Accepted Manuscript

Title: Evaluation of the biotechnological potential of a novel

purified protease BS1 from Bacillus safensis S406 on the
chitin extraction and detergent formulation

Authors: Samiha Mhamdi, Intidhar Bkhairia, Rim Nasri,

Tahar Mechichi, Moncef Nasri, Alya Sellami Kamoun

PII: S0141-8130(17)30250-7
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.062
Reference: BIOMAC 7742

To appear in: International Journal of Biological Macromolecules

Received date: 18-1-2017

Revised date: 10-6-2017
Accepted date: 12-6-2017

Please cite this article as: Samiha Mhamdi, Intidhar Bkhairia, Rim Nasri, Tahar
Mechichi, Moncef Nasri, Alya Sellami Kamoun, Evaluation of the biotechnological
potential of a novel purified protease BS1 from Bacillus safensis S406 on the
chitin extraction and detergent formulation, International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.062

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Evaluation of the biotechnological potential of a novel purified protease BS1 from Bacillus safensis
S406 on the chitin extraction and detergent formulation

Samiha Mhamdi, Intidhar Bkhairia, Rim Nasri*, Tahar Mechichi, Moncef Nasri and Alya
Sellami Kamoun.

Laboratoire de Génie Enzymatique et de Microbiologie - Université de Sfax- Ecole Nationale

d’Ingénieurs de Sfax. B.P. 1173-3038-Sfax – Tunisia.

*Corresponding author: e-mail: ryma47@hotmail.fr

Tel: +216 97915603

Highlights:
 - Novel protease from Bacillus safensis S406 was purified (SB1) and characterized.
 - BS1 with a molecular mass of 29 kDa
 - Optimum pH and temperature values for activity were pH 10 and 60 °C, respectively.
 - BS1 is a potential candidate for peptide synthesis and detergent formulations.
 - BS1 was found to be effective in the deproteinization of shrimp waste.

Abstract

The aim of this study is the production of High-Added Value enzyme for Industrial Uses. An
extracellular alkaline stable protease BS1 was purified from a newly bacterium, Bacillus safensis
S406, isolated from the Sfax Solar Saltern. BS1 was purified by a process involving sulfate
d’ammonium sulfate precipitation, sephadex G-75, Mono-Q anion-exchange chromatography and
ultrafiltation. The enzyme has a molecular weight of 24 kDa and appeared as a single band on native-
PAGE. The optimum pH and temperature values of its proteolytic activity were pH 11.0 and 60 °C,
respectively. The enzyme was then tested for its potential applications in chitin extraction from
shrimp waste. A ration enzyme-protein of 10 allows to eliminate 93% of protein linked to the chitin
after 3 h hydrolysis at 45 °C. In addition FT-IR spectra of the extracted chitin display a series of
narrow absorption bands, typical of crystalline polysaccharide samples. Being very active in alkaline
pH, the potential application of BS1 in laundry formulation was investigated. High stability of BS1
activity in the presence of non-ionic surfactants and some commercial liquid and solid detergents,
was detected, suggesting its eventual use in detergent formulations.

Keywords: Bacillus safensis; Alkaline protease; Detergent formulations; Chitin extraction.

Biochemical characterization

1. Introduction
Proteases are a particularly important group of enzymes that account for 60% of the total
worldwide sales of industrial enzymes [1]. These enzymes have various industrial applications
including detergents [2], foods, pharmaceuticals and leathers [3]. The majority of commercial
proteases are produced by bacteria, especially Bacillus spp [4]. Among them, alkaline proteases are

1
particularly important because they are both stable and active at high pH solutions and in the
presence of surfactants and oxidizing agents [3, 5]. Their major application is in detergent industry,
where the pH of laundry detergents is generally in the range of 9.0–12.0. The performance of
detergent proteases is influenced by several factors such as the pH of washing solution, wash
temperature and detergent composition. Alkaline proteases from high yielding strains have been
extensively studied. However, bleach-stable enzymes are not generally available except for few
reports [6, 7].

Many alkaline proteases have been purified and characterized [8-11]. Subtilisin Carlsberg
produced by Bacillus licheniformis [10] and Subtilisin Novo produced by Bacillus amyloliquefaciens
[11] have been the enzymes of choice in detergent formulations. These enzymes showed maximum
activity at pH values between 8.0–10.0 [9].

Another application of proteolytic enzymes is the deproteinization of shrimp wastes to

produce chitin [12, 13]. Chitin is the abundant renewable natural resource after cellulose [14].

Chitin and mainly its deacetylated form (chitosan) are biomolecules of a great potential (food
industry, pharmacy, cosmetics,…), due to their numerous biological activities as well as to their
biodegradability and biocompatibility [15].

In this study, we describe the isolation and identification of a new bacterial strain B. safensis
S406 with high protease activity and purification of new potent alkaline protease. The compatibility
of the purified enzyme with several laundry liquid and solid detergents, as well as its potential in
deproteinization of shrimp wastes to extract chitin, were also investigated.

2. Materials and methods

2.1. Bacterial strain and growth conditions for B. safensis S406 proteases production
B. safensis S406, an alkaline protease producing strain, was isolated from the pond M2 of the
Sfax Solar Saltern with average salinity of 190 p.s.u. The Sfax Solar Saltern is located in the central
eastern cost of Tunisia, about 34° 39’ N and 10° 42’ E.

Production of proteases by B. safensis S406 was carried out in medium containing (g/l):
chickpea 40, faba bean 30, NaCl 2.0, KH2PO4 1, K2HPO4 1, CaCl2, 0.1 and MgSO4 0.1, pH 8.0. Inocula
were routinely grown in Luria-Bertani (LB) broth medium composed of (g/l): peptone 10, yeast
extract 5, NaCl 5, pH 7.0 [16]. The Media were autoclaved at 120 °C for 20 min and the flasks were
incubated for 72 h at 30 °C with continuous shaking at 150 rpm. The culture medium was centrifuged
at 10,000 g for 35 min at 4 °C, and the cell-free supernatant was used as crude protease.

2.2. Protease assay

2
 Protease activity was measured by the method described by Kembhavi et al. [17] using casein
as a substrate. One unit of protease activity was defined as the amount of enzyme required to
liberate 1µg of tyrosine per minute under the used experimental conditions. Protease activities
represent the mean of at least two determinations carried out in duplicate.

