oxi-
dizing agents at different concentrations on activity of the
purified protease of B. lichenifomis UV-9 was studied after
pre-incubation at 40°C for 1 h (Table 3). The residual activity
of B. licheniformis UV-9 protease were found 90.45%,
90.15% and 97.56% at 1% each of Tween-20, Tween-45 and
Triton-X-405, respectively. While 1% of Tween-65 showed
121.06% residual activity. However, a minimum residual
activity of 45.83% was measured at 1% of SDS. These find-
ings indicated that Tween-65 might improve the interaction
of enzyme with substrate and this could result in increased
residual activity. Similar effects of SDS on alkaline proteases
of various Bacillus species have been reported in earlier
investigations (Matta and Punj, 1998; Banerjee et al., 1999).
Oxidizing agents such as peroxide and perborates are
common ingredients of modern bleach-based detergent
formulations. The enzymes have significant importance for
detergent industries and show extreme stability towards
oxidizing agents. Interestingly, 1% of H2O2 and sodium per-
borate stimulated the activity and enzyme expressed 108 and
115% residual activities, respectively. Joo et al. (2003)
reported that Bacillus clausii 1-52 protease exhibited resi-
dual activity of up to 114% after treatment with 1% H2O2.
Haddar et al. (2009) reported alkaline protease from B.
mojavensis A21 showing residual activity of up to 79.40%
and 35% after incubation with 1% of H2O2 and sodium perbo-
rate. However, the present enzyme showed >100% residual
activities at 5% of H2O2 and sodium perborate indicating its
eventual use in detergent formulations.
3.2.4 Substrate specificity
Activity of the purified alkaline protease against vari-
ous proteinaceous substrates was examined (Table 4). The
enzyme showed a high level of catalytic activity against
casein, hemoglobin and albumin (bovine) indicating its
ability to hydrolyze several proteins. Substrate diversity is an
important criterion to analyze the potency of a protease. In
Table 3. Effect of different oxidizing agents and surfactants
on alkaline protease activity produced by B. liche-
niformis UV-9
Surfactants/ Concentration Residual activity
Oxidizing agents (% ) (%)
Control - 100
Tween 20 0.5 105.80
1.0 90.45
Tween 45 0.5 98.46
1.0 90.15
Tween 65 0.5 128.40
1.0 121.06
Triton X 405 0.5 102.72
1.0 97.56
5.0 15.32
SDS 0.5 54.64
1.0 45.83
5.0 25.71
H2O20.5 110.96
1.0 108.56
5.0 100.47
Sodium perborate 0.5 118.78
1.0 115.56
5.0 101.92
Residual activity was expressed as a percentage of the activ-
ity level in the absence of surfactants and oxidizing agents.
Separate blank was processed for each agent
Table 4. Effect of various substrates on alkaline protease ac-
tivity of B. licheniformis UV-9
Substrate Relative activity(%)
Casein 100
Albumin (bovine) 52
Hemoglobin 72
Gelatin 11
Albumin (egg) 42
Keratin 8
Collagen 2

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M. Nadeem et al. / Songklanakarin J. Sci. Technol. 35 (2), 187-195, 2013
contrast, the enzyme showed very poor hydrolysis against
collagen, keratin and gelatin corresponding 2, 8 and 11%
relative activities, respectively. Adinarayana et al. (2003)
reported similar finding that casein was a good substrate for
serine protease enzyme produced by B. subtilis.
3.2.5 Kinetic studies
The kinetic parameters Km and Vmax of the alkaline
protease produced by B. licheniformis UV-9 were estimated
by Lineweaver-Burk Plot employing various concentrations
(2.5-20 mg/ml) of casein as substrate (Figure 5). The Line-
weaver-Burk Plot for the proteolytic reaction of the casein
revealed that the Km and Vmax values of the reaction were 5
mg/ml and 61.58 uM/ml/min, respectively. Kaur et al. (1998)
reported Km of 3.7 mg/ml for B. polymyxa protease, while
Thangam and Rajkumar (2002) reported a Km and Vmax of 1.66
mg/ml and 526 U/min/mg, respectively, for alkaline protease
of Alcaligenes faecalis using casein as a substrate. Jaswal
and Kocher (2007) plotted a double reciprocal plot (Line-
weaver-Burk Plot) and estimated an apparent Km of 5 mg/ml
and Vmax of 1000 mol tyrosine/min/ml for protease of B.
circulans. The Km value represents the dissociation constant
(affinity for substrate) of the enzyme-substrate (ES) complex.
Low values of Km indicate that the ES complex is held
together tightly and dissociates rarely before the substrate is
converted to product. The value of Km further proved that
the enzyme may preferably be used for protein hydrolysis
due to its catalytic efficiency.
4. Conclusion
The alkaline protease enzyme of mutan t Bacillus
licheniformis UV-9 was purified up to homogeneity level by
employing ammonium sulphate precipitation (60-70%) and
gel filtration through Sephadex G-100 chromatography. After
final purification step, the enzyme was purified 36.83 fold
with a specific activity of 12102.96 U/mg and 11% recovery.
The molecular weight of the enzyme was estimated to be
36.12 kDa by SDS-PAGE. PMSF completely inhibited the
enzyme activity, suggesting its serine nature. The purified
enzyme showed desirable properties such as high activity and
stability at broad ranges of pH and temperature. The enzyme
was also found compatible and stable with most of surfac-
tants and oxidizing agents tested and retained its more than
100% residual activity. These properties indicate the possi-
bilities of commercial exploitation of the alkaline protease in
detergent formulations.
Acknowledgement
The authors are thankful to the Ministry of Science
and Technology (MoST), Islamabad, Govt. of Pakistan, for
providing financial support under PSDP project No. 31 to
carry out this research work.
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