Biocatalysis and Agricultural Biotechnology 26 (2020) 101631

Contents lists available at ScienceDirect

Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Production of detergent stable thermophilic alkaline protease by Bacillus

licheniformis ALW1
Mohamed A. Emran , Shaymaa A. Ismail , Amal M. Hashem *
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries, National Research Centre, El Behouth Street, Dokki, Giza,
12622, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: The growing industrial applications of alkaline proteases urged the production of highly active stable enzymes. In
Alkaline protease the current study, the enzyme production was achieved by submerged fermentation using the bacterial strain
Bacillus licheniformis ALW1 Bacillus licheniformis ALW1 that was further optimized to reach 22.903U/mL. The overall optimization fold was
Submerged fermentation
46.7, achieved under the optimized fermentation medium composed of (%) molasses; 8, (NH4)2SO4; 0.45, wheat
Optimization
Detergent additive
bran; 5, MgSO4⋅7H2O; 0.2, KH2PO4; 0.4, NaCl; 0.2 adjusted at pH 8 and incubated for 7days at 37 � C and 280
rpm. Additionally, the enzyme activity after partial purification was optimized by studying the effect of pH and
temperature as well as the substrate concentration. The results indicated that the enzyme optimum activity was
achieved at pH 9 and 70 � C with Km, Vmax and Kcat values of 3.846 mg/mL, 76.923U/mL/min and 1.206min 1
respectively. Thermal stability study of the partial pure enzyme indicated the half live times of the enzyme as
693.15, 231.05 and 57.76 min 1 at 55, 60 and 65 � C respectively, confirming its thermo-stability. Finally, the
efficacy of the partial pure enzyme as a detergent additive was examined. The enzyme retained more than 80% of
its activity with an efficient washing performance in the removal of blood stain after 30 min at 50 � C in addition
to a commercial detergent.

1. Introduction
Proteases are a complex group of enzymes that occupy a pivotal
position among different classes of enzymes owing to their physiological
and commercial roles. They are capable of the cleavage of the peptide
bond in the large protein molecules converting them to amino acids or
small peptides. They are ubiquitous in nature, found in plants, animals,
and microbes (Barrett and McDonald, 1986). Microbes especially bac­
teria are considered to be the main source for their production as they
possess several advantages including rapid and easy production pro­
cesses in addition to the high production yield and their genetic
manipulation feasibility (Breithaupt, 2001). Proteases occupy more than
60% of the industrial global production of enzymes in which 35% are
alkaline proteases. Alkaline proteases are those enzymes active over a
pH range from neutral to alkaline pH. Although they are applicable in
different industries including detergent, food, cosmetics and leather
(Dias et al., 2008), their industrial application is restricted by their
limited activity and stability at high temperature, extreme pH, the
addition of detergent ingredients and organic solvents (Olajuyigbe and
Falade, 2014). Therefore, the microbial production of highly active
alkaline proteases with novel properties has attracted the research focus
(Ahmad et al., 2020; Gulmus and Gormez, 2020; Hakim et al., 2018;
Harer et al., 2018; Lakshmi et al., 2018; Qureshi et al., 2018; Ramkumar
et al., 2018; Zheng et al., 2020).
In industry, the overall production cost of the applied enzyme is a
major obstacle against its successful use. Since about 40% of the overall
production cost is attributed to the cost of the growth medium (Joo
et al., 2002). Therefore, there is a growing need for the adjustment of a
low-cost production medium that works effectively and efficiently for
the production of the enzyme. The optimization of the fermentation
medium, as well as the fermentation conditions, has been performed
initially by using the conventional one variable at a time approach.
Consequently, statistical techniques have been employed for saving time
and effort in addition to examining the interactive effects between
various parameters (Desai et al., 2008). Response surface methodology
(RSM) is a statistical technique that has been described for the first time
by Box and Wilson (1951). Currently, it is the most popular technique
applied in the optimization of various microbial and biotechnological
processes (Hashem et al., 2018a,b, Ismail, 2019 and Ismail et al., 2019a,
b,c) and it has been used occasionally in the optimization of the bacterial

* Corresponding author.
E-mail address: amal_mhashem@yahoo.com (A.M. Hashem).