2.3. BS1 purification process

The culture supernatant containing the extracellular enzyme preparation was first fractionated
by precipitation with ammonium sulfate. Fractions of 0-20%, 20-60% and 60–80% (w/v) were
centrifuged at 10,000 g for 30 min. Then, the precipitates were collected and suspended in a
minimum volume of Tris–HCl buffer (100 mM), pH 8.0. The 20–60% (w/v) ammonium sulfate fraction
was subsequently subjected to gel filtration on a Sephadex G-75 column (2.5 cm x 80 cm) which had
been equilibrated with 25 mM Tris–HCl buffer (pH 8.0) containing 0.05% Triton X-100. Fractions of 5
ml were collected at a flow rate of 30 ml/h with the same buffer. Protein contents (Abs 280 nm) and
protease activity were determined. Fractions of the peak exhibiting protease activity were pooled
and subjected to a Mono Q-Sepharose column (2 cm x 10 cm) pre-equilibrated with Tris–HCl (25 mM,
pH 8.0) buffer. Fractions of 5 ml each were eluted with the same buffer containing an increasing
concentration of NaCl from 0 to 0.5 M at a rate of 35 ml/h. Fractions were analyzed for protease
activity and protein concentrations. Finally, the active fractions from Mono Q-Sepharose were
collected and applied to a stirred ultrafiltration cell (Millipore 8400; Millipore, Billerica, MA) through
a 10-kDa molecular weight cut-off membrane (PBGC membrane, Millipore). The concentrated
enzyme was stored at -20 °C for further analyses. All the purification steps were conducted at
temperatures not exceeding 4 °C.

2.4. Polyacrylamide gel electrophoresis

SDS-PAGE was carried out to determine the purity and the molecular mass of the enzyme as
described by Laemmli [18] using 5% (w/v) stacking and 15% (w/v) separating gels. Sample was
prepared by mixing the purified enzyme at a ratio of 1:5 (v/v) with distilled water containing 10 mM
Tris–HCl (pH 8.0), 2.5% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.002% bromophenol blue.
Sample was heated at 100 °C for 5 min before electrophoresis. After electrophoresis, the gel was
stained with 0.25% Coomassie brilliant blue R-250 in 45% ethanol-10% acetic acid and destained with
5% ethanol-7.5% acetic acid. The molecular mass of the proteolytic enzyme was estimated using a
low-molecular mass calibration kit as markers (Sigma), consisting of: phosphorylase b (97 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor
(20.1 kDa) and bovine α-lactoalbumin (14.4 kDa).

2.5. Detection of protease activity by zymography

3
 Zymography was performed in conjunction with SDS–PAGE according to the method described
by Garcia-Carreno et al. [19]. The sample was not heated before electrophoresis. After
electrophoresis, the gel was submerged in 100 mM glycine-NaOH buffer (pH 10.0) containing 2.5%
Triton X-100 for 30 min, with constant agitation to remove SDS. Triton X-100 was then removed by
washing the gel three times with 100 mM glycine–NaOH buffer (pH 10.0). The gel was then incubated
with 1% (w/v) casein in 100 mM glycine–NaOH buffer (pH 10.0) for 20 min at 50 °C. Finally, the gel
was stained with Coomassie brilliant blue R-250. The development of clear zone on the blue
background of the gel indicated the presence of protease activity.

2.6. Protein content determination

Protein concentration was determined according to the method described by Bradford [20]
using bovine serum albumin as a standard. During the course of enzyme purification, protein content
was estimated by measuring the absorbance at 280 nm.

2.7. Determination of the N-terminal amino acids sequence of B. safensis protease

The purified enzyme, from the last purification step, was subjected to SDS–PAGE and
electrophoretically transferred to a polyvinylidiene difluoride (PVDF) membrane. After brief staining
with Coomassie brilliant blue R-250, the PVDF band corresponding to the protease was excised and
the N-terminal amino acids sequence was determined by the Edman degradation method on an ABI
Procise 494 protein sequencer (Applied Biosystems, Foster City, CA).

2.8. Biochemical properties of BS1

2.8.1. Kinetic studies
The activity of the purified protease was determined using different concentrations of casein
ranging from 4 to 80 µM. The assays were repeated twice and the respective kinetic parameters,
including the apparent Michaelis Menten constant (Km) and the maximum velocity (Vmax), were
calculated from Lineweaver–Burk plots [21]. The value of the turnover number (kcat) was calculated
from the equation: kcat = Vmax / [E], where [E] is the enzyme concentration in the reaction mixture.

2.8.2. Effect of pH on activity and stability of the enzyme

The effect of pH on the purified enzyme activity was studied over a pH range of 6.0–13.0 at 60
°C using casein 1% (w/v) as a substrate. For the study of pH stability, the enzyme was pre-incubated
in buffers at different pH values in the range of 5.0–13.0 for 1 h at 40 °C. Aliquots were withdrawn
and residual proteolytic activities were determined under standard assay conditions. The following
buffer systems were used: 100 mM sodium acetate for pH 5.0-6.0; 100 mM potassium phosphate for
pH 7.0; 100 mM Tris-HCl for pH 8.0; 100 mM glycine-NaOH for pH 9.0-11.0 and 100 mM KCl-NaOH for
pH 12.0-13.0. The non-incubated enzyme was considered as control (100%).

4
2.8.3. Optimum temperature and thermal stability of the protease
The protease activity was tested at different temperatures between 40 and 100 °C for 15 min
in 100 mM glycine–NaOH buffer, pH 11.0. Thermostability of the protease BS1 was examined by
incubating the enzyme for 48 h at different temperatures ranging from 40 °C to
80 °C in the absence or presence of 5 mM CaCl2. Aliquots were withdrawn at desired time intervals to
test the remaining activity under standard conditions. The non-heated enzyme was considered as
control (100%).

2.8.4. Effects of metal ions and enzyme inhibitors

The effects of various metal ions on protease activity were investigated by adding monavalent
(Na+, K+) or divalent (Ca2+, Mn2+, Zn2+, Ba2+, Mg2+, Hg2+, Cu2+) metal ions to the reaction mixture at a
final concentration of 5 mM. The activity of the enzyme in the absence of metal ions was considered
as control.

The effects of various enzyme inhibitors on the protease activity were studied using
phenylmethylsulfonyl fluoride (PMSF), ethylene-diamine-tetraacetic acid (EDTA), β- mercaptoethanol
and dithio-bis-nitrobenzoic acid (DTNB). The purified enzyme was pre-incubated with inhibitors for
30 min at 30 °C and then the remaining enzyme activity was determined at pH 10.0 and 55 °C using
casein as a substrate. The activity of the enzyme assayed in the absence of inhibitors was taken as
100%.

