Applied Biochemistry and Biotechnology (2019) 189:87–102

https://doi.org/10.1007/s12010-019-02983-6

Production, Partial Purification, and Biochemical

Characterization of a Thermotolerant Alkaline
Metallo-protease from Staphylococcus sciuri

Rasha Abu-Khudir 1,2 1 3

& Maha M. Salem & Nanis Gamal Allam & Ehab M. M. Ali
1,4

Received: 31 October 2018 / Accepted: 1 March 2019 /

Published online: 14 March 2019
# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Protease-producing Staphylococcus sciuri was isolated from poultry soil samples and culture
conditions for protease production were optimized. The isolated protease showed a maximum
activity of 235.1 U/ml. Enzyme purification procedure involved ammonium sulphate precip-
itation and Sephacryl S-200 HR gel filtration chromatography (GFC). The purification process
resulted in the production of three protease fractions namely protease І (metallo-alkaline
protease), II, and IІІ. The metallo-alkaline protease was purified to 25.49-fold with specific
activity of 982.22 U/mg and 3.76% recovery. The partially purified metallo-protease was
optimally active at pH 10.0 and 70 °C and exhibited thermal stability up to 50 °C. The protease
activity was enhanced by Ca2+ and Mg2+, completely inhibited by Hg2+ and Cu2+, and
significantly reduced by EDTA. The protease showed significant stability towards various
surfactants, including SDS. The Km and Vmax values were 0.68 mg/ml and 166.66 nmol of
azocasein/ml/h, respectively, while the activation energy (Ea) was 3.07 Kcal/mol. Hence, it is
evident that the produced protease possesses unique characteristics and could be a plausible
candidate for various industrial and biotechnological applications.

Keywords Protease . Bacterial isolates . Poultry waste . Purification . Characterization

* Rasha Abu-Khudir
rabukhudir@kfu.edu.sa; rasha_hammad@hotmail.com

1
Chemistry Department, Biochemistry Branch, Faculty of Science, Tanta University, Tanta 31527,
Egypt
2
Chemistry Department, College of Science, King Faisal University, P.O. Box 380, Al-Hofuf, Al-Ahsa
31982, Saudi Arabia
3
Botany Department, Microbiology Unit, Faculty of Science, Tanta University, Tanta 31527, Egypt
4
Department of Biochemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203,
Jeddah 21589, Saudi Arabia
88 Applied Biochemistry and Biotechnology (2019) 189:87–102

Introduction

Proteases or peptidases (EC 3.4) are a prominent group of catalytic enzymes known to hydrolyze
peptide bonds in other proteins. On the basis of the position of the peptide bond to be cleaved,
proteases are subdivided into two major groups: endopeptidases (EC 3.4.21-99) and exopeptidases
(EC 3.4.11-19) [1]. Proteases are involved in numerous vital biological processes in all living
organisms [2]. In addition, they are involved in different industries and biotechnological applications
including food technology, leather processing, detergent, and pharmaceutical industries [3]. More-
over, proteases and their inhibitors are in use for treatment of several disorders [4, 5]. Also, light has
been shed on the importance of the catalytic action of plant-derived keratinases in the fields of
essential amino acid production and wastewater system blockage prevention [1].
Proteases of microbial origin or microbial proteases account for approximately 40% of the
total worldwide enzyme sales [6]. Microbial proteases show preference over those obtained
from animal or plant sources, as they are characterized by various features essential for
biotechnological applications [7, 8]. Microbial proteases can be either extracellular or intra-
cellular and their production is highly affected by various factors including the strain as well as
nutritional and physicochemical factors [7].
Commercial proteases, chiefly neutral and alkaline ones, are produced by bacteria that
belong to the genus Bacillus, a specific producer of extracellular proteases with a wide range of
industrial applications [1]. Actually, neutral bacterial proteases are catalytically active over a
narrow range of pH (pH 5.0 to 8.0) and they have a comparatively low ability to withstand
high temperatures, a character allowing the control of their reactivity during their use in food
industry [1, 9]. On the other hand, alkaline bacterial proteases are characterized by being
highly active at alkaline pH (pH 10.0), having their optimal temperature around 60 °C, and by
having broad substrate specificity. Hence, such characteristics of alkaline bacterial proteases
render them suitable for use in the detergent industry [10].The present study aimed to screen
poultry soil samples, collected from a number of local feather processing areas (El-Gharbia
governorate, Egypt), for the most protease-producing bacterial isolates. In addition, we aimed
to identify the most promising bacterial strain, optimize the culture conditions for its growth
and protease production, and moreover to purify and characterize the produced protease.