https://doi.org/10.1016/j.bcab.2020.101631
Received 13 March 2020; Received in revised form 2 April 2020; Accepted 26 April 2020
Available online 3 May 2020
1878-8181/© 2020 Elsevier Ltd. All rights reserved.
M.A. Emran et al.
production of alkaline proteases (Adetunji and Olaniran, 2020; Ham­
mami et al., 2020; Lakshmi and Hemalatha, 2016; Limkar et al., 2019;
Mian et al., 2018; Ramkumar et al., 2018).
The current study concerned with the bacterial production of alka­
line protease using submerged fermentation technique as well as the
optimization of the fermentation process. The produced enzyme was
partially purified and its kinetic parameters were determined. Finally,
the efficiency of the partial pure enzyme as a detergent additive was
examined.
2. Materials and methods
2.1. Materials
Casein from bovine milk and tryptic soy broth were obtained from
Sigma-Aldrich, Saint Louis, USA. Wheat bran and molasses were pur­
chased from the local market. Other chemicals were of analytical or
HPLC grade.
2.2. Microorganism and production of the enzyme
The bacterial strain applied in this study was formerly isolated from
leather during a program aimed at the isolation of proteolytic bacteria
(Abdel-Fattah et al., 2018). Bacillus licheniformis ALW1 (Accession
number LC315920) was found to be the most powerful isolate able to
produce alkaline protease under submerged fermentation condition in
250 mL Erlenmeyer flasks holding 50 mL of the production medium. The
production medium described by Qureshi et al. (2011) composed of (%)
glucose; 1, peptone; 0.5, MgSO4⋅7H2O; 0.2, KH2PO4; 0.3, NaCl; 0.2 and
adjusted by 1 N NaOH and HCL to pH 7, was cultured by 2.5 mL of 24 h
inoculum (pre-cultured on tryptic soy broth) and incubated at 37 � C,
180 rpm for 2 days. At the end of the fermentation period, the cultured
medium was centrifuged at 4 � C for 10 min at 5000 rpm and the clear
supernatant was used further as the crude enzyme.
2.3. Determination of the enzyme activity
The activity of the enzyme was determined according to the method
of McDonald and Chen (1965) using 1% casein dissolved in 50 mM
Tris-HCl buffer pH8. A reaction mixture of 1 mL substrate added to 1 mL
of the suitably diluted enzyme was incubated at 40 � C for 30min. At the
end of the reaction time, 2 mL of 15% (w/v) trichloroacetic acid was
added to terminate the reaction. The reaction mixture was centrifuged
for 10 min at 10000 rpm at 4 � C and the protein content liberated in the
clear supernatant was assayed according to Lowry et al. (1951). One unit
of protease activity was defined as the amount of enzyme needed to
release 1 μmol of tyrosine per minute under the assay conditions.
2.4. Optimization of the fermentation process
2.4.1. One-variable at a time
It includes classical approaches of replacement and supplementation
in which each experiment was done under the optimized conditions. The
effect of the incubation period (1–6days), carbon sources with equiva­
lent carbon content to the constituent glucose (dextrin, maltose, starch,
lactose, and molasses), nitrogen sources with equivalent nitrogen con­
tent to the constituent peptone (soya bean, casein, corn steep liquor,
whey, urea, yeast extract, NaNO3, (NH4)2SO4 and NH4H2PO4) and the
addition of wheat bran at concentrations ranging from 0.2 to 6% were
examined.
2.4.2. Statistical optimization
The interactive effect between multiple fermentation parameters and
their impact on the enzyme production in a flask culture was investi­
gated by the two-phase model. In the first step (Plackett-Burman
design), variables that significantly influence the productivity were
2
Biocatalysis and Agricultural Biotechnology 26 (2020) 101631
identified then Box-Behnken design was applied in the second phase.
2.4.2.1. Plackett-Burman design. In this study, seven independent vari­
ables (initial pH, agitation speed, volume per flask, the concentration of
carbon source, the concentration of nitrogen source, fermentation
period and the concentration of K2HPO4) were evaluated at two levels
(high (þ1) and low ( 1) values) in eight experimental runs (Plackett
and Burman, 1946) as shown in Table (2). Each generated response was
calculated as described by the following first-order linear equation:
Y¼ B0 þΣ Bi Xi (1)
where Y is the response (protease activity), B0 is the model intercept and
Bi is the linear coefficient and Xi is the level of the independent variable.
The variables main effects were calculated by the following equation:
E(Xi) ¼ 2(Σ Miþ Mi )/N (2)
where E(Xi) is the effect of the tested variable. Miþ and Mi represent
protease activity of the experimental runs where the independent vari­
able (Xi) measured was present at high and low values respectively and
N is the number of runs.
2.4.2.2. Box-Behnken design. Box-Behnken design (Box and Behnken,
1960) was applied to determine the optimum level of the three highest
influencing variables at three different levels (low ( ), high (þ) and
control or basal (0) values) in the experimental design of 15 run and 3
central points (Table 3). The correlation between the variables and the
response (protease activity) was interpreted by the following second
order polynomial equation:
Y¼ B0 þΣ Bi Xi þΣ Bii X2i þΣ BijXiXj (3)
where Y is the predicted protease activity; β0 is the model intercept, ßi is
linear coefficient, ßii is a quadratic coefficient and ßij is a cross product
coefficient where Xi and Xj are the coded levels of the independent
variables.
2.5. Partial purification of the enzyme
The crude enzyme was precipitated using ethanol or acetone and
salted out using ammonium sulfate at 30–90% concentration with 10%
intervals. The resulted fractions were assayed for enzyme activity and
protein content (Hashem et al., 2018a).
2.6. pH and temperature effect
The effect of pH was estimated by determining the activity of the
partially purified enzyme at different pH range from 7 to 8 using 0.1 M
Tris/HCl buffer and from 9 to 10 using 0.1 M borax/NaOH buffer. The
optimum pH of the partial pure enzyme was determined as the pH value
at which the enzyme represents the highest activity. At this value, the
enzyme was incubated without a substrate to determine its stability by
estimating the enzyme residual activity every 30min up to 2 h. The
enzyme activity without pre-incubation was considered as 100%
activity.
To study the effect of temperature, the activity of the partial pure
enzyme was determined at the optimum pH for different temperatures
ranged from 40 to 75οC. Arrhenius plot was used to calculate the acti­
vation energy (Ea) of the produced enzyme as prescribed in the following
equation:
Slope ¼ Ea /R (4)
where R is the gas constant (8.3145 J/mol/K).
At the estimated optimum conditions, the residual activity of the
partial pure enzyme was determined in order to study its thermal sta­
bility after pre-incubation of the enzyme at different temperatures (55,
M.A. Emran et al. Biocatalysis and Agricultural Biotechnology 26 (2020) 101631