2.8.5. Effect of NaCl on activity and stability of BS1 protease

The effect of NaCl on the activity of the purified enzyme was carried out using casein as a
substrate, at pH 10.0 and 55 °C by the addition of NaCl (0-60%; w/v) to the reaction mixture. To
assess its stability against salts, the enzyme was pre-incubated for 1 h with different concentrations
of NaCl (w/v) in the range of 0-60%, and the residual activity was measured under standard assay
conditions. The activity of the enzyme determined without NaCl was used as a control.

2.8.6. Stability of BS1 enzyme in the presence of bleaches and surfactants

The effects of some surfactants (Triton X-100, Tween 80 and SDS) and oxidizing agents
(hydrogen peroxide and sodium perborate) on the enzyme stability were studied by pre-incubating
enzyme for 1 h at 30 °C. The residual activity was measured at pH 10.0 and 55 °C. The activity of the
purified enzyme pre-incubated without any additive was taken as 100%.

2.8.7. Stability and compatibility of BS1 with laundry detergents

The stability of the alkaline protease in the presence of commercial solid (7 mg/ml) and liquid
laundry detergents (1%; v/v), diluted in tap water to simulate wash conditions, was examined by pre-
incubation the enzyme preparation for 1 h at 30, 40 and 50 °C and then the remaining activities were
determined under standard assay conditions. The enzyme activity of a control sample (without

5
detergent), incubated under the same conditions, was taken as 100%. The solid detergents used
were Dixan (Henkel-Spain), Nadhif (Henkel-Alki-Tunisia), Ariel (Procter and Gamble, Switzerland),
New Det (Sodet, Tunisia) and OMO (Colgate–Palmolive, France). The endogenous enzymes contained
in the detergents were inactivated by heating the diluted solution for 1 h at 65 °C prior to the
addition of the purified enzyme. The liquid detergents used were: Lav+ (Best LAV, Tunisia), Textile
(Mc Bride, Belgique), Carrefour (Henkel-Alki, Tunisia) and Ariel (Procter and Gamble, Switzerland)

2.9. Effect of organic solvents on stability of BS1 protease

The purified protease was incubated separately in the absence or presence of organic solvents
(50%, v/v) at 30 °C and 150 rpm for 120 days. The residual activities were estimated by the assay
procedure described above. Each experiment was carried out three times and the difference in the
individual results in each set of experiments was less than 5%.

2.10. Chitin extraction and characterization

Shrimp waste was washed thoroughly with tap water, and then cooked in distilled water at a
ratio of 1:2 (w/v) for 20 min at 90 °C to inactivate endogenous enzymes. The cooked sample was
drained and homogenized in a Moulinex® blender for about 2 min. The pH of the mixture was
adjusted to 11.0, then, the shrimp waste proteins were digested with the purified protease BS1 using
different Enzyme/Substrate ratios (Unit of enzyme/mg of protein) during 3 h. The reactions were
stopped by heating the mixtures at 90 °C for 20 min to inactivate enzymes. The shrimp waste protein
hydrolysates were then centrifuged for 20 min at 5000×g to separate insoluble and soluble fractions.
The solid phases were washed with distilled water and dried for 1 h at 60 °C. Deproteinization degree
(DP) was expressed as percentage and computed by the following equation as described by Rao et al.
[22].

where PO and PR are protein concentrations (%) before and after hydrolysis respectively; while, O and
R represent the masses (g) of original sample and hydrolyzed residue on dry weight basis,
respectively.

Demineralization was carried out in HCl solution according to the method developed by
Madhavan and Nair [23]. Solid fractions obtained after BS1 protease hydrolysis were treated with 1.5
M HCl solution at a ratio of 1:10 (w/v) for 6 h at 25 °C under constant stirring (150 rpm). The chitin
products were filtered, using a vacuum pump, through four layers of gauze, washed to neutrality
with deionized water and then dried for 12 h at 60 °C. Demineralization efficiency was determined
from the ash content percentage in the chitin sample.

Infrared (IR) spectra of the extracted and commercial chitins were determined using a Fourier
transform (FT) spectrophotometer (Perkin Elmer®, Spectrum™ 100, Singapore) equipped with
attenuated total reflection (ATR) accessory containing a diamond/ZnSe crystal. An extra accessory

6
plate for powdered samples with a conic awl was used without need of previous sample preparation.
ATR-FTIR spectra were obtained in the 4000-600 cm-1 range at room temperature, using 10 scans and
4 cm-1 resolution.

2.11. Statistical analysis

The data were analyzed by Microsoft Excel 2007 (Microsoft Corporation), and the values
represent the mean standard deviation (mean ± SD) of three independent replicates. The results
were considered statistically significant for P values less than or equal to 0.05.

3. Results and discussion

3.1. Isolation and identification of the microorganism
The strain used in this study was isolated from the Sfax solar saltern. Samples were collected
and plated onto skim-milk agar. Plates were incubated for 24-48 h at 37 °C. Clear halos around
colonies on skim-milk agar gave indication on protease-producing strains. Individual colonies were
purified through repeated streaking on fresh agar plates. Among ten strains, isolated in the
laboratory and screened for proteolytic activity, S406 strain was selected to be identified. Thus, the
internal transcribed spacer region of the 16S rDNA (1484 bp) was amplified and sequenced. The
nucleotide sequence was analyzed with the GenBank database using BLAST program. The isolate was
identified as B. safensis and designated B. safensis S406.

3.2. Enzyme purification

The alkaline protease from B. safensis was purified by the four-step procedure described in
Materials and Methods. In the first step, the cell-free supernatant was precipitated with ammonium
sulfate (20–60%). The precipitate was then successively subjected to Sephadex G-75 gel filtration,
Mono Q-Sepharose anion exchange chromatography and finally ultrafiltration (Data not shown).
After the final purification step, the enzyme was purified 12.70-fold, with a recovery of 20.29% and a
specific activity of 39744.28U/mg of protein using casein as a substrate (Table 1). The purified
protease was homogenous on SDS–PAGE and its molecular mass was estimated to be 29 kDa (Fig.
1A), corresponding to that determined by gel filtration. In addition, the zymogramme analysis (Fig.
1B) of the cell-free supernatant shows six clear bands indicating the presence of at least six proteases
(lane 3). However, the purified protease shows a unique clear band of casein hydrolysis indicating its
homogeneity (lane 4).