Materials and Methods

Sample Collection

The soil samples were collected from a number of feather processing areas on January 2014 in
Kotor city, El-Gharbia governorate, Egypt. The collected samples were transferred in sterile
plastic bags for further processing.

Isolation of Protease-Producing Bacteria

Bacteria were isolated using the serial dilution method described by Uyar et al. [11] with few
modifications. For each soil sample, 1 g of fresh weight was transferred into 10 ml of sterilized
distilled water. Heat shock activation of such initial dilutions was carried out at 70 °C for
15 min. In order to reduce the initial number of microorganisms, heat treated samples were
serially diluted (up to 10−9). Out of each diluted sample, an aliquot of 0.1 ml was inoculated on
Applied Biochemistry and Biotechnology (2019) 189:87–102 89

skimmed milk agar plates containing (g/l) peptone, 1; glucose, 1; beef extract, 5; skim milk
powder, 8; and agar, 15. The plates were incubated at 30 ± 2 °C for 2 days. Subsequently,
bacterial isolates were purified on nutrient agar medium followed by maintenance of selected
pure isolates on slants of nutrient agar medium (NAM) at 4 °C for further studies.

Identification and Characterization of Bacterial Isolates

Identification was attained using Gram staining as well as biochemical identification via VITEK 2C
system. Bacterial isolates were inoculated in basal medium enriched with skimmed milk and
adjusted to pH 7.0. Incubation of the inoculated media was done at 30 °C for 48 h on a rotary
shaker (180 rpm). Subsequently, bacterial cells were removed by centrifugation at 3000 rpm for
10 min and the cell-free supernatants were collected and enzyme activity was determined.

Assay of Proteolytic Activity and Protein Content

Proteolytic activity with azocasein (Sigma-Aldrich) as a substrate was determined according to the
method of Leighton et al. [12]. Briefly, the protease activity was measured using a reaction mixture
containing 150 μl enzyme solution and 250 μl of 5 mg/ml azocasein in 0.05 M acetate buffer, pH
5.6. The mixture was incubated for 30 min at 37 °C and the reaction was terminated by adding 1 ml
of trichloroacetic acid (TCA; 10%, v/v). The reaction mixture was incubated at 4 °C for 30 min and
then centrifuged (6,000 rpm for 10 min at 4 °C). Subsequently, 500 μl of 1.8 N NaOH was added to
neutralize 1 ml of supernatant, and absorbance was read at 339 nm. The amount of enzyme that
cleaved 1 nmol of azocasein/ml/h represented one protease unit. Protein content was determined
according to the method of Lowry et al. [13], using bovine serum albumin (BSA) as the standard.

Factors Affecting Enzyme Production

To detect the best cultivation conditions for protease secretion, a cultivation time course
experiment was conducted using 100 ml of the bacterial culture incubated at 37 °C. At
different time points, samples were withdrawn until the enzyme activity decreased.
To determine the optimum medium contents, bacterial growth was carried out in liquid basal
medium skimmed milk supplemented gradually with various components to be assessed. The major
components being assessed included carbon sources (glucose, sucrose, fructose, galactose, maltose,
or lactose) added at a concentration of 2% (w/v), nitrogen sources (yeast extract, ammonium sulfate,
peptone, ammonium chloride, beef extract, or urea) added at a concentration of 1.2% (w/v), and
inorganic salts (MnCl2, CaCl2, MgCl2, HgCl2, or CuCl2) added at a concentration of 1 mM.
Components were added and assessed one after the other using 100 ml of bacterial culture
grown in a 250-ml Erlenmeyer flask at 37 °C for 48 h under aerobic conditions. Subsequently,
the supernatant was used for determination of protease activity after centrifugation at 3000 rpm for
10 min. With the use of the optimal culture composition, the effects of different temperatures ranged
from (20 to 60 °C) and pH values (3.6 to 10.6) on the production of protease were similarly
investigated in the same manner until the optimal conditions were attained.