Table 1
One-variable at a time optimization.
Incubation period 1 2* 3 4 5 6
(days)
Activity (U/mL) 0.26 � 0.058 0.49 � 0.052 0.98 � 0.069 1.05 � 0.046 1.91 � 0.01 1.67 � 0.04
Carbon source Glucose* Dextrin Maltose Starch Lactose Molasses
Activity (U/mL) 1.9 � 0.027 1.61 � 0.116 1.61 � 0.311 1.92 � 0.138 1.35 � 0.33 3.24 � 0.118
Molasses 2 5* 8 11 14 16
concentration
(%)
Activity (U/mL) 2.81 � 0.065 3.08 � 0.072 4.68 � 0.177 4.53 � 0.174 4.46 � 0.011 4.09 � 0.313
Nitrogen source Soya Casein Corn steep liquor Whey Urea Yeast extract Peptone* NaNO3 (NH4)2SO4 NH4H2PO4
bean
Activity (U/mL) 2.12 � 1.99 � 2.48 � 0.111 2.05 � 2.61 � 2.8 � 0.148 4.68 � 4.43 � 5.16 � 4.75 �
0.358 0.051 0.249 0.002 0.014 0.037 0.125 0.222
Addition of wheat 0* 0.2 0.5 1 1.5 2 2.5 3 3.5 4 5 6
bran (%)
Activity (U/mL) 5.23 � 6.03 � 7.11 � 7.64 � 9.2 � 10.03 � 10.21 � 11.39 � 13.34 � 14.95 � 17.53 � 15.17 �
0.189 0.297 1.698 0.453 0.245 0.036 1.423 0.029 1.257 0.251 0.797 0.323

*control.

Table 2
Plackett – Burman design.
Run number pH Agitation speed Volume/flask Molasses concentration (NH4)2SO4 Incubation period K2HPO4 (%) Protease (U/
(rpm) (mL) (%) (%) (days) mL)

1 (6) - (180) - (25) - (11) þ (0.45) þ (7) þ (0.2) - 16.78431

2 (8) þ (180) - (25) - (5) - (0.15) - (7) þ (0.4) þ 9.970142
3 (6) - (260) þ (25) - (5) - (0.45) þ (3) - (0.4) þ 8.228886
4 (8) þ (260) þ (25) - (11) þ (0.15) - (3) - (0.2) - 17.65632
5 (6) - (180) - (75) þ (11) þ (0.15) - (3) - (0.4) þ 0.24792
6 (8) þ (180) - (75) þ (5) - (0.45) þ (3) - (0.2) - 0.201445
7 (6) - (260) þ (75) þ (5) - (0.15) - (7) þ (0.2) - 18.03714
8 (8) þ (260) þ (75) þ (11) þ (0.45) þ (7) þ (0.4) þ 20.04515

Table 3
Box-Behnken design.
Run number Independent variable Observed protease (U/ Predicted protease (U/ Residual
mL) mL)
X1 Molasses concentration (%) X2 Agitation speed (rpm) X3 Incubation period (days)

1 8( ) 240( ) 7(0) 7.145275 6.985518 0.159757

2 14(þ) 240( ) 7(0) 3.179798 6.132294 2.9525
3 8( ) 280(þ) 7(0) 22.90302 20.53252 2.370499
4 14(þ) 280(þ) 7(0) 15.21041 15.95209 0.74168
5 8( ) 260(0) 5( ) 10.41159 12.68703 2.27544
6 14(þ) 260(0) 5( ) 10.35685 9.519098 0.837755
7 8( ) 260(0) 9(þ) 16.42689 17.84325 1.41636
8 14(þ) 260(0) 9(þ) 17.27437 15.57753 1.69684
9 11(0) 240( ) 5( ) 2.925555 1.593742 1.331813
10 11(0) 280(þ) 5( ) 11.68103 12.73602 1.05499
11 11(0) 240( ) 9(þ) 7.132916 6.65995 0.472966
12 11(0) 280(þ) 9(þ) 16.97069 18.88447 1.91378
13 11(0) 260(0) 7(0) 20.124 20.33443 0.21042
14 11(0) 260(0) 7(0) 19.9651 20.33443 0.36932
15 11(0) 260(0) 7(0) 20.04632 20.33443 0.28811

60, 65οC) for 2 h without the addition of the substrate. The enzyme by using different casein concentrations (5–25 mg/mL). The kinetic
activity without pre-incubation was considered as 100% activity. The constants for the partial pure enzyme were determined from
enzyme thermal inactivation kinetics was calculated as follow: Lineweaver-Burk plot (Lineweaver and Burk, 1934), plotted on the base
of the following equation:
Slope of Arrhenius plot (lnKd versus 1/T) ¼ -Ed/R (5)
1/V ¼ (1/Vmax) þ (Km/Vmax)(1/S) (8)
T1/2 ¼ ln(2)/Kd (6)
Kcat ¼ Vmax/e (9)
Decimal reduction time (D-value) ¼ ln(10)/ Kd (7)
in which V is the activity of the partial pure enzyme (U/mL), Vmax is the
in which Kd is the thermal deactivation rate constant, Ed is the decay
maximal activity, Km is Michaelis-Menten constant, S is the casein
activation energy (KJmol 1) and T is the temperature (K). concentration (mg/mL), Kcat is the turnover number and e is the enzyme
concentration.
2.7. Effect of substrate (casein) concentration