The N-terminal sequence of the first 10 amino acid residues of the purified enzyme BS1 was
determined to be AQTVPYGIPQ (Fig. 2). As shown in Fig. 2, the N-terminal amino acid sequence
alignment analysis of protease BS1 showed 100% identity with Subtilisin from B. pumilus [24, 25],
whereas, it differs from the Subtilisin Carlsberg of B. subtilis [26] and Subtilisin DY of B. licheniformis
[27] by only one amino acid. However, there are 2 amino acid residues in the BS1 terminal sequence

7
that differ from Subtilisin NAT of B. subtilis, Natto [28] and Subtilisin E from B. subtilis [29]. Thus BS1
protease could be a subtilisin enzyme.

3.3. Determination of kinetic parameters

The kinetic parameters (Km and Vmax) values of the purified BS1 protease were 0.48 mM and
52.63×103 U/mg, respectively, using casein as a substrate. The Km value was similar to that of
alkaline serine-protease from Caldicoprobacter guelmensis [30]. The values recorded for the deduced
turnover number (kcat) and catalytic efficiency (kcat/Km) were 7.87 x 103 min−1 and 16.39 x 103
min−1mM−1, respectively.

3.4. Effect of pH on protease activity and stability

The effect of pH on the protease activity was examined over a pH range of 6.0 –13.0 at 60 °C
(Fig. 3a). The purified BS1 protease exhibited optimum activity at pH 11.0. The relative activities at
pH 9.0, 10.0 and 12.0 were about 81.9%, 91% and 83.12% of that recorded at pH 11.0. As detergents-
solution’s pH are generally between 9.0 and 10.5, the observed high activity of BS1 enzyme at
alkaline pH is an important criterion required in almost all detergent-enzymes, such as those
described from B. licheniformis NH1 [31] and Bacillus alcalophilus TCCC11004 [32].

The pH stability profile reported in Fig. 3b shows that the protease was highly stable over a
wide pH range, maintaining 100% of its original activity at pH values from 7.0 to 11.0, whereas about
70% of its initial activity was last at pH 13.0.

3.5. Effect of temperature on protease activity and stability

The optimum temperature for protease activity assessed at pH 11.0 was 60 °C in the absence
of CaCl2 and 65 °C in the presence of 5 mM Ca2+, using casein as a substrate (Fig. 4a). In addition, BS1
optimum proteolytic activity was significantly (p<0.05) enhanced to reach 225 % in the presence of 5
mM Ca2+. In agreement to the current study, serine-proteases from B. mojavensis A21 [33] and from
Bacillus subtilis PF1 [34] showed the same optimum temperature of 60 °C.

The thermal stability profiles of the purified protease are reported in Fig. 4b. The half-life
times of BS1 in the absence of any additive were 30, 21, 15, 9, and 6 h at 40, 50, 60, 70, and 80 °C,
respectively. However, the addition of 5 mM of CaCl2 increased its thermostability (p<0.05).

Similarly, Ca2+ was reported to improve the activity and stability of many Bacillus proteases
[24]. In fact, serine-proteases are known to contain two calcium binding sites, and the removal of
Ca2+ from the strong binding site is associated with a significant decrease in thermal stability. The role
of Ca2+ could presumably be attributed to the stabilization of the activated form of the BS1 and the
preservation of its structure against autolysis.

3.6. Effects of enzyme inhibitors and metal ions on protease activity

8
 In order to classify the BS1 protease, enzyme activity was measured in the presence of a
variety of enzyme inhibitors, such as chelating agent and specific group reagents (Table 2). The
enzyme was completely inhibited by the serine protease inhibitor (PMSF) indicating that the purified
BS1 enzyme is a serine-protease. Thiol reagent (DTNB) at 2 mM showed practically no influence on
the activity of the purified enzyme. Furthermore, EDTA
(5 mM), a metalloprotease inhibitor, inhibited the enzyme by 22.8% (p<0.05). Partial inhibition of
many serine enzymes by chelating agents has been reported [31, 35, 36] indicating the probable
interaction of ions on the stabilization or activation of conformational structure of enzymes.

The effects of different metal ions, at a concentration of 5 mM, on protease activity were
studied at 60 °C and pH 11.0. The results in Table 2 show that Zn2+ did not affect protease activity,
whereas Ca2+, Mn2+, Ba2+, Na+ and Mg2+ increased protease activity by 226, 163, 142, 132 and 123%,
respectively(p<0.05). However, Hg2+ decreased the protease activity by 29%. These results are quite
similar to those reported for alkaline serine-protease from B. licheniformis NH1 [31].

3.7. Effect of NaCl concentration on protease activity

As B. safensis S406, was isolated from a hypersaline biotope, the effect of NaCl concentration
on the protease BS1 activity was checked (Fig. 5). This investigation revealed that the purified
protease BS1 was active over a wide range of salt concentrations. In fact, it showed a great stability
in the presence of high salt concentrations, retaining approximately 100 and 93 % of its initial activity
at sodium chloride concentrations of 55 and 60 % (w/v), respectively (p<0.05). The halostability of
the purified protease BS1 from B. safensis S406 was higher than that observed for Bacillus aquimaris
protease which was stable at NaCl concentrations below 2 M (11.68%) [37].

In addition to being pH and temperature stable, the purified enzyme has functional activity
and stability in the presence of saline conditions, making it effective for application using ground
water with a high salt content [38].

3.8. Effects of surfactants and oxidizing agents on protease stability

The stability of BS1 enzyme as a detergent additive was assessed using different oxidant and
surfactant agents. As shown in Table 3, the alkaline protease BS1 is highly stable in the presence of
nonionic surfactants. These results are consistent with those reported for alkaline protease from
Bacillus clausii [39] and Bacillus subtilis [40]. Interestingly, BS1 protease showed relative stability
against the strong anionic surfactant (SDS) and retained 87.83% and 34;45% in the presence of 0.1%
and 1% SDS, respectively. The stability of the enzyme against SDS was similar to CP-2 protease from
Serratia rubidaea, which retained about 80% of its initial activity after incubation 1-4 h at 20 °C in the
presence of 0.5% SDS [41].

9
 In addition, BS1 protease was little influenced by oxidizing agent, retaining 58.24 % of its initial
activity after incubation for 1 h at 30 °C in the presence of 5% H2O2, and 71% in the presence of 2%
sodium perborate. These are very important characteristics for the eventual use of the enzyme in
detergent formulations.

3.9. Compatibility of BS1 with commercial liquid and solid detergents

The high activity and stability of the purified BS1 enzyme in the pH range from 7.0 to 11.0, and
its relative stability towards oxidizing agents are very useful for its application as a detergent
additive. To check the compatibility and stability of the alkaline protease with washing detergents,
the enzyme was pre-incubated in the presence of various commercial laundry detergents for 1 h at
30, 40, and 50 °C.

The data presented in Fig. 6a show that the alkaline protease BS1 was highly stable in all
tested solid laundry detergents when incubated at 30 and 40 °C retaining 100% of its initial activity.
However, the protease was found to be less stable at 50 °C, retaining between 70% (Dixan) and 90%
(Ariel) of the initial activity. Singh et al. [42] reported that the alkaline protease from Bacillus sp. SSR1
retained 37% of its initial activity after 1 h of incubation at 40 °C in the presence of Ariel detergent at
a concentration of 5 mg/ml.

The data presented in Fig. 6b show that the enzyme was also highly stable in the presence of
liquid detergents, retaining 100% of its activity after 1 h incubation at 30 and 40 °C. The enzyme was,
however, less stable at 50 °C than 30 and 40 °C; although it retained more than 85% of its initial
activity in the presence of all tested detergents. These results are consistent with those reported for
alkaline proteases from B. licheniformis MP1 [43] and B. mojavensis A21 [44] and provide further
support for the usefulness of this purified protease for future industrial application as a cleaning
bioadditive in detergent formulations [3].

3.10. Effects of organic solvents on protease stability

The purified BS1 protease was incubated with various organic solvents 50% (v/v) at 30 °C with
constant shaking. The enzyme activity was found stable in many solvents even after incubation for
120 days (Table 4). The enzyme showed remarkable stability in the presence of cyclohexane, hexane,
decane with log P above 3.0 and DMSO (logP = -1.35). Nevertheless, BS1 protease was comparatively
less stable in the presence of toluene, chloroform, butanol and acetone, while it wasn’t stable in
hydrophilic solvents tested such as methanol (log P = -0.76) and ethanol (log P = -0.24).

Generally, solvents with log P values below 4.0 are considered to be extremely toxic because
their degrees of partitioning into the aqueous layer are high [45, 46]. However, BS1 protease showed
improved stability against a major part of the tested solvents, including hydrophilic solvents.

10
3.11. Chitin extraction
For efficient chitin extraction, associated proteins should be removed as a first stage. Many
reports have demonstrated the application of proteolytic enzymes for the deproteinization of marine
crustacean wastes to produce chitin [47, 48]. Deproteinization tests were conducted for 3 h at 45 °C
and pH 10.5. E/S ratios between 5 and 20 (U/mg of proteins) were assayed to compare the
deproteinization efficiency. As shown in Fig. 7a, the deproteinization rate of shrimp shells without
enzyme was 32%. Indeed, some proteins associated to chitin by electrostatic forces or hydrogen
bonds, could be dissociated by thermal treatment. The other proteins are probably linked to the
chitin by covalent bonds; their removal requires additional chemical or enzymatic treatment [49].
The addition of the purified alkaline protease BS1 improved the deproteinization rate. The DP rate
with E/S = 5 U/mg was 80%, the percentage of protein removal increased with increasing E/S ratio
and reached about 93% with E/S ratio of 10 U/mg. Further increase in enzyme concentration did not
increase significantly the deproteinization rate which reached 95% at E/S ratio of 20 U/mg.

3.12. Chitin characterization by FTIR

The spectra of the extracted chitin display a series of narrow absorption bands, typical of
crystalline polysaccharide samples (Fig. 7b). In the FTIR spectra of BS1 chitin, two separate peaks
were observed at 1662 and 1618 cm−1 and were attributed to the occurrence of the intermolecular
hydrogen bond CO· · ·HN and the intramolecular hydrogen bond CO· · ·HOCH2, respectively. The
characteristic carbonyl C=O stretching of chitin at 1618 cm-1 is attributed to the vibrations of the
amide I band. The sharp bands at 1427 cm-1 and 1551 cm-1 correspond to a symmetrical deformation
of the CH3 group and N-H deformation of amide II, respectively, which is in agreement with the
results described by Al Sagheer et al. [50]. These bands can be seen clearly in the shrimp chitin
spectrum and the commercial chitin. An additional band observed at about 1378 cm−1 was assigned
to C–O stretching vibration region. Another characteristic marker is the CH deformation of the β-
glycosidic bond, was shown at 895 cm−1. A broad absorption band in the range 3437 cm-1 is attributed
to the OH stretching vibrations [51]. The band due to NH of the amide group observed at 3263 cm-
1
, is assigned to the vibrational modes involved in intermolecular hydrogen bond CO…HN groups.
Consequently, the BS1 chitin sample displayed an IR spectrum typical to that of the commercial
chitin.

Conclusion
The purification to homogeneity of alkaline protease BS1 was achieved by precipitation with
ammonium sulfate, gel filtration through Sephadex G-75, anion exchange chromatography and
ultrafiltration. The purified protease was homogenous on SDS-PAGE and its molecular mass was
estimated to be 29 kDa. The enzyme showed an optimum pH of 11.0 and optimum temperature at

11
60 °C. The protease showed stability in the presence of a wide range of organic solvents and
compatibility with a wide range of commercial solid and liquid detergents. Furthermore, BS1
protease was highly effective in the deproteinization of shrimp waste to extract chitin. These results
suggest that the B. safensis BS1 protease could be a potential candidate for several processing
operations, as a laundry detergents additive, or in the chitin extraction from shrimp wastes.

Acknowledgement
This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia.

References
[1] M.B. Rao, A.M. Tanksale, M.S. Ghatge, V.V. Deshpande, Molecular and biotechnological aspects
of microbial proteases, Microbiol Mol Biol Rev. 62 (1998) 597–635.

[2] K.H. Maurer, Detergent proteases, Curr. Opin. Biotechnol. 15 (2004) 330-334.

[3] R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline proteases: molecular approaches and industrial
applications, Appl Microbiol Biotechnol. 59 (2002) 15-32.

[4] N. Erikson, Industrial Enzymology, 2nd ed, The Macmillan Press Ltd, London (1996) pp1-8.

[5] R. Gupta, K. Gupta, R.K. Saxena, S. Khan, Bleach-stable, alkaline protease from Bacillus sp,
Biotechnol. Lett. 21 (1999) 135-138.

[6] C. Mei, X. Jiang, A novel surfactant- and oxidation-stable alkaline protease from Vibrio
metschnikovii DL 33-51, Process Biochem. 40 (2005) 2167-2172.

[7] B.B. Samal, B. Karan, Y. Stabinsky, Stability of two novel serine proteinases in commercial
laundry detergent formulations, Biotechnol. Bioeng. 35 (1990) 650-652.

[8] N. Bhaskar, E.S. Sudeepa, H.N. Rashmi, A.T. Selvi, Partial purification and characterization of
protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its
antibacterial activities, Bioresour. Technol. 98 (2007) 2758–2764.

[9] K. Horikoshi, Enzymes of alkaliphiles, in: E. Ichishima (Ed.), Microbial enzymes and
biotechnology, Elsevier Applied Science, Amsterdam, (1990), pp. 275-94.

[10] M. Jacobs, M. Eliasson, M. Uhlén, J.I. Flock, Cloning, sequencing and expression of subtilisin
Carlsberg from Bacillus licheniformis, Nucleic Acids Res. 13 (1985) 8913-8926.

[11] J.A. Wells, E. Ferrari, D.J. Henner, D.A. Estell, E.Y. Chen, Cloning, sequencing, and secretion of
Bacillus amyloliquefaciens subtillisin in Bacillus subtilis, Nucleic Acids Res. 11 (1983) 7911-7925.

12
[12] O. Ghorbel-Bellaaj, S. Hajji, I. Younes, M. Chaabouni, M. Nasri, K. Jellouli, Optimization of chitin
extraction from shrimp waste with Bacillus pumilus A1 using response surface
methodology, Int. J. Biol. Macromol. 61 (2013) 243-250.

[13] L. Manni, K. Jellouli, O. Ghorbel-Bellaaj, R. Agrebi, A. Haddar, A. Sellami-Kamoun, M. Nasri, An

oxidant- and solvent-stable protease produced by Bacillus cereus SV1: Application in the
deproteinization of shrimp wastes and as a laundry detergent additive, Appl. Biochem.
Biotechnol. 160 (2010) 2308-2321.

[14] M.A. Abdel-Naby, S.A. Ahmed, H.R. Wehaidy, S.A. El-Mahdy, Catalytic, kinetic and
thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease, Int. J.
Biol. Macromol. 96 (2017) 265–271.

[15] C. Lopes, L.T. Antelo, A. Franco-Uría, A.A. Alonso, R. Pérez-Martín, Chitin production from
crustacean biomass: Sustainability assessment of chemical and enzymatic processes, J. Clean.
Prod (2017). (in press)

[16] J.H. Miller, Experiments in Moleculer Genetics, Cold Spring Harbor Laboratory Press, NY (1972)
466.

[17] A. Kembhavi, A. Kulkarni, A. Pant, Salt-tolerant and thermostable alkaline protease from
Bacillus subtilis NCIM No. 64, Appl. Biochem. Biotechnol. 38 (1993) 83-92.

[18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage
T4, Nature, 227 (1970) 680-685.

[19] F.L. Garcia Carreno, L.E. Dimes, N.F. Haard, Substrate-gel electrophoresis for composition and
molecular weight of proteinases or proteinaceous proteinase inhibitors, Anal. Biochem. 214
(1993) 65-69.

[20] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-254.

[21] H. Lineweaver, D. Burk, The Determination of enzyme dissociation constants, J. Am. Chem. Soc.
56 (1934) 658-666.

[22] S.M. Rao, J. Muñoz, F.W. Stevens, Critical factors in chitin production by fermentation of shrimp
biowaste, Appl. Microbiol. Biotechnol. 54 (2000) 808-813.

[23] P. Madhavan, and K.G.R. Nair. Utilization of prawn waste: Isolation of chitin and its conversion to
chitosan. Fish. Technol. 11(1974) 50-53.

13
[24] B. Jaouadi, S. Ellouz-Chaabouni, M. Rhimi, S. Bejar, Biochemical and molecular characterization
of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic
efficiency, Biochimie. 90 (2008) 1291-1305.

[25] Q. Huang, Y. Peng, X. Li, H. Wang, Y. Zhang, Purification and characterization of an extracellular
alkaline serine protease with dehairing function from Bacillus pumilus, Curr. Microbiol. 46
(2003) 0169-0173.

[26] C.A. McPhalen, H.P. Schnebli, M.N.G. James, Crystal and molecular structure of the inhibitor
eglin from leeches in complex with subtilisin Carlsberg, FEBS Letters. 188 (1985) 55-58.

[27] S. Eschenburg, N. Genov, K. Peters, S. Fittkau, S. Stoeva, K.S. Wilson, C. Betzel, Crystal structure
of subtilisin DY, a random mutant of subtilisin Carlsberg, Eur J Biochem. 257 (1998) 309-318.

[28] T. Nakamura, Y. Yamagata, E. Ichishima, Nucleotide sequence of the Subtilisin NAT gene, aprN,
of Bacillus subtilis (natto), Biosci. Biotechnol. Biochem. 56 (1992) 1869-1871.

[29] S.C. Jain, U. Shinde, Y. Li, M. Inouye, H.M. Berman, The crystal structure of an autoprocessed
Ser221Cys-subtilisin E-propeptide complex at 2.0 A resolution1, J. Mol. Biol. 284 (1998) 137-
144.

[30] K. Bouacem, A. Bouanane-Darenfed, H. Laribi-Habchi, M.B. Elhoul, A.d. Hmida-Sayari, H.

Hacene, B. Ollivier, M.L. Fardeau, B. Jaouadi, S. Bejar, Biochemical characterization of a
detergent-stable serine alkaline protease from Caldicoprobacter guelmensis, Int. J. Biol.
Macromol. 81 (2015) 299-307.

[31] N.E. Hadj-Ali, R. Agrebi, B. Ghorbel-Frikha, A. Sellami-Kamoun, S. Kanoun, M. Nasri, Biochemical

and molecular characterization of a detergent stable alkaline serine-protease from a newly
isolated Bacillus licheniformis NH1, Enzyme. Microb. Technol. 40 (2007) 515-523.

[32] K. Cheng, L. Fu-Ping, L. Ming, L. Li-Li, L. Xiao-Mei, Purification and biochemical characterization
of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in
detergent formulations, Afr. J. Biotechnol. 9 (2010) 4942–53.

[33] A. Haddar, R. Agrebi, A. Bougatef, N. Hmidet, A. Sellami-Kamoun, M. Nasri, Two detergent

stable alkaline serine-proteases from Bacillus mojavensis A21: Purification, characterization and
potential application as a laundry detergent additive, Bioresour. Technol. 100 (2009) 3366-
3373.

[34] K. Bhange, V. Chaturvedi, R. Bhatt, Simultaneous production of detergent stable keratinolytic

protease, amylase and biosurfactant by Bacillus subtilis PF1 using agro industrial waste,
Biotechnol. Rep. 10 (2016) 94-104.

14
[35] N. Fakhfakh-Zouari, A. Haddar, N. Hmidet, F. Frikha, M. Nasri, Application of statistical
experimental design for optimization of keratinases production by Bacillus pumilus A1 grown
on chicken feather and some biochemical properties, Process Biochem. 45 (2010) 617-626.

[36] L.D. Kluskens, W.G.B. Voorhorst , R.J. Siezen , R.M. Schwerdtfeger, G. Antranikian, J. van der
Oost, W.M. de Vos, Molecular characterization of fervidolysin, a subtilisin-like serine protease
from the thermophilic bacterium Fervidobacterium pennivorans, Extremophiles. 6 (2002) 185-
194.

[37] P. Shivanand, G. Jayaraman, Production of extracellular protease from halotolerant bacterium,

Bacillus aquimaris strain VITP4 isolated from Kumta coast, Process Biochem. 44 (2009) 1088-
1094.

[38] D. Jain, I. Pancha, S.K. Mishra, A. Shrivastav, S. Mishra, Purification and characterization of
haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: A
potential additive for laundry detergents, Bioresour. Technol. 115 (2012) 228-236.

[39] H.S. Joo, C.G. Kumar, G.C. Park, S.R. Paik, C.S. Chang, Oxidant and SDS-stable alkaline protease
from Bacillus clausii I-52: production and some properties, J. Appl. Microbiol. 95 (2003) 267-
272.

[40] S.K. Rai, A.K. Mukherjee, Statistical optimization of production, purification and industrial
application of a laundry detergent and organic solvent-stable subtilisin-like serine protease
(Alzwiprase) from Bacillus subtilis DM-04, Biochem. Eng. J. 48 (2010) 173-180.

[41] K.K. Doddapaneni, R. Tatineni, R.N. Vellanki, B. Gandu, N.R. Panyala, B. Chakali, L.N.
Mangamoori, Purification and characterization of two novel extra cellular proteases from
Serratia rubidaea, Process Biochem. 42 (2007) 1229-1236.

[42] J. Singh, N. Batra, R.C. Sobti, Serine alkaline protease from a newly isolated Bacillus sp. SSR1,
Process Biochem. 36 (2001) 781-785.

[43] K. Jellouli, O. Ghorbel-Bellaaj, H.B. Ayed, L. Manni, R. Agrebi, M. Nasri, Alkaline-protease from
Bacillus licheniformis MP1: Purification, characterization and potential application as a
detergent additive and for shrimp waste deproteinization, Process Biochem. 46 (2011) 1248-
1256.

[44] A. Haddar, A. Bougatef, R. Agrebi, A. Sellami-Kamoun, M. Nasri, A novel surfactant-stable

alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and
characterization, Process Biochem. 44 (2009) 29-35.

15
[45] V. Dandavate, J. Jinjala, H. Keharia, D. Madamwar, Production, partial purification and
characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its
application for ester synthesis, Bioresour. Technol. 100 (2009) 3374-3381.

[46] R. Rahman, S. Mahamad, A. Salleh, M. Basri, A new organic solvent tolerant protease from
Bacillus pumilus 115b, J. Ind. Microbiol. Biotechnol. 34 (2007) 509-517.

[47] K. Jellouli, A. Bayoudh, L. Manni, R. Agrebi, M. Nasri, Purification, biochemical and molecular
characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp
wastes, Appl. Microbiol. Biotechnol. 79 (2008) 989-999.

[48] H. He, X. Chen, C. Sun, Y. Zhang, P. Gao, Preparation and functional evaluation of oligopeptide-
enriched hydrolysate from shrimp (Acetes chinensis) treated with crude protease from Bacillus
sp. SM98011, Bioresour. Technol. 97 (2006) 385-390.

[49] C. Jeuniaux, J.C. Bussers, M.F. Voss-Foucart, M. Poulicek, Chitin Production by Animals and
Natural Communities in Marine Environment, in: R. Muzzarelli, C. Jeuniaux, G.W. Gooday (Eds.),
Chitin Nat. Technol., Springer US, 1986: pp. 515–522

[50] F.A. Al Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Extraction and characterization of
chitin and chitosan from marine sources in Arabian Gulf, Carbohydr Polym. 77 (2009) 410-419.

[51] F.G. Pearson, R.H. Marchessault, C.Y. Liang, Infrared spectra of crystalline polysaccharides. V.
Chitin, J. Polym. Sci. 43 (1960) 101-116.

Figure Caption

16
Figr-1Fig. 1

Fig. 2
Protease BS1 (B.safensis S406) A Q T V P Y G I P
Q
Subtilisin SAPB (B. pumilus CBS) CAO03040 A Q T V P Y G I P
Q
Subtilisin (B. pumilus) UN-31-C-42 A Q T V P Y G I P
Q
Subtilisin Carlsberg (B.subtilis) 1CSE_E GI:229772 A Q T V P Y G I P
L
Subtilisin DY (B. licheniformis) 1BH6_A GI:3891521 A Q T V P Y G I P
L
Subtilisin NAT (B.subtilis. Natto) 4DWW_A GI:380259250 A Q S V P Y G I S
Subtilisin E (B. subtilus) 3WHI_A GI:568786416 Q
Subtilisin BPN’ (B.amyloliquefaciens) 1SPB_S GI:1311236 A Q S V P Y G I S
Selenosubtilisin Bpn (B.amyloliquefaciens) 1UBN_A GI:5542495 Q
A Q S V P Y G V S
Q
X Q S V P Y G V S

Fig. 3
(a)

(b)
Relative protease activity (%)

Fig. 4
(a)

(b)
Fig.5
Fig. 5
Fig.6
(a) pH

(b)
Fig.7
(a)

17
 (b)

Fig. 1. SDS–PAGE and zymography of the purified protease from B. safensis S406. Lane 1:

standard protein markers; lane 2: purified protease; lane 3:zymography of the cell free

supernatant, lane 4: zymography of the purified protease.

Fig. 2. Alignment of the N-terminal amino acid sequence of the purified alkaline protease BS1

from B. safensis S406 with the sequences of other proteases. Residues not identical with BS1

protease are shaded. BS1 B. safensis S406 protease (present study); Subtilisin SAPB Subtilisin

(B. pumilus CBS) (Jaouadi et al., 2008); Carlsberg (B.subtilis 1CSE-E) (Phalen et al., 1985);

Subtilisin DY (B. licheniformis 1402) (Eschenburg et al., 1998); Subtilisin NAT (B. subtilis

(natto)) (Nakamura et al., 1992); Subtilisin E (B. subtilis 1SCJ-A) (Jain et al., 1998); Subtilisin

BPN’ (B. amyloliquefaciens) (Wells et al., 1983); Selenosubtilisin Bpn ( B. amyloliquefaciens

1UBN-A) (Dinakarpandian et al., 1999).

Fig. 3. Effect of pH on the activity (a) and stability (b) of the purified S406 protease. Protease

activity was evaluated in the pH range of 6.0–13.0 using buffers of different pH values at 60

°C. The maximum activity obtained at pH 11.0 was considered as 100%. The pH stability of

the purified enzyme was determined by incubating enzyme in different buffers for 1 h at

40 °C and the residual activity was measured at pH 11.0 and 60 °C. The activity of enzyme

before incubation was taken as 100%. Buffer solutions used for pH activity and stability are

presented in Section 2. Values are means of three independent experiments, deviations were ±

SD.

Fig. 4. Effect of temperature on the activity (a) and thermal stability (b) of the purified protease.

The temperature profile was determined by assaying protease activity at temperatures between

40 and 100 °C in the absence and presence of CaCl2 (5 mM). The activity of the purified enzyme

at pH 11.0 60 °C in the absence of CaCl2 was taken as 100%.

18
The temperature stability was determined by incubating the purified enzyme at different

temperatures for 48 h at pH 11.0, in the absence and presence of CaCl2 (5 mM). The residual

enzyme activity was estimated at regular intervals under standard conditions. The non-heated

enzyme was taken as 100%.

Fig. 5. Effect of salinity on the activity and stability of the purified enzyme. For protease

activity, the enzyme was assayed in the presence of NaCl (0-60 %). For enzyme stability, it was

preincubated for 1 h at different NaCl concentrations, and the residual activity was measured

under standard assay conditions. The enzyme activity determined without NaCl was taken as a

control.

Fig. 6. Stability of the purified alkaline protease in some solid (a) and liquid (b) laundry

detergents. The purified protease was incubated 1 h at 40 °C in tap water containing 7 mg/ml

of solid detergents, and 1% (v/v) of various liquid laundry detergents. The remaining activity

were determined at pH 11.0 at 60 °C, using casein as a substrate. Enzyme activity of control

sample pre-incubated in the same conditions without any detergent was taken as 100%.

Fig. 7. Effects of increasing enzyme concentration on the deproteinization of shrimp shell

waste. Protein hydrolysis was performed for 3 h at 45 °C (a). FTIR spectra of commercial and

BS1 chitins (b).

19
Table 1: Summary of the purification of BS1 protease from B. safensis S406
Total Specific
Total activity Recovery Purification
Purification steps protein activity (U/mg
(U) (%) (fold)
(mg) protein)
Culture
137050 43.8 3128.99 100 1
supernatant
(NH4)2SO4
precipitation 111695.75 11.88 9401.99 81.5 3.0
(20%-60%)
Sephadex G-75 59975 4.1 14628.04 43.76 4.67

Mono-Q 48672 1.9 25616.84 35.51 8.18

Ultrafiltration 27821 0.7 39744.28 20.29 12.70

All operations were carried out at 4 °C.

Table 2: Effects of enzyme inhibitors and various metal ions on S406 protease activity.

Inhibitors /metallic ions Concentration (mM) Relative activity (%)

None 0 100 ± 1.74

PMSF 2 77.2 ± 2.15

20
 5 0 ± 0.00

EDTA 2 93 ± 1.54

5 74 ± 1.75

DTNB 2 100 ± 2.03

β-Mercaptoethanol 2 100 ± 2.15

Cu2+ 5 97 ± 1.71

Hg2+ 5 71 ± 2.33

Na+ 5 132 ± 2.05

Mn2+ 5 163 ± 2.52

K+ 5 104 ± 3.26

Ba2+ 5 142 ± 4.02

Zn2+ 5 103 ± 1.63

Ca2+ 5 226 ± 1.48

Mg2+ 5 123 ± 2.10

The purified enzyme was preincubated with various enzyme inhibitors for 30 min at 30 °C, and the
remaining activity was determined at pH 11.0 and 60 °C. Enzyme activity measured in the absence
of any inhibitor was taken as 100%. The effects of metal ions on the activity of the protease
were determined by incubating the enzyme in the presence of various metal ions for 15 min at
60 °C and pH 11.0.

Table 3: Stability of the purified alkaline protease in the presence of various surfactants and

oxidizing agents.

Surfactant/oxidizing agents Concentration (%) Residual activity (%)

None 0 100 ± 1.65

SDS (w/v) 0.1 87.83 ± 3.21

21
 0.5 51.01 ± 1.26

1 34.45 ± 2.04

Triton X-100 (v/v) 1 123 ± 2.67

5 109 ± 2.48

Tween 80 (v/v) 1 121 ± 2.31

5 102.5 ± 1.33

Tween 20 (v/v) 1 138 ± 2.52

5 115 ± 2.73

H2O2 (v/v) 1 97.31 ± 2.17

5 58.24 ± 3.14

Sodium perborate (w/v) 1 99 ± 1.09

2 71 ± 2.62

The enzyme was incubated with different surfactants and oxidizing agents for 1 h at 30 °C and the
remaining activity was measured under standard conditions.

Table 4: Stability of the purified alkaline protease in organic solvents.

Organic solvent log P Residual activity (%)

None ─ 100

DMSO -1.35 91.86 ± 0.67

22
Methanol - 0.76 10.23 ± 2.17

Ethanol - 0.24 7.06 ± 1.69

Acetone - 0.23 35.67 ± 1.54

Acetonitrile - 0.15 18.38 ± 1.95

Butanol 0.8 46.74 ± 2.45

Chloroform 2 63.29 ± 1.56

Toluene 2.5 72.05 ± 1.70

Cyclohexane 3.2 100.46 ± 1.65

Hexane 3.5 98.40 ± 2.07

Decane 5.6 100.21± 1.03

23