Partial Purification of Protease from Staphylococcus sciuri

After incubation of the culture broth at the optimal growth conditions, cells were
harvested by centrifugation (3000 rpm for 10 min at 4 °C) and the collected cell-free
90 Applied Biochemistry and Biotechnology (2019) 189:87–102

supernatant (CFS) was deemed as the starting material for protease purification.
Precipitation of protease in the CFS was done using 80% saturation of ammonium
sulphate. Thereafter, the precipitate was recovered by centrifugation (3000 rpm for
15 min at 4 °C), dissolved in the least volume of 50 mM sodium acetate buffer,
pH 5.6, and dialyzed against the same buffer for overnight. After centrifugation, the
dialysate was applied to Sephacryl S200 HR gel filtration chromatography (GFC)
column (20 × 3 cm) washed several times with water. Proteins were eluted with 2 to 3
bed volumes of 50 mM acetate buffer, pH 5.6. Fractions were collected in labeled test
tubes at a flow rate of 3 ml/5 min. The total protein content in each fraction was
measured at 280 nm and fractions enriched in proteins were pooled and assayed for
protease activity. The proteolytic activity was determined spectrophotometrically and
fractions containing high protease activity were pooled together [14].

Characterization of Partially Purified Protease

Effect of pH on the Proteolytic Activity

The partially purified protease was diluted in the following buffers at different ranges of pH:
50 mM acetate buffer (pH 3.6 to 5.6); 50 mM phosphate buffer (pH 6.0 to 8.5); 50 mM
glycine-NaOH buffer (pH 9.0 to 10.6). Protease activity was determined at 37 °C for 30 min in
the above mentioned buffers.

Effect of Temperature on Proteolytic Activity and Stability

The optimal reaction temperature for the partially purified protease was determined by
incubating the reaction mixture at optimum pH value and different temperatures in the range
of 20 to 90 °C under standard assay conditions as previously described. For thermal stability
assessment, enzyme solution in 50 mM acetate buffer (pH 5.6) was allowed to stand for 1 h at
various temperatures (10–90 °C). Afterwards, the residual enzymatic activities were assayed as
previously described.

Effect of Various Metals Ions, Inhibitors, and Surfactants on Proteolytic Activity

The effects of various divalent cations (Mg2+, Ca2+, Hg2+, and Cu2+ at 1, 2, 5, and 10 mM/
each), a couple of protease inhibitors (ethylenediaminetetraacetic acid (EDTA) and dithiothre-
itol (DTT) at 1, 2, 5, and 10 mM/each), and different surfactants (SDS, Triton X-100, and
Tween 20 at 0.1, 0.2, and 0.5%/each) on the activity of protease were determined. The activity
of the partially purified protease was determined by pre-incubating the enzyme with the above
mentioned additives, one at a time, for 15 min at 37 °C. Under optimum assay conditions, the
relative protease activity (%) was calculated with respect to no-additive control.

Estimation of Kinetic Parameters

Determination of Km and Vmax Values

The kinetic parameters, Michaelis-Menten constant (Km) and maximum reaction velocity
(Vmax), of the partially purified protease were determined by measuring the enzyme activity
Applied Biochemistry and Biotechnology (2019) 189:87–102 91

at different concentrations of azocasein. The Km and Vmax values were determined using the
double-reciprocal plot graphical method of Lineweaver and Burk [15].

Determination of Activation Energy (Ea)

The activation energy (Ea) of the partially purified protease was determined by measuring the
maximal initial rate (V) at different temperatures (T) using azocasein as a substrate. The Ea was
calculated from the slope of linear Arrhenius plot of logV against 1/T (K−1), where Ea =
−slope × 2.3 R, R (gas constant) = 1.987 × 10−3 kcal/mol [16].