The activity of the enzyme was estimated at the optimum conditions

3
M.A. Emran et al.
2.8. The scope on the efficacy of the produced enzyme in detergent
formulation
2.8.1. Effect of surfactants
The effect of different surfactants (non-ionic, anionic and cationic) at
the concentration of 1 g/L on the activity of the partial pure enzyme was
determined. The enzyme activity after pre-incubation of the enzyme
with the surfactants for 30 min at 50οC and 60οC was estimated under
the optimum conditions. The enzyme activity without pre-incubation
was considered as 100% activity.
2.8.2. Compatibility with a commercial detergent
The applicability of the partial pure enzyme as a commercial deter­
gent additive was initially estimated by determining its stability with
one of the commonly used liquid laundry detergent (Arial, power gel,
P&G Italia S.p.A., Palomba-Pomezia Roma, Italy). Initially, the com­
mercial detergent was boiled for 30min to denature the indigenous en­
zymes then the produced enzyme under investigation was added to the
detergent solution after cooling (in the ratio 1:5 enzyme to heat inacti­
vated detergent solution). The enzymatic preparation was incubated for
30 min at 50οC then the residual activity of the enzyme was determined
under the optimum conditions in which the enzyme activity without
detergent was considered as 100% activity (Niyonzima and More,
2015).
2.8.3. Washing performance
The efficacy of the partial pure enzyme in the removal of protein-rich
stain (blood) from cotton fabrics was examined according to the method
described by Ismail et al. (2019a) in which 0.1 mL of fresh blood was
transferred to the surface of each piece of cotton fabrics (3 cm2) and in
order to fix the stain, cotton fabrics were left for 1 h in the oven (70οC) to
dry. The dried cotton pieces were dipped separately in 50 mL
screw-capped bottles with final volume of 20 mL, each containing 4 mL
of heat-inactivated commercial detergent, 4 mL of the partially purified
enzyme (50U/mL determined under the optimum conditions) or 4 mL of
heat-inactivated detergent þ 1 mL of the partially purified enzyme. The
addition of 4 mL of detergent (without inactivation) was used as a
positive control and tape water only was used as a negative control. In a
shaking water bath, all the bottles were incubated at 50οC for 30min
then rinsed with tap water and dried at 70οC for 1 h in the oven.
2.9. Statistical analysis
All data reported in this study represented the average of the results
obtained from three measurements of each experiment which originally
performed in triplicates � standard deviation.
3. Results
3.1. Enzyme activity
The bacterial isolate Bacillus licheniformis ALW1 under the described
fermentation conditions produced enzyme activity of 0.49U/mL. The
total protein content was 3.275 mg/mL and consequently, the specific
activity was calculated to be 0.151U/mg protein.
3.2. Optimization of the productivity
The enzyme production was optimized by applying one variable at a
time experiments then by applying statistical designs.
3.2.1. One-variable at a time
The results illustrated in Table (1) indicated that the optimum
enzyme activity was obtained after 5 days of fermentation and the use of
molasses (8%) and (NH4)2SO4 (0.28%) were the most effective carbon
and nitrogen sources. Additionally, the presence of wheat bran at the
4
Biocatalysis and Agricultural Biotechnology 26 (2020) 101631
concentration of 5% increased the enzyme productivity up to 17.53U/
mL while the increase in the concentration adversely affected
productivity.
3.2.2. Statistical optimization
3.2.2.1. Placket-Burman design. The results illustrated in Table (2)
indicated a variation in the observed mean values of the enzyme activity
(from 0.201U/mL to 20.045U/mL). The maximum productivity was
observed at trial number 8 in which 75 mL of the fermentation medium
composed of (%) molasses; 11, (NH4)2SO4; 0.45, wheat bran; 5,
MgSO4⋅7H2O; 0.2, KH2PO4; 0.4, NaCl; 0.2 at pH 8 was incubated for
7days at 260 rpm.
Multiple regression analysis of the resulted data was illustrated in the
supplementary Table (1), indicating that all of the tested variables
except the concentration of (NH4)2SO4 significantly affected the enzyme
productivity with R2 value of 0.999 for the applied model. The analysis
of variance (ANOVA) has been calculated for the selected model, indi­
cating the overall significance of the model since the Prob > F value was
5.96 E 12.
The correlation between the examined seven independent variables
and the enzyme activity could be presented by the first order equation as
follows:
Y ¼ 6.75052 þ 0.142962X1 þ 0.028722X2 – 0.01764X3 þ 0.190584X4
–0.13578X5 þ 0.601596X6 – 4.43348X7 (10)
where Y is the enzyme activity and X1, X2, X3, X4, X5, X6, X7 are the pH,
agitation speed, volume per flask, molasses concentration, ammonium
sulfate concentration, incubation period and dipotassium hydrogen
orthophosphate concentration respectively.
The calculated main effect of each variable was graphically repre­
sented in the supplementary Fig. (1), indicating that the fermentation
period, agitation speed and concentration of molasses were the variables
that possessed the highest main effects and all of them had positive
signs. So these three variables were examined for further optimization in
the second phase.
3.2.2.2. Box-Behnken design. The data in Table (3) represented the
mean values of protease activity (both experimental and predicted re­
sults) by applying Box-Behnken design. The optimum productivity of
protease (22.90302U/mL) was observed in trial no. 3 in which the in­
dependent variables optimized level was indicated to be: the fermen­
tation period, 7days; molasses, 8% and agitation speed, 280 rpm.
The result of the multiple regression analysis was illustrated in the
supplementary table (2) in which the R2 value was 0.933617 and
consequently, the predicted protease activity was calculated from the
following second order polynomial equation:
Y ¼ 1173.96 þ 8.205412X1 þ 8.132367X2 þ 14.73426X3 - 0.22198X21
