International Journal of Biological Macromolecules 56 (2013) 20–27

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Characteristics and thermodynamics of a thermostable protease from

a salt-tolerant alkaliphilic actinomycete
S.D. Gohel, S.P. Singh ∗
Department of Biosciences, Saurashtra University, Rajkot 360 005, Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history: An alkaline serine protease from a newly isolated salt-tolerant alkaliphilic actinomycetes, Brachys-
Received 8 November 2012 treptospora xinjiangensis OM-6 was purified with 35- and 26-fold purification and 47% and 22% yield
Received in revised form 23 January 2013 employing two steps and one step methods, respectively. The enzyme was quite stable at 80 ◦ C in 30%
Accepted 24 January 2013
Na-glutamate with the deactivation rate constant (Kd ) 8.66 and half life (t1/2 ) 80.04 min. The activation
Available online 5 February 2013
energies (E), enthalpy (H*), entropy (S*) and change in free energy (G*) for the protease deactiva-
tion were calculated in the presence of 30% Na-glutamate and correlated with the enzyme stability.
Keywords:
The thermodynamic analysis corresponded the trends of the enzyme stability and inactivation. The
Salt-tolerant alkaliphilic actinomycetes
Thermostable alkaline protease
enzyme retained high activity and significant stability at higher salt, temperature, range of pH and metal
Single step purification ions. The enzyme was extremely resistant against urea denaturation, oxidizing and reducing agents and
Enzyme kinetics surfactants, a finding which is rather unique and restricted to only few proteins.
Thermodynamics © 2013 Elsevier B.V. All rights reserved.

1. Introduction Most of the halophilic proteases belong to serine protease fam-

Proteases are established in enzyme catalysis and have been
the subject of numerous texts and reviews. The majorities of
halophilic proteases, however, have been studied from halophilic
archaea and bacteria [1–5], while representation from haloalka-
liphilic actinomycetes in this context is quite restricted [6–11].
Despite their advantages, the application of halophilic proteases
is further constrained because of their limited stability under
extremes of temperature, pH and ionic strength. The available array
is still not sufficient to meet the ever increasing demand for suitable
proteases. Halophilic proteases have several applications in food,
leather, detergent and antifouling coating industries as well as pep-
tide synthesis in organic media. Haloarchaeal proteases catalyze
the reaction at 4–5 M NaCl, losing activity rapidly when exposed
to low salt concentrations [12]. This property of enzyme severely
restricts the choice of purification methods, making the majority of
the conventional purification procedures unsuitable. The methods
used for purification of haloarchaeal proteases include concentrat-
ing the enzyme by ethanol precipitation or ultra filtration followed
by affinity chromatography and gel filtration. However, many steps
make the method more cumbersome and hence, single step purifi-
cation by hydrophobic interaction chromatography is a better
choice [13–15].
∗ Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419.
E-mail address: satyapsingh@yahoo.com (S.P. Singh).
ily, dependent on high salt concentrations for structural stability
and display optimum activity at high salt, neutral to basic pH and
temperatures 37–50 ◦ C. However, literatures describing halophilic
proteases with alkaliphilic and thermophilic properties are quite
limited, especially from haloalkaliphilic actinomycetes [16]. The
activity and stability of enzymes are important parameters which
codetermine the economic feasibility of applying protease in indus-
trial processes. High stability is generally considered an economic
advantage because of reduced enzyme turnover [17]. A number of
strategies are applied to increase the stability of protease enzymes
such as mutation, chemical modification at active site, introduc-
tion of disulfide bridges, the optimization of helices and helix
caps, immobilization, entropic stabilization, changing pH condi-
tion and using various salts. Studies on the thermodynamic stability
of enzymes have provided insight into the factors that determine
enzyme stability [17]. However, thermodynamic properties of puri-
fied proteases, particularly from actinomycetes, are not described.
Thus, the present investigation addresses upon the thermodynamic
approaches (deactivation kinetics, H*, S*, E and G*) to under-
stand the behavior of these enzymes at different temperatures and
salts [18].
Halophilic eubacteria accumulate organic compatible solutes
such as sucrose, mannitol, trehalose, glycerol, betaine, proline, glu-
tamate, etc. to maintain the protein structure. Vidyasagar et al. [19]
studied an extracellular protease from Halogeometricum brorin-
quense strain TSS101 in the presence of compatible solutes; sucrose,
mannitol, glycerol and betaine in absence of NaCl. However, no fur-
ther information is available on the stability of this enzyme in these

0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2013.01.028
 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27 21

solutes. Kinetic studies on the behavior of haloalkaliphilic pro- were collected by BIO-RAD fraction collector (BIO-RAD, California,
teases from actinomycetes in NaCl and compatible solutes would USA) and analyzed for protease activity. The purity of the enzyme
provide insight into their unique properties. The present investiga- was judged on sodium dodecyl sulphate polyacrylamide gel elec-
tion focused on the single step purification in conjunction with the trophoresis (SDS-PAGE) [22]. The active fractions were pooled and
elucidation of the biochemical, thermodynamic and kinetic proper- used for further characterization.
ties of extracellular protease. The characterization of the protease
under different conditions of salt, pH, denaturants, osmolytes and 2.4. Effect of NaCl and Na-glutamate on temperature optima,
detergents from a salt-tolerant alkaliphlic actinomycete, Brachys- protease activity and stability
treptospora xinjiangensis OM-6 was under taken. The findings hold
significance in developing novel enzymes for potential industrial The effect of salt on alkaline protease activity and stability was
applications and understanding their behavior. assessed. The reaction mixtures (pH 10.0) were separately supple-
mented with 0–4 M NaCl and 30% Na-glutamate and incubated for
2. Materials and methods 10 min at 37 ◦ C–90 ◦ C. The thermal stability of the purified enzyme
was studied by incubating the enzyme with 20 mM Borex–NaOH
2.1. Strain, media and cultivation and identification buffer (pH 10.0) containing 0–4 M NaCl and 30% Na-glutamate at
37 ◦ C–80 ◦ C for 5 h. The residual activities were expressed as % of
A halo tolerant and alkaliphilic actinomycete strain OM-6 was the initial activity.
isolated by enrichment culture and standard serial dilution and
plating methods from the Okha Madhi along the Gujarat Coast, 2.5. Estimation of deactivation rate constant
India. The media used for the screening and cultivation of OM-6
consisted of 0.5% (w/v) gelatin, 0.5% (w/v) peptone, 0.5% (w/v) yeast The deactivation rate of alkaline protease was calculated by first
extract and 5% (w/v) NaCl. The pH of the medium was adjusted to order expression:
9.0 with 20% (w/v) Na2 CO3 . The organisms were cultivated aero-
dE
bically at 37 ◦ C under shake flask conditions at 120 rpm. For 16S = −KdE (1)
dt
rRNA gene sequencing, the genomic DNA of OM-6 was subjected to
PCR with consensus universal primers for 16S rRNA amplification. So that
The PCR product was sequenced by using pair of forward, reverse
E 
t
ln = −Kdt (2)
and internal primers. Sequence data was aligned and analyzed for E0
closest homologous actinomycetes using Mega 3.1 using Neighbor
The Kd (deactivation rate constant or first order rate constant)
Joining method.
values were calculated from a plot of ln[Et /E0 ] vs. t at a particular
temperature and apparent half lives were estimated using Eq. (3).
2.2. Protease assay
2
t½ = ln (3)
Alkaline protease activity was measured by modified Anson- Kd
Hagihara’s method [20]. The enzyme (0.5 ml) was added to 3.0 ml
hammarsten casein (0.6%, w/v in 20 mM Borex–NaOH buffer, pH 2.6. Estimation of thermodynamic parameters for protease
10.0) and the reaction mixture was incubated at 60 ◦ C for 10 min. deactivation
The reaction was terminated by the addition of 3.2 ml of TCA mix-
ture (0.11 M trichloro acetic acid, 0.22 M sodium acetate, and 0.33 M In order to obtain energies and entropies of protease deactiva-
acetic acid) and incubated at room temperature for 20 min. The tion, absolute rates of reaction theory were used [23] where the rate
precipitates were removed by filtration through Whatman-1 filter of any reaction at a given temperature depends only on the concen-
paper and absorbance of the filtrate was measured at 280 nm. One tration of an energy rich activated complex. Thermodynamic data
unit was defined as the amount of enzyme liberating 1 ␮g of tyro- were calculated by rearranging the Eyring absolute rate equation
sine per minute at 60 ◦ C under the standard assay conditions. The [24].The Eyring absolute rate equation is
protein content was measured according to the method of Brad-  K T   S∗   −H ∗ 
b
ford [21], using bovine serum albumin (10 ␮g/ml) as a standard Kd = .e .e (4)
h R RT
(4–20 ␮g/ml).
where h (Plank constant) = 6.63 × 10−34 J s, R (Gas con-
2.3. Purification of the protease from streptomycete strain OM-6 stant) = 8.314 J/K mol, H* (change in enthalpy), S* (change
in entropy) and Kb (Boltzman constant [R/N]) = 1.38 × 10−23 J/K
Purification was achieved by two-step and a single step purifica- where N (Avogadros no.) = 6.02 × 10–23 mol-1 .To calculate H*
tion method using hydrophobic interaction chromatography (HIC) and S* the Eyring absolute rate equation is rearranged to give
on a phenyl sepharose 6 fast flow. The HIC column (1 cm × 6.5 cm) K   H ∗   1    K  S ∗

d b
was equilibrated with 0.1 M sodium phosphate buffer (pH 8.0) con- ln =− + ln + (5)
T R T h R
taining 1 M ammonium sulfate. The crude protease preparation
[20 ml crude containing 1 M (NH4 )2 SO4 ] was loaded onto column H* and S* values were calculated from the slope and intercept
in case of single step purification, while partially purified protease of a ln [Kd /T] vs. 1/T plot respectively.So that
[1 ml of 70% (NH4 )2 SO4 saturated enzyme sample] was loaded onto
H ∗ = −(slope)R (6)
the column in two step purification. Since in single-step purifica-   K 
tion, we directly load crude enzyme preparation onto to the HIC S ∗ = R intercept − ln b
(7)
column, we refer it as single step purification. While in two-step h
purification approach, the first step of the purification was ammo- Free energy change (G) for inactivation of protease was calcu-
nium sulfate fractionation followed by the application of the HIC lated by using the following relationship.
column. The bound enzyme was eluted by 0.1 M sodium phos-   K ∗ T 
phate buffer, pH 8.0 containing a decreasing gradient of ammonium G∗ = −RT ln d
(8)
sulfate; 1000–100 mM. Fractions at a flow rate of 0.8 ml min−1 Kb ∗ h
22 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27

Energy of deactivation was estimated using the Arrhenius equation,

 E

Kd = Ae − (9)
RT

So that

E
ln[Kd ] = − + lnA (10)
RT

Energy involved in this process was calculated from the slope of

a linear plot of ln[Kd ] vs. 1/T [18]. Thermalstability of protease in the
presence of different salts and pH was determined by incubating the
enzyme in the presence of each salt in sealed tubes at 37 ◦ C, 50 ◦ C,
60 ◦ C, 70 ◦ C and 80 ◦ C. Enzyme activity was measured before and
after incubation at respective temperature to determine residual
activity under each condition. All experiments were conducted in
triplicate, results shown are mean values.

2.7. Substrate specificity and determination of Km and Vmax

Protease activity with various protein substrates including BSA,

casein, egg albumin and gelatin (0.6%, w/v) was assayed at 60 ◦ C
under standard assay condition. The specific protease activity
toward casein was taken as a control. Km and Vmax values of the
pure enzyme were determined by measuring the activity with vari- Fig. 1. SDS-PAGE of the purified protease using 12% cross linked polyacrylamide
gel stained with coomassie brilliant blue. Lane 1: protein marker; Lanes 2 and 3:
ous concentrations of casein substrate (0.025–1.0 g/100 ml). Kinetic
electrophoretic separation of purified protease by two step purification; Lane 4: elec-
constants were calculated from Lineweaver–Burke plot. trophoretic separation of purified protease by one step purification method using
hydrophobic interaction chromatography; Lane 5: partially purified protease.

2.8. Effect of pH on protease activity and stability

The effect of pH on alkaline protease activity was deter-
mined by assaying the enzyme activity at different pH values
from 6.0 to 13.0 using buffers: sodium phosphate (pH 6.0–7.0),
Tris–HCl (pH 8.0–9.0), glycine–NaOH (pH 9.0–11.0), Borex–NaOH
(pH 10.0–12.0) and KCl–NaOH (pH 12.0–13.0) at 20 mM concen-
trations and remaining activity was evaluated at Borex–NaOH pH
10.0. Similarly, for enzyme stability, the enzyme was inoculated
with respective buffers at 40 ◦ C and activity was measured after
1 h, 6 h and 24 h of incubation at Borex–NaOH pH 10.0. The residual
activities were measured by considering the initial activity as 100%.
3. Results and discussion
The actinomycete, OM-6, isolated from coastal region of Gujarat
(India), was gram positive with filamentous structure. Based
on nucleotides homology and phylogenetic analysis, OM-6 was
related to B. xinjiangensis (GenBank accession number: AF251709).
The nearest homolog genus–species was Nocardiopsis kunsanen-
sis (accession no. AB368716). The GenBank accession number for
OM-6 isolate was EU710555.1.
3.1. Enzyme purification

The results of the protease purification are summarized in

2.9. Effect of denaturing agents on protease activity and stability
The effect of chemical denaturants; urea and guanidine
hydrochloride (GH) on the stability of purified protease was studied
at 0–8 M. The enzyme was incubated with urea and GH at different
temperatures for 5 h followed by the measurement of the residual
activities to find out loss in enzyme activity.
2.10. Effect of osmolytes, inhibitors, metal ions, oxidizing agents,
reducing agents and surfactants
The reaction mixture was incubated with various osmolytes
(5–30%): NaCl, KCl, glycerol, mannitol, trehalose, sucrose and
Na-glutamate; metal ions (5 mM); Ca2+ , Cd2+ , Co2+ , Cu2+ , Fe2+ ,
Sr2+ , Ni2+ , Ba2+ , Hg2+ ; different inhibitors (1 mM, 5 mM and
10 mM): phenyl methyl sulfonyl fluoride (PMSF), dihiortitol (DTT),
ethylene diamine tetraacetic acid (EDTA), thiourea (TU), and p-
chloromercuribenzoic (p-CMB); oxidizing and reducing agents
(0–50 mM): H2 O2 and ␤-mercaptoethanol; surfactants (0.5%):
sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide
(CTAB), Tween 80 and Triton X-100; the effect was assessed by
considering the control (without effectors) as 100%.
Table 1. The two-step purification method yielded 35-fold purifica-
tion with a yield of 47% and specific activity of 22938 U/mg protein.
On the other hand, single-step purification method resulted in
26-fold purification with 22.30% yield and specific activity at
18,590 U/mg protein. The results are quite encouraging in compar-
ison to an alkaline protease from Thermoactinomycetes, in which
only limited purification was achieved by DEAE SepharoseCL-6B
and Toyopearl 650 column chromatography [15]. SDS PAGE analy-
sis revealed a single band with a molecular mass of 25 kDa (Fig. 1).
The value was lower than other halophilic alkaline proteases where
molecular weight ranged from 40 to 130 kDa [14,16,25,26]. How-
ever, there are some reports in the literature on lower molecular
weight corresponding to our values [27].
3.2. Effect of NaCl and Na-glutamate on temperature optima and
protease activity
The effect of temperature on caseinolytic activity of the puri-
fied enzyme was assessed in presence of 0–2 M NaCl and 30%
Na-glutamate at pH 10.0. Interestingly, temperature optima shifted
from 60 ◦ C to 70 ◦ C in the presence of 4 M NaCl and 30% Na-
glutamate and % maximum activity increased more than two fold.
 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27 23

Table 1
Purification of OM-6 alkaline protease with salt precipitation and hydrophobic interaction chromatography (using both one and two step purification methods).

Purification steps Total activity (U) Total protein (mg) Specific activity Purification fold Yield (%)

Crude 307,314 470.53 653.12 1 100

70% NH4 SO4 247,802.4 24.64 10,056.91 15.39 80.63
Two step purification (phenyl sepharose 6FF) 144,510.75 6.3 22,938.21 35.12 47.02
Crude 6829.2 9.62 709.89 1 100
One step purification (phenyl sepharose 6FF) 1524 0.082 18,589.87 26.18 22.32

Table 2
Deactivation rate constant (Kd ) and half life (t1/2 ) of purified protease in presence of 0 M NaCl, 2 M NaCl, 4 M NaCl and 30% Na-glutamate at the range of 37 ◦ C–80 ◦ C temperature.

T (◦ C) 0 M NaCl 2 M NaCl 4 M NaCl 30% Na-glutamate

Kd (×10−3 ) t1/2 (min) Kd (×10−3 ) t1/2 (min) Kd (×10−3 ) t1/2 (min) Kd (×10−3 ) t1/2 (min)

37 1.24 558.98 0.73 949.51 0.51 1359.11 0.42 1650.35

50 4.94 140.31 2.52 274.62 0.91 761.70 0.73 949.51
60 27.51 25.19 10.92 63.47 1.68 412.58 1.14 608.02
70 35.86 19.32 27.29 25.39 10.75 64.47 2.41 287.61
80 41.09 16.86 33.30 20.81 28.50 28.50 8.66 80.04

These trends revealed halophilic and thermophilic nature of the
extracellular protease from B. xinjiangensis strain OM-6. NaCl and
Na-glutamate dependence of the enzyme for its activity at higher
temperatures was well evident. The temperature optimum of the
purified protease was 70 ◦ C at higher salt concentration (Fig. 2), a
trend comparable to alkaline proteases from Bacillus subtilis NCIM
2713 [28] and Streptomyces clavuligerus [16]. The optimum tem-
perature for OM-6 protease was nearly 2-fold higher than those
recently reported from haloalkaliphilic bacteria [14,29]. In addi-
tion to the temperature profile of the activity, the enzyme had
greater stability at higher temperatures, a feature quite desirable
for biotechnological applications.
3.3. Thermal stability and deactivation rate constants for protease
Deactivation rates and half-lives of purified protease were cal-
culated at 37 ◦ C–80 ◦ C in presence of 0 M, 2 M and 4 M NaCl and
30% Na-glutamate (Fig. 3). The deactivation rate constant (Kd )
increased and half-life (t1/2 ) decreased with increasing tempera-
tures (Table 2). Highest stability of the enzyme was observed in
Na-glutamate followed by 4 M NaCl, 2 M NaCl and 0 M NaCl. The
enzyme was least stable in the absence of NaCl. The trend was
also apparent from the Kd and t1/2 values of protease at 60 ◦ C in
presence of 30% Na-glutamate (Kd = 1.14 × 10−3 ; t1/2 = 608.02), 4 M
NaCl (Kd = 1.68 × 10−3 ; t1/2 = 412.58), 2 M NaCl (Kd = 10.92 × 10−3 ;
t1/2 = 63.47) and 0 M NaCl (Kd = 27.51 × 10−3 ; t1/2 = 25.19). The
enzyme was stable even at 80 ◦ C in 30% Na-glutamate (Kd = 8.66
and t1/2 = 80.04 min). The results revealed the role of Na-glutamate
in stabilizing the enzyme at higher temperatures with significant
800
% Maximal activity
activities at higher temperatures. Further, the reduced NaCl from
4 to 2 M led to the decrease in the stability of the enzyme at all
examined temperatures, suggesting the significant role of salt in
the conformational stability. The OM-6 protease exhibited high-
est stability in 30% Na-glutamate followed by 4 M NaCl. Enzyme
had minimum stability in NaCl up to 2 M, as evidenced from
the apparent high deactivation rate constants (Kd ) and low t1/2
values. The results indicated that the effect of salt on enzyme sta-
bility was related not only to the salt concentration. The higher
stability of enzyme at 30% Na-glutamate and 4 M NaCl may be
due to decrease in unfavorable electrostatic repulsion. In addi-
tion, halophilic enzymes have highly negative surface charge with
hydrated carboxyl groups which are shielded by high salt concen-
tration. This avoids unfolding and maintains the solubility of these
proteins [30,31].
3.4. Thermodynamic parameters for protease deactivation
The thermal denaturation of enzymes is accompanied by the
disruption of non-covalent linkages, including hydrophobic inter-
actions, with concomitant increase in the enthalpy of activation
[32,33]. The opening up of the enzyme structure is accompa-
nied by an increase in the disorder, randomness or entropy of
activation [34]. Therefore, the thermodynamic properties of the
enzyme are necessary to understand its behavior under differ-
ent physiological conditions [18]. We studied thermodynamics
to understand the behavior of extracellular alkaline protease
from B. xinjiangensis OM-6 at various temperatures and salts
(Table 3). The activation energies (E) for protease deactivation
calculated in presence of Na-glutamate, 4 M NaCl, 2 M NaCl and
0 M NaCl were 37.14 kJ/mole, 44.01 kJ/mole, 99.57 kJ/mole and
113.92 kJ/mole respectively. While change in enthalpy (H*) and

700 entropy (S*) for deactivation of protease in presence of 30% Na-

600 glutamate, 4 M NaCl, 2 M NaCl and 0 M NaCl were 34.47 kJ/mole,
−196.37 J/mole; 41.34 kJ/mole, −175.17 J/mole; 96.91 kJ/mole,
500
6.48 J/mole; 111.26 kJ/mole, 57.03 J/mole respectively. The change
400
300
Table 3
200 Values of H*, S* and activation energy for protease deactivation in presence of
100 0 M NaCl, 2 M NaCl, 4 M NaCl and 30% Na-glutamate at the range of 37 ◦ C–60 ◦ C
temperature.
0
37 50 60 70 80 Treatment H* (kJ/mole) S* (J/mole) E (kJ/mole)

Temp (OC) 0 M NaCl 111.26 57.03 113.92

2 M NaCl 96.91 6.48 99.57
4 M NaCl 41.34 −175.17 44.01
Fig. 2. Effect of 0 M NaCl (), 2 M NaCl (), 4 M NaCl () and 30% Na-glutamate (•)
30% Na-glutamate 34.47 −196.37 37.14
on temperature optima and enzyme activity of purified protease.
24 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27

Fig. 3. Thermal stability of the purified protease in the presence of 0 M NaCl (A), 2 M NaCl (B), 4 M NaCl (C) and 30% Na-glutamate (D) at 37 ◦ C (), 50 ◦ C (), 60 ◦ C (), 70 ◦ C
(•) and 80 ◦ C (*) temperature.

in free energy (G*) for protease deactivation at 60 ◦ C temperature
in presence of 30% Na-glutamate, 4 M NaCl, 2 M NaCl and 0 M NaCl
was 101.62 kJ/mole, 99.54 kJ/mole, 94.36 kJ/mole, 91.80 kJ/mole
respectively (Table 4). Low values of H* and S* in presence
Na-glutamate followed by 4 M NaCl revealed a higher thermal sta-
bility of protease in presence of Na-glutamate. Moreover, negative
values of S* designate ordered transition state of protease in pres-
ence of Na-glutamate followed by NaCl. Similar results have been
observed for the protease from Halobacterium sp. SP(1) [30]; chiti-
nase of Pantoea dispersa [18]; ascorbate oxidase of Cucubita maxima
[35], amylase of Bacillus lichiniformis [36]. The results indicated
that high salt/solute concentration resulted in a stable conforma-
tion of the protease leading to decreased entropy of unfolding
which favored the thermodynamic stability. Furthermore, increase
in G* revealed that the thermal stabilization of protease was due
to the higher free energy (functional energy) which enabled the
enzyme to resist against unfolding of its transition state [18]. Javed
et al. [37] had reported thermal stabilization of endoglucanase
from Aspergillus oryzae cmc-1 due to high free energy. Indeed, the
lowest G* value for the heat labile enzyme corresponds to the
largest H* and S* contributions and conversely the high G*
corresponds to the low H* and S* for heat stable enzyme [38].
Interestingly, proteases from actinomycetes are not characterized
thermodynamically so far. In addition, enormous stability of the
purified protease at higher temperatures in the presence of salt,
Table 4
Values of G* for deactivation of protease in presence of 0 M NaCl, 2 M NaCl, 4 M
NaCl and 30% Na-glutamate at the range of 37 ◦ C–80 ◦ C temperature.
T (◦ C) G* (kJ/mole) for deactivation of protease
0 M NaCl 2 M NaCl 4 M NaCl 30% Na-glutamate
revealed by thermodynamic analysis, hold significance for under-
standing the properties of the enzyme.
3.5. Hydrolysis of protein substrate and determination of Km and
Vmax
When assayed with native proteins as substrates, the protease
showed a high level of hydrolytic activity against casein (100%)
and poor to moderate hydrolysis of BSA (66.66%), gelatin (58.09%),
and egg albumin (15.23%). The kinetic parameters (Km and Vmax )
of extracellular protease for hydrolysis of casein at 60 ◦ C and pH
10.0 were determined by double reciprocal Lineweaver–Burk plot.
Values for Km and Vmax were 0.18 mg/ml and 2414.22 U/ml respec-
tively. The estimated Km values indicated the affinity of enzyme
toward the substrate. Vmax is an indication of the catalytic activity
of an enzyme which is usually desired to be as high as possible.
3.6. Effect of pH on protease activity and stability
The effect of pH on caseinolytic activity of the purified enzyme
was determined in the range of 6.0–13.0 pH using different
buffer systems. Protease exhibited highest activity in the pH
range 10.0–11.0 (Fig. 4A) with Borex–NaOH buffer system like-
wise maximum stability of enzyme was found at the same range
of pH reflecting alkaliphilic nature of the extracellular protease
from B. xinjiangensis strain OM-6. The pH findings are in accor-
dance with several recent reports showing pH optima of 10.0–11.0
[14,16,29,40]. The OM-6 alkaline protease was stable at broad range
of pH from 6.0 to 12.0 after 6 h while stability was found in the range
of 10.0–11.0 pH even after prolong incubation (Fig. 4B) reflecting
alkaliphilic nature of the enzyme.

37 93.27 94.63 95.56 96.06

50 93.58 94.38 98.12 98.71 3.7. Effect of denaturing agents on protease activity and stability
60 91.80 94.36 99.54 101.62
70 93.89 94.67 97.32 101.59 Purified protease was subjected to denaturation at 2 M, 4 M
80 96.31 96.93 99.85 100.88
and 8 M urea and guanidine hydrochloride (GH) (Fig. 5A and B ).
 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27 25

Fig. 4. pH optimum (A) of the purified alkaline protease. The pH stability (B) of the purified alkaline protease after 1 h (), 6 h () and 24 h () of incubation. The buffers used
were sodium phosphate (pH 6.0–8.0), Tris–HCl (pH 8.0–9.0), glycine–NaOH (pH 9.0–11.0), Borex–NaOH (pH 10.0–12.0) and KCl–NaOH (pH 12.0–13.0).

Purified enzyme found resistant to 8 M urea and GH and retain
almost 50% of original activity even after 1 h of incubation at 60 ◦ C.
While at 2 M and 4 M concentration enzyme retain more than 90%
of initial activity in presence of urea. The resistance of OM-6 pro-
tease toward urea denaturation was in contrast with one of the
previous studies from our own laboratory, where an alkaline pro-
tease from a haloalkaliphilic Bacillus sp. was highly sensitive to urea
denaturation [29]. The phenomenon of extreme resistance against
chemical denaturation appears to be quite rare among the alkaline
proteases reported from extremophilic actinomycetes [39].
Urea and guanidine hydrochloride denaturation curves are gen-
erally used to obtain an estimate of the conformational stability
of proteins by measuring the differences in conformational sta-
bilities between the native (folded) and the denatured (unfolded)
states. Interactions of urea with polar solutes enhance hydrogen
bond formation between the peptide amide units and urea than
with water because urea itself is a soluble amide [41]. Urea will
adhere on the surface of charged residues leading to the repul-
sion between residues that will induce denaturation and provoke
the unfolding. This explains the need of high urea concentration to
achieve denaturation [42,43].
3.8. Effect of osmolytes, inhibitors, metal ions, oxidizing agents,
reducing agents and surfactants
The effect of different concentrations of various osmolytes NaCl,
KCl, glycerol, mannitol, trehalsoe, sucrose and Na-glutamate on
the activity of purified enzyme was examined (Fig. 6A). Halophiles
respond to increase in osmotic pressure in different ways. The
accumulation of the compatible organic solutes such as sucrose,
mannitol, glycerol, trehalose, betaine, glutamate and proline by
the halophilic bacteria helps to maintain an internal environment
isotonic with the growth medium. These substances also help to
protect cells and enzymes against stress due to high tempera-
ture, desiccation and freezing. To date these compatible solutes are
known to serve as significant osmolytes among halophilic bacteria.
As a second alternate, halophilic organisms use KCl as the internal
inorganic compatible solute with their enzymes being stable only in
the presence of high KCl or NaCl concentrations (4–5 M). However,
when we examined the effect of various osmolytes on the activ-
ity of the purified protease in the absence of NaCl, the osmolytes
supported the enzyme catalysis. Maximum activity in glycerol was
observed at 5%, while, the activity in presence of NaCl and KCl was
comparable with control up to 30%. Among the osmolytes, Na-
glutamate (30%) enhanced the activity by 84% whereas; sucrose
(30%) maintained the activity without any enhancement. The
activity in Na-glutamate increased with increasing concentrations
indicating that osmotic pressure or reduced water activity was
important in maintaining enzyme. A significant increase in the
enzyme activity by 30% Na-glutamate, indicated its role in main-
taining high osmolarity required for the stability of this enzyme
in the absence of NaCl. The enzyme was completely inhibited by
10 mM PMSF, a serine protease inhibitor and there was no signifi-
cant effect of EDTA and DTT, indicating that the enzyme was a serine
protease (Fig. 6B). Our results correspond with the reported alkaline
serine proteases from halophilic and haloalkaliphilic Bacillus sp. as

120 A 140
B
100 120
% Residual Activity

80 100

80
60
60
40
40
20
20

0 0
0 10 20 60 12 0 18 0 24 0 144 0 0 10 20 60 120 180 240 1440
Time(min)
Fig. 5. Denaturation of the alkaline protease in the presence of urea (A) and guanidine hydrochloride (B) at 0 M (), 2 M (), 4 M () and 8 M (×) concentrations up to 5 h of
incubation.
26 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27

Fig. 6. Effect of osmolytes (A): NaCl ( ), KCl ( ), glycine ( ), mannitol ( ), trehalose ( ), sucrose ( ) and Na-glutamate ( ); inhibitors (B): at 1 mM ( ), 5 mM ( ), and
10 mM ( ) concentration; metal ions (C): at 5 mM ( ) concentrations; oxidizing and reducing agents (D): H2 O2 ( ), ␤-mercaptoethanol ( ) from 0 to 50 mM concentration;
surfactants (E): SDS ( ), CTAB ( ), Tween 80 ( ), Triton X-100 ( ) up to 2% concentrations.

well as alkaliphilic Streptomyces clvuligens strain Mit-1 [15,16,29]. 4. Conclusion

The metal ions did not have any significant effect on enzyme activity
except Cd2+ , Cu2+ and Hg2+ (Fig. 6C). The findings are in agreement
with a recent report on two novel halotolerant extracellular pro-
teases form B. subtilis strain FP-133 [31]. The % activity of OM-6
protease significantly increased in the presence of 10 mM H2 O2
and 5 mM ␤-mercaptoethanol (Fig. 6D). The results indicated that
H-bond and disulfide bonds are not directly involved with protein
activity or stability [8]. Joo and chang [40] reported the existence of
oxidant stable alkaline protease in a halo-tolerant Bacillus clausii l-
52. The OM-6 protease also had high % maximal activity in different
surfactants at 0.2% concentration; SDS (122.94%), CTAB (109.32),
Tween 80 (101.47) and Triton X-100 (137.05) (Fig. 6E). Haddar
et al. [27] reported a surfactant stable alkaline serine–protease
from a newly isolated Bacillus mojavensis A21. High activity in
oxidizing agent, reducing agent and different surfactants high-
lights toward the enzymatic activity and stability under multitude
of extremity and hence possible potential applications in varied
industries.
The present report focused on single step purification of a novel
alkaline protease from salt tolerant alkaliphilic actinomycetes,
B. xinjiangensis strain OM-6. Since the halophilic proteases are
difficult to purify, development of a simple purification proce-
dure in the present study would be quite useful for purification
of other extremozymes. In addition, the present research was
an attempt to understand the biochemical, thermodynamic and
kinetic properties of extracellular protease. Moreover, such a
detailed characterization of protease from actinomycetes have
not been conducted till date and to the best of our knowledge
this is the first report on the analysis of the thermodynamic and
kinetic parameters of protease from salt tolerant alkaliphilic acti-
nomycetes. The findings would add to the understanding of the
behavior of extremozymes. Besides, the enzyme had high activity
and significant stability at higher salt, temperature, pH and a range
of metal ions. The enzyme displayed extreme resistance against
urea denaturation, oxidizing and reducing agents and surfactants,
 S.D. Gohel, S.P. Singh / International Journal of Biological Macromolecules 56 (2013) 20–27
a finding which is rather unique and restricted to only few proteins.
The results, therefore, would open a new horizon for the biotechno-
logical applications of enzymes from less attended haloalkaliphilic
actinomycetes.
Acknowledgements
Dr. Sangeeta Gohel greatly acknowledges UGC (Govt. of India,
New Delhi) for providing Meritorious Fellowship and Research
Associateship. The work was sponsored by Saurashtra University,
Rajkot, India and UGC, New Delhi.
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