Results

Isolation, Identification, and Characterization of Protease-Producing Bacteria

Bacterial colonies with different morphology were observed on skimmed milk agar plates.
Colonies surrounded by hydrolytic clear zones were designated as protease producers. Totally,
33 proteolytic bacterial isolates were isolated from the collected soil samples. Among the 33
isolates, eight exhibited the largest clear zones of casein hydrolysis around colonies and were
designated as protease producers, among which isolate no. 2 exhibited the largest clear zone
(12 mm) in skim milk agar. Comparatively less proteolytic zones were observed for the other
isolates ranging between 1 and 5.1 mm (Table 1). The eight different isolates under investi-
gation secreted proteases at various levels. Using azocasein assay, a maximum protease
activity of 219.96 U/ml was obtained after 48 h at 37 °C from isolate no. 2. Other three
bacterial isolates (no. 4, 5, and 8) showed high levels of extracellular protease activities of
111.06, 150.35, and 177.65 U/ml, respectively. On the other hand, isolates no. 1, 3, and 6
exhibited moderate protease activities of 56.48, 57.02, and 81.85 U/ml, respectively, while the
lowest extracellular protease activity (6.54 U/ml) was exhibited by isolate no. 7 (Table 1). The
investigated bacterial isolates were identified by Gram staining as Gram-positive bacteria. In
addition to Gram staining, the isolates were identified biochemically by VITEK 2C. As shown
in Table 1, the isolates were identified as Staphylococcus lentus, Staphylococcus sciuri,
Streptococcus cristatus, Lactococcus garvieae, Enterococcus avium, Streptococcus anginosus,
Streptococcus pyogenes, and Bacillus sciuri.
Since it was found to yield the highest protease activity, the purity of Staphylococcus sciuri
(S. sciuri) was confirmed by repeated streaking on agar plates and it has been selected for
further characterization.

Table 1 Protease activity obtained from eight different bacterial isolates

Name of bacteria Isolate number Clear zones of casein hydrolysis (mm) Enzyme activity (U/ml)

Staphylococcus lentus Isolate 1 2.5 56.48 ± 2.42

Staphylococcus sciuri Isolate 2 12 219.96 ± 2.18
Streptococcus cristatus Isolate 3 2.6 57.02 ± 2.16
Lactococcus garvieae Isolate 4 3.5 111.06 ± 1.71
Enterococcus avium Isolate 5 4 150.35 ± 1.96
Streptococcus anginosus Isolate 6 3 81.85 ± 2.62
Streptococcus pyogenes Isolate 7 1 6.54 ± 2.44
Bacillus sciuri Isolate 8 5.1 177.65 ± 2.52
92 Applied Biochemistry and Biotechnology (2019) 189:87–102

Factors Affecting Protease Production by S. sciuri

To determine the optimum cultivation time for protease secretion by S. sciuri, a cultivation
time course experiment was conducted at 37 °C. Samples were withdrawn at different time
intervals (4–84 h). The protease activity was increased till it reached its maximum (219.8 U/
ml) after 48 h of incubation then decreased to reach its minimum after 84 h.
A group of experiments was carried out to study the effect of medium composition on the
protease production. To determine the influence of carbon sources on the production of
protease, S. sciuri was grown on skimmed milk liquid medium, either comprising no addi-
tional carbon sources (control) or 2% of glucose, sucrose, fructose, galactose, maltose, or
lactose. Protease production by S. sciuri was increased by the addition of 2% lactose (220 U/
ml) compared to control (189.04 U/ml), while it was completely inhibited by glucose.
In addition to carbon sources, the impact of different nitrogen sources on the protease
production was tested in a medium containing 1.2% of yeast extract, ammonium sulfate,
peptone, ammonium chloride, beef extract, or urea. The production of protease was enhanced
by the addition of yeast extract (229.03 U/ml), while it was decreased upon addition of peptone
(110.40 U/ml) compared to control (189.04 U/ml).
Among all the investigated inorganic salts, CaCl2 increased the protease production
(235.18 U/ml) compared to control (189.045 U/ml). On the contrary, the protease production
was inhibited by HgCl2 (81.130 U/ml), CuCl2 (26.475 U/ml), and extensively inhibited by
MnCl2 (2.129 U/ml). It is most likely that S. sciuri uses Ca2+ ions for enzyme secretion.
To determine the effect of temperature and pH on protease production, the bacterial cells
were grown in the optimal medium at various temperatures (20–60 °C) and pH values (3.6–
10.6) for 48 h. The maximum protease activity was reached at 37 °C (235.1 U/ml) and was
drastically reduced at 60 °C (11.67 U/ml). The strain S. sciuri produced higher protease
activity (217.20 U/ml) at pH 8.0, while minimal activity (77 U/ml) was detected at pH 3.6.
The optimized medium composition and growth conditions for protease production by
S. sciuri are shown in Table 2.

Partial Purification of Protease from S. sciuri

Under optimized medium composition and growth conditions, S. sciuri secreted protease into
the culture fluid from which it was partially purified. Outcomes of the partial purification
procedure are represented in Table 3. The crude enzyme was precipitated with 80% saturation
of ammonium sulphate, dialyzed, and then subjected to gel filtration on a Sephacryl S-200
High Resolution (HR) column. As shown in Table 3, the specific activity of the enzyme
increased from 38.52 to 79.56 U/mg, (2.06-fold enrichment) at 16.9% yield. After dialysis, the
specific activity of the enzyme increased from 79.56 to 154.34 U/mg (4.00-fold enrichment) at
15.8% yield. The elution profile from the Sephacryl S-200 HR column is shown in Fig. 1. Two

Table 2 Optimized medium contents and culture conditions for protease production by S. sciuri

Optimum medium contents Optimum culture conditions

Carbon source 2% lactose Time 48 h

Nitrogen source 1.2% yeast extract Temperature 37 °C
Inorganic metal ions 1 mM CaCl2 pH 8.0
Applied Biochemistry and Biotechnology (2019) 189:87–102 93

Table 3 Purification table of protease obtained from S. sciuri

Step Total activity Total protein Specific activity Purification Recovery

(U) (mg) (U/mg) (fold) (%)

Crude extract 2350.1 61 38.52 1 100

(NH4)2SO4 397.82 5 79.56 2.06 16.9
precipitation
Dialysis 371.52 2.407 154.34 4.00 15.80
Sephacryl S-200 HR
Protease I 88.40 0.09 982.22 25.49 3.76
Protease II 123.76 1 123.76 3.21 5.26
Protease III 118.05 1.1 107.31 2.78 5.02

protein peaks and three with proteolytic activity were observed (representing proteases I, П,
and III), with specific activity of 982.22, 123.76, and 107.31 U/mg and 25.49, 3.21, and 2.78-
fold purification, respectively (Table 3). Based on its higher purification fold, fractions
corresponding to form I protease were pooled, dialyzed, and subjected to further
characterization.

Characterization of the Partially Purified Protease

Effect of pH on the Proteolytic Activity

The effect of pH on the catalytic activity of the partially purified protease was assessed using
azocasein as a substrate under standard assay conditions. No activity was observed below or at
pH 4.4. However, the protease was significantly active over a broad range of higher pH values,
showing maximum catalytic activity (100%) at pH 10.0, above which the activity was
decreased (Fig. 2). Hence, it was indicated that the partially purified protease from S. sciuri
is a typical alkaline protease.

Fig. 1 Elution profile of protease from S. sciuri (gel filtration chromatography on Sephacryl S-200 HR)
94 Applied Biochemistry and Biotechnology (2019) 189:87–102

Fig. 2 Effect of pH on the activity of partially purified protease from S. sciuri

Effect of Temperature on Proteolytic Activity and Stability

The partially purified protease remained catalytically active over a range of temperatures varying
from 20 to 90 °C, at pH 5.6 using azocasein as a substrate. The optimum reaction temperature of the
protease was observed at 70 °C, above which the activity began to decline sharply where it reached
less than 40% at 90 °C (Fig. 3a). In addition, thermal stability assessment showed that the partially
purified protease was 100% stable up to 50 °C. However, the protease retained about 87% of its
original activity at 60 °C, which was gradually decreased till it reached 51% at 90 °C (Fig. 3b).

Effect of Various Metals Ions, Inhibitors, and Surfactants on the Protease Activity

The impact of various divalent cations on the activity of the partially purified protease from
S. sciuri was examined at 1, 2, 5, and 10 mM concentrations, and the results are represented in
Table 4. Among the assessed divalent cations, the partially purified protease was activated by

Fig. 3 Effect of temperature on activity (a) and stability (b) of the alkaline protease from S. sciuri
Applied Biochemistry and Biotechnology (2019) 189:87–102 95

Table 4 Effect of metal ions/inhibitors on the activity of protease obtained from S. sciuri

Metal ions/inhibitors Relative activity (%)

1 mM 2 mM 5 mM 10 mM

Control 100
Ca2+ (CaCl2) 95.2 97.35 101.74 189.6
Mg2+ (MgCl2) 94.7 96.74 97.23 112.23
Hg2+ (HgCl2) 71.82 52.86 34.16 0
Cu2+ (CuCl2) 57.85 46.88 18.70 0
EDTA 71.57 44.38 41.40 39.6
DTT 128.92 117.45 107.73 103.6

Ca2+ and Mg2+ at a concentration of 10 mM, where it exhibited 189.6% and 112.23% activity
relative to control, respectively. In contrast, both Cu2+ and Hg2+ at 10 mM concentration
completely inhibited the protease activity (Table 4).
In an attempt to determine the nature of the partially purified protease, the effect of the
metallo-protease inhibitor EDTA and the thiol reagent DTT (at 1, 2, 5, and 10 mM) on enzyme
activity was investigated. The protease from S. sciuri was significantly inhibited in the
presence of EDTA with 39.6% residual activity observed at 10 mM concentration, which
suggested that metal cofactors were required for its function and that the enzyme is a metallo-
protease. On the other hand, the protease activity was rather increased up to 28.9 and 17.5%
relative to control at 1 and 2 mM concentrations of DTT, respectively (Table 4).
The effect of the anionic detergent SDS and the two non-ionic ones Triton X-100 and
Tween 20 (at concentrations of 0.1, 0.2, and 0.5%) on protease activity is shown in Table 5. It
was noticed that the activity of the protease was increased in the presence of 0.1% of all the
investigated detergents compared to control. However, the partially purified protease was
stable towards the non-anionic detergents at concentrations of 0.2 and 0.5%. On the other
hand, the enzyme retained 58.2% and 52.73% of its activity at 0.2 and 0.5% SDS concentra-
tions, respectively.

Estimation of Kinetic Parameters

Determination of Km and Vmax Values For further characterization of the protease from
S. sciuri, its kinetic parameters were determined using azocasein as a substrate. As estimated
using Lineweaver-Burk double-reciprocal plot, the Km and Vmax values were 0.68 mg/ml and
166.66 nmol/ml/h, respectively (Fig. 4).

Table 5 Effect of surfactants on the activity of protease obtained from S. sciuri

Surfactant Relative activity (%)

0.1% 0.2% 0.5%

Control 100
Triton X100 112.5 106.2 93.53
Tween 20 131.3 102.4 100.4
SDS 126.6 58.2 52.73
96 Applied Biochemistry and Biotechnology (2019) 189:87–102

Fig. 4 Lineweaver-Burk plot for S. sciuri protease under varying azocasein concentrations indicating the Km and
Vmax values

Determination of Activation Energy (Ea) The activation energy (Ea) of the partially purified
protease was calculated from the slope of Arrhenius plot, which showed a linear variation with
increasing temperature (Fig. 5). The activation energy (Ea) of the partially purified alkaline
protease was 3.07 Kcal/mol for azocasein hydrolysis.

Discussion

It is widely accepted that proteases can be used in a wide range of industrial applications including
food, detergents, production of nutritionally important amino acids, and pharmaceutical

Fig. 5 Arrhenius plot to calculate activation energy (Ea) of protease from S. sciuri
Applied Biochemistry and Biotechnology (2019) 189:87–102 97

manufacture applications [17]. Hence, we aimed to search for a locally available, inexpensive, and
high-quality source for the isolation of microbial proteases from poultry soil.
In the present study, 33 isolates, representing different types of bacteria isolated from
poultry soil, were screened for their protease-production capacities. Only eight isolates, of
five different genera, showed a high level of enzyme activity, among which S. sciuri was the
best protease producer.
In an attempt to characterize S. sciuri, the optimum cultivation time for protease secretion
by the isolated bacterial strain was estimated to be 48 h at 37 °C with maximum activity of
219.96 U/ml. Similar to our findings, maximum protease activity (44 U/ml) was observed at
37 °C after 48 h by Pseudomonas aeruginosa PAO1 [18]. Akram et al. [19] reported the
production of a protease from Staphylococcus aureus S-2 isolated from chicken waste with
maximum enzyme activity of 360 U/ml after 12 h at 37 °C. Moreover, maximum protease
production by Staphylococcus sp. (198 U/ml) and S. auricularis p18 (0.038 U/ml) was
obtained after incubation for 48 and 72 h at 30 °C and 45 °C, respectively [20, 21].
Components of bacterial growth media are of great importance for the production of
alkaline proteases as they play crucial role in the biosynthesis and generation of energy.
Among media components, carbon sources are known to exhibit significant effects on protease
production [10]. In this study, protease production by S. sciuri was increased by adding lactose
(220 U/ml), while it was completely inhibited by glucose. Similar results were reported for
Yersinia sp. [20] and Pseudomonas aeruginosa K-187 [22], where protease activity was
maximal (194 U/ml and 21.2 U/ml, respectively) using lactose as compared to glucose. On
the other hand, it has been shown that Staphylococcus sp. [20] exhibited maximum protease
production of 198 U/ml in presence of glucose rather than other carbon sources. The current
study also showed that among nitrogen sources, yeast extract was the most significant nitrogen
source for protease production (229.03 U/ml). Our findings are in agreement with other reports
on proteases produced from various bacterial strains including Bacillus subtilis (3600
U/ml) [23] and Bacillus infantis SKS1 [24]. On the other hand, maximum protease production
of 198 U/ml was obtained when tryptone and beef extract were used as the sole nitrogen source
by Pseudomonas aeruginosa PAO1 [18] and Staphylococcus sp. [20], respectively. Previously,
it has been reported that protease production was greatly enhanced in the presence of Ca2+ [18,
25, 26]. In accordance with these previous reports, we have shown that the optimal protease
production of 235.18 U/ml was observed when CaCl2 was supplemented at a 1 mM
concentration.
The results of the present investigation showed that maximum protease production of
217.20 U/ml was observed at pH 8.0, which is consistent with that described for protease
production (186 U/ml) by Staphylococcus sp. [20]. On the other hand, the highest protease
production of 44 U/ml by Pseudomonas aeruginosa PAO1 [18] and 123.5 U/ml by Bacillus
subtilis NS [27] was observed at pH values 7.0 and 9.0, respectively.
The present protease produced by S. sciuri was partially purified by the combination of
several steps. The dialyzed suspension after ammonium sulfate precipitation was applied on
Sephacryl S-200 HR column and resolved into three protease fractions designated as proteases
I, II, and III with specific activity of 982.22, 123.76, and 107.31 U/mg and 25.49, 3.21, and
2.78-fold purification, respectively. The protease I exhibited higher specific activity compared
to previously reported proteases from various bacterial strains including Bacillus infantis SKS1
(21.27 U/mg), Pseudomonas thermaerum GW1 (137.54 U/mg), Bacillus licheniformis LHSB-
05 (185.6 U/mg), B. aquimaris VITP4 (424 U/mg), and halophilic AH10 (659.57 U/mg) [24,
28–31].
98 Applied Biochemistry and Biotechnology (2019) 189:87–102

Generally, alkaline proteases are effective at pH values ranging from 7.0 to 11.0 with an
optimum at pH 10.0 [32]. Our results showed that the protease produced by S. sciuri exhibited
maximum activity at pH 10.0 and 70 °C. It has been previously reported that a protease
produced by Bacillus mojavensis SA [33] was active at pH 12.0 (60 °C), while Staphylococcus
aureus S2 [19] and Streptomyces sp. M30 [34] produced proteases which showed maximum
activity at pH 8.0 (50 °C) and pH 9.0 (80 °C), respectively.
For industrial applications, proteases must possess stability under relatively unfavorable
conditions, including extremes in temperature [35]. Our data showed that the partially purified
protease from S. sciuri exhibited thermal stability up to 50 °C for 1 h and it lost around 50% of
its initial activity at 90 °C. Comparable thermal stability has been previously reported for
proteases from Pseudomonas aeruginosa PAO1 [18], Bacillus sp. KG5 [25], and Staphylo-
coccus saprophyticus [36]. Collectively, the obtained results showed that the protease pro-
duced by S. sciuri is thermostable under alkaline conditions, thus rendering its applicability in
industrial applications (Table 6). The activity of proteases in the presence of several metal ions
at different concentrations is considered one of the beneficial features of such enzymes. Our
results showed that the partially purified protease displayed increased activity in presence of
the divalent cations Ca2+ and Mg2+, whereas its proteolytic activity was completely inhibited in
presence of Cu2+ and Hg2+. Similar to our findings, enhancement of protease activity in
presence of the divalent cations Ca2+ and Mg2+ and inhibition in presence of Cu2+ and Hg2+
has been previously reported [25, 39]. On the contrary to our findings, it was observed that
either Mg2+, Ca2+, or both significantly repressed the activity of proteases derived from Ps.
aeruginosa PAO1 [18], Bacillus cereus MCM B-326 [43], and Stenotrophomonas maltophilia
strain SK [44], respectively. It has been stated that Ca2+ acts as an activator for proteases [45].

Table 6 Comparison of alkaline proteases produced from various bacterial strains

Name of bacteria Optimum temperature Optimum pH Optimum Optimum pH Ref.

for production (°C) for production temperature for for activity
activity (°C)

Aeribacillus pallidus – – 60 9.0 [37]

C10
Bacillus caseinilyticus 37 9.0 60 8.0 [38]
Bacillus circulans – – 60 10.0 [39]
MTCC 7942
Bacillus infantis 40 10.0 50 10.0 [24]
SKS1
Bacillus licheniformis – – 70 9.0 [40]
A10
Bacillus mojavensis – – 60 12.0 [33]
SA
Bacillus nealsonii – – 65 10.0 [41]
PN-11
Micromonospora 70 8.0 [42]
chaiyaphumensis
S103
Ps. aeruginosa PAO1 37 7.0 40–50 8.0 [18]
Staphylococcus 37 7.0 50 8.0 [19]
aureus S2
Streptomyces sp. M30 – – 80 9.0 [34]
Staphylococcus sciuri 37 8.0 70 10.0 Present
stud-
y
Applied Biochemistry and Biotechnology (2019) 189:87–102 99

Indeed, the Ca2+ ion-dependent enhancement in enzyme activity observed in the present study
is in accordance with what has been previously reported about the enhancement of proteases,
generally, by Ca2+ ions [46].
The partially purified protease from S. sciuri was significantly inhibited in the presence of
EDTA, suggesting that metal cofactors were required for its function and that the enzyme is a
metallo-protease. Similar findings have been previously reported for proteases obtained from
Pseudomonas aeruginosa PAO1 [18], Streptomyces sp. M30 [34], and several Bacillus sp. [25,
40, 47]. Conversely, the activity of proteases from Staphylococcus saprophyticus [36] and
Bacillus caseinilyticus [38] was significantly improved in the presence of EDTA.
On the other hand, DTT slightly increased the proteolytic activity of the partially purified
protease. Previously, a large increase in the activity of a protease from Staphylococcus
saprophyticus [36] was observed in the presence of DTT, indicating it to be a thiol-
dependent protease. However, the proteolytic activity of enzymes obtained from Streptomyces
sp. M30 [34] and Bacillus amyloliquefaciens SP1 [47] was inhibited by DTT.
The partially purified alkaline protease showed high stability in the presence of the non-
ionic detergents Tween 20 and Triton X-100. Similar results were obtained by Xin et al. [34]
and Chatterjee et al. [48]. Comparable to proteases from Ps. aeruginosa PAO1 [18] and
Streptomyces sp. M30 [34], the protease from S. sciuri was highly stable in the presence of the
strong anionic detergent SDS at 0.1%, above which the activity was decreased. The stability of
alkaline proteases towards SDS is important because as per previous reports, many of the
available proteases displayed low activity and stability in presence of SDS [25, 49].
Further characterization of the partially purified protease was extended with determining its
Km and Vmax towards azocasein as a substrate. The obtained data indicated that the protease
from S. sciuri exhibited high affinity for its substrate compared to alkaline proteases purified
from Bacillus thuringiensis MC28 strain [50] and Bacillus cereus [51] which exhibited higher
Km value of 1.57 and 14.5 mg/ml, respectively. In addition, activation energy (Ea) of the
alkaline protease for azocasein hydrolysis was calculated from the slope of an Arrhenius linear
plot. The calculated Ea (3.07 kcal/mol) was lower than that previously reported for an
alkaline protease from Bacillus amyloliquefaciens SP1 [47]. Hence, an effective hydrolytic
capacity of the investigated protease is highly suggested based on its low Ea which indicated
that less energy is needed for the formation of the activated complex of azocasein hydrolysis
[52].

Conclusion

In the present study, the optimal production, partial purification, and characterization of a new
thermostable metallo-protease were studied. The enzyme was produced by a locally protease-
producing bacterial strain isolated from poultry soil samples, identified as S. sciuri. The
bacterial isolate was characterized on the basis of carbon and nitrogen sources utilization,
pH and temperature requirements, and chemical sensitivity assays. The partially purified
protease showed desirable stability at high alkaline pH and extreme temperature, thereby
permitting its wide biotechnological and industrial applications. As per our knowledge, this is
the first report of a thermostable alkaline metallo-protease production from S. sciuri locally
isolated from poultry soil samples.

Acknowledgements No funding sources had been involved in the conduction and/or preparation of this article.
100 Applied Biochemistry and Biotechnology (2019) 189:87–102

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest.

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