-0.01484X22 - 1.10747X23- 0.01553X1X2 þ 0.037592X1X3 þ
0.006764X2X3 (11)
where Y is the protease activity and X1, X2 and X3 are molasses con­
centration, agitation speed and incubation period respectively.
The ANOVA indicated the overall significance of the model since the
Prob > F was 5.68 E 18. Moreover, the residual plot obtained by plotting
of the residuals versus the observed protease activity (Supplementary
Fig. 2) indicated that the values of residuals were spread symmetrically
and constantly throughout the observed range.
3.3. Partial purification
The produce enzyme under the optimized fermentation conditions
possessed activity of 22.9U/mL with total protein content of 19.4 mg/
mL and consequently, the specific activity was calculated to be 1.18U/
mg protein. The ethanol fractional precipitation led to an increase in the
M.A. Emran et al.
specific activity by 3 fold since the fraction of 60–70% ethanol possessed
specific activity of 3.49U/mg protein with 55.4% activity recovery yield.
3.4. Effect of pH and temperature
The enzyme activity at different pH was shown in Fig. 1A indicating
that the partially purified enzyme was optimally active at pH 9 using 0.1
M borax/NaOH buffer and the enzyme possessed 96% of its optimum
activity at pH 10. The stability of the enzyme at pH 9 and 10 was
determined by its pre-incubation at that pH. The enzyme retained 100%
of its activity up to 2 h for both pH 9 and 10 (Fig. 1B).
The enzyme activity was measured as a function of the reaction
temperature (Fig. 2A) indicating that the enzyme optimum activity was
70 � C and at 75 � C the enzyme possessed more than 80% of its initial
activity. The thermal stability of the enzyme indicated its ability to
retain 83% of its activity after its pre-incubation without substrate at 55
�
C for 2 h. Additionally, it retained about 90 and 50% of its activity at 60
and 65 � C for 1 h respectively (Fig. 2B).
Arrhenius plot which obtained by drawing the relation between ln of
the relative activity (%) versus the reciprocal of the temperature (in
Kelvin) is used mainly to illustrate the impact of the temperature on the
reaction rate (Fig. 3A). The slope of the observed straight line in the
figure is equal to the value of -Ea/R in which Ea is the activation energy
and R is the gas constant. The Ea value for the partial pure enzyme was
calculated to be 7.8655 � 0.8637 kJ mol 1 for casein hydrolysis be­
tween 45 and 70 � C at pH 9.
The catalytic rate of the enzyme, as well as its inactivation rate, was
activated by the temperature so the heat inactivation rate of the partial
pure enzyme was investigated at 55, 60 and 65 � C and consequently the
Kd, T1/2 and D-values were calculated and illustrated in Table (4). By
drawing the relation between lnKd versus the reciprocal of the temper­
ature (in Kelvin), the Ed value for the partial pure enzyme was calculated
to be 229.065 kJ mol 1 (Fig. 3B). The z value was calculated to be 9.346
�
C by plotting log of the D-values against the temperature (� C).
3.5. Effect of casein concentration
The activity of the partial pure enzyme was determined using
different casein concentration and the maximum activity was estimated
by using 20 mg/mL casein concentration (Fig. 4A). Additionally, the
kinetic constants were determined from Lineweaver-Burk plot (Fig. 4B).
The values for Km,Vmax and Kcat were calculated to be 3.846 mg/mL,
76.923U/mL/min and 1.206min 1 respectively.
Fig. 1. The pH effect on (A) the activity and (B) the stability of the enzyme.
5
Biocatalysis and Agricultural Biotechnology 26 (2020) 101631
3.6. The scope on the use of the produced enzyme in detergent
formulation
3.6.1. Effect of surfactants
The effect of different surfactants on the activity of the produced
enzyme was examined. The results illustrated in Table (5) indicated that
the enzyme retained more than 80 and 70% of its activity after its pre-
incubation with different surfactants for 30 min at 50 and 60 � C
respectively.
3.6.2. Compatibility with a commercial detergent
The stability of the produced enzyme against commercial detergents
was estimated by determining the residual activity of the enzyme after
its pre-incubation with Arial for 30 min at 50 � C. The result indicated
that the enzyme retained more than 80% of its initial activity.
3.6.3. Washing performance
The activity of the partial pure enzyme in the removal of protein-rich
stain (blood) was examined. The result indicated that the addition of the
enzyme with the heat-inactivated detergent had the ability to remove
the stain efficiently as that of the commercial detergent (Fig. 5).
4. Discussion
Alkaline proteases have been indicated to be an essential group of
commercial industrial enzymes as they can be exploited in various in­
dustrial processes including waste management (Delgado-García et al.,
2019), food production (Lemes et al., 2016), detergent (Ramkumar
et al., 2018; Priya et al., 2019) and leather industry (Ammasi et al.,
2020; Hakim et al., 2018). In the current study, the enzyme production
was achieved using the bacterial strain Bacillus licheniformis ALW1 by
submerged fermentation technique in which the productivity was
initially indicated to be 0.49U/mL and it was further optimized to reach
22.90302U/mL. Among all protease producing bacteria, Bacillus sp. is
the most popular commercial source for the production of alkaline
proteases but great interest is mainly focused on those with high pro­
ductivity yield (Razzaq et al., 2019). Apart from this, the optimization of
the fermentation process is necessary to maximize the yield and conse­
quently reduce the cost.
In the current study, the optimization of the fermentation process
was achieved by single factor optimization followed by applying sta­
tistical designs. By studying the influence of different carbon and ni­
trogen sources, the fermentation medium composed of (%) molasses; 8,
(NH4)2SO4; 0.3, wheat bran; 5, MgSO4⋅7H2O; 0.2, KH2PO4; 0.3, NaCl;
0.2 was conducted to be the suitable medium for the cultivation of
M.A. Emran et al. Biocatalysis and Agricultural Biotechnology 26 (2020) 101631

Fig. 2. The thermal effect on (A) the activity and (B) stability of the enzyme.

Fig. 3. Arrhenius plot for thermal (A) activation and (B) denaturation of the enzyme.

which the enzyme productivity influencing variables were initially

Table 4
indicated by Plackett–Burman design. The multiple regression analysis
The Kd, T1/2 and D-values for the enzyme.
of the results indicated the significance of all of the examined variables
Temperature (� C) Kd (min 1) T1/2 (min) D-value (min) except the concentration of (NH4)2SO4 in which the R2 value was 0.999,
55 1 � 10 3 693.1472 2302.585 indicating the high accuracy of the applied model as it suggested a
60 3 � 10 3 231.0491 767.5284 variation of 99.9% in the enzyme activity was occurred due to the
65 12 � 10 3 57.76227 191.8821
examined variables. Edwards et al. (2008) reported that the model ac­
curacy was confirmed when the R2 value was greater than 0.9. The
Bacillus licheniformis ALW1 and the production of alkaline protease. calculated main effects for the examined variables indicated that the
Molasses is a readily available byproduct of sugar industry that has been fermentation period, agitation speed and the concentration of molasses
reported to be an efficient inexpensive substrate used as a carbon source were the variables that possessed the highest main effects with positive
in the production of numerous microbial enzymes and it was used oc­ signs for all of them. The positive value indicates that the variable under
casionally in alkaline protease production (Lakshmi et al., 2018; Mian investigation exerts more effect as it adjusted at the high level so these
et al., 2018). Additionally, wheat bran has been found to be an efficient three variables were maintained at the positive level and subjected to
low-cost substrate for the production of alkaline proteases (Ahmad et al., further optimization by applying Box-Behnken design. The multiple
2020; Hammami et al., 2020; Limkar et al., 2019). The production of regression analysis of the results indicated that the R2 value for the
enzymes was also influenced by the nitrogen source (Ward, 1995). So by model was 0.933617, confirming its accuracy. Moreover, the residual
examining the effect of different nitrogen sources in the current study, plot expressed that the model was correct on average for all of the
the highest activity was achieved with inorganic salts especially observed results. The optimum productivity of the crude enzyme was
ammonium sulfate. Similar result was indicated by Lakshmi et al. (2014) 22.903U/mL, achieved under the optimized fermentation medium
for alkaline protease production by Bacillus licheniformis MTCC NO. composed of (g%) molasses; 8, (NH4)2SO4; 0.45, wheat bran; 5,
7053. In a subsequent step, statistical optimization was carried out in MgSO4⋅7H2O; 0.2, KH2PO4; 0.4 and NaCl; 0.2 with initial pH 8 incu­
bated for 7days at 37 � C and 280 rpm. The achieved optimum

6
M.A. Emran et al. Biocatalysis and Agricultural Biotechnology 26 (2020) 101631

Fig. 4. The profile of the enzyme activity by using different casein concentrations (A) and Lineweaver-Burk plot (B).

so the optimum temperature of the produced enzyme suggested it as a

Table 5
suitable candidate in numerous applications. The thermal stability study
The effect of surfactants.
indicated the ability of the enzyme to retain 83% of its activity after
Surfactant Residual activity (%) pre-incubation at 55 � C for 2 h. Additionally, it retained about 90 and
50 � C 60 � C 50% of its activity after pre-incubation at 60 and 65 � C for 1 h respec­
Non-ionic 85.168 � 1.557 70.870 ± 4.877
tively. Thermal stability studies for alkaline proteases indicated that the
Anionic 86.625 � 1.097 71.178 ± 1.038 activity of the enzyme produced by Bacillus invictae AH1 decreased to
Cationic 83.826 ± 1.645 76.059 � 3.884 10.48% of its maximal activity after pre-incubation at 60 � C for 1 h
(Hammami et al., 2020) while the enzyme produced by Geobacillus
SBS-4S (a subgroup of Bacillus) retained 50% of its activity after
productivity of the crude enzyme was quite high as that reported by
pre-incubation at 60 � C for 110min (Ahmad et al., 2020) and Gaonkar
Hammami et al. (2020) for Bacillus invictae AH1 (38.633U/mL) but
and Furtado (2020) reported that the produced enzyme by Halococcus
extremely higher than that reported for other Bacillus sp., 1.023U/mL
agarilyticus GUGFAWS-3 retained 50.26% of its activity after
reported for Bacillus licheniformis MTCC NO. 7053 (Lakshmi and
pre-incubation at 70 � C for 75min. Additionally, in the current study, the
Hemalatha, 2016), 1.032U/mL reported for Bacillus cereus FT1 (Asha
calculated half lives were 693.15, 231.05 and 57.76 min 1 at 55, 60 and
and Palaniswamy, 2018), 1.407U/mL reported for Bacillus subtilis
65 � C respectively. Harer et al. (2018) reported that the half lives for
MN173351 (Priya et al., 2019) and 0.571U/mL reported for Bacillus
heat-stable alkaline proteases were greater than 200min at 50 � C and in
cereus HP-RZ17 (Jadhav et al., 2020).
the range from 2 to 22min at 60 � C, confirming the thermo-stability of
From all the fractions precipitated using ethanol or acetone and
the enzyme produced in the current study. The pH and the thermal
salted out using ammonium sulfate, the fraction obtained by 60–70%
stability profiles of Bacillus licheniformis ALW1 protease support its
ethanol precipitation with specific activity 3.49U/mg protein and 55.4%
applicability in detergents. Hammami et al. (2020) and Mhamdi et al.
activity recovery yield was partially characterized, the partial pure
(2017) reported that the detergent compatible enzymes have to possess
enzyme was indicated to be optimally active at pH 9 at which the
high pH and thermal stability properties.
enzyme was completely stable for 2 h. This result is consistent with other
By the drawing of Arrhenius plot for the enzyme thermal activation
researches concerned with proteases from Bacillus sp. (Ahmad et al.,
and denaturation, Ea and Ed values for the partial pure enzyme were
2020; Hakim et al., 2018; Hammami et al., 2020). By measuring the
calculated to be 7.8655 and 229.065 kJ mol 1respectively. The lower
enzyme activity at different reaction temperatures, the optimum activity
the value of the Ea indicates the lower energy required for the confor­
was indicated at 70 � C. This result was higher than 40 � C (Hakim et al.,
mation of the enzyme active sites for the complex formation with the
2018; Ramkumar et al., 2018), 45 � C (Harer et al., 2018), 55 � C (Gulmus
substrate that consequently led to the reduction of the overall cost of the
and Gormez, 2020; Zheng et al., 2020) and 60 � C (Ahmad et al., 2020;
enzyme. Additionally, the higher the value of the Ed indicates the
Hammami et al., 2020; Iqbalsyah et al., 2019) reported for bacterial
thermal adaptation of the enzyme as a result of the decrease in the rate
alkaline proteases but the similar result was reported by Gaonkar and
of the unfolding of the enzyme (Jana et al., 2013). The kinetic constants
Furtado (2020) and Lakshmi et al. (2018). In some biotechnological
for the enzyme were calculated on the base of the Lineweaver-Burk plot.
applications high temperatures are required to achieve optimal results

Fig. 5. Washing performance for a stain of blood (A) using tap water as a negative control (B), commercial Arial as a positive control (C), heat-inactivated Arial (D),
the produced partial pure enzyme (E), a mixture of the heat-inactivated Arial and the produced partial pure enzyme (F).

7
M.A. Emran et al.
The Km and Vmax values were calculated to be 3.846 mg/mL and
76.923U/mL/min respectively. The Km and Vmax values depend mainly
on the source of the enzyme reflecting the sensitivity of the produced
enzyme toward the substrate i.e. the sensitivity of the produced enzyme
toward the substrate increases as the Km increase and the Vmax decrease
(Horn et al., 2006). Lakshmi et al. (2018) reported that Km and Vmax
values were 0.25 mg/mL and 310U/mL respectively for Bacillus cereus
strain S8 alkaline protease. Ramkumar et al. (2018) reported that Km
and Vmax values were 0.4384 mg/mL and 88.57U/mL respectively for
the enzyme produced by Bacillus licheniformis NK while Gaonkar and
Furtado (2020) reported that Km and Vmax values were 3.99 mg/mL and
41.49U/mL respectively for the enzyme produced by Halococcus agar­
ilyticus GUGFAWS-3.
The partially purified enzyme retained more than 80% of its activity
after pre-incubation for 30min at 50 � C with different surfactants as well
as with one of the popular commercial detergents (Arial). The charac­
teristics of the enzyme produced by Bacillus licheniformis ALW1 sug­
gested its applicability in detergent formulations. So its contribution to
the removal of blood stain was examined, confirming that the addition
of the produced enzyme to the detergent formulation improved its
washing performance. Similar behavior has been observed for other
Bacillus sp. proteases (Harer et al., 2018; Priya et al., 2019).
5. Conclusion
In this study, low-cost production of alkaline protease was achieved
under submerged fermentation technique using the bacterial strain Ba­
cillus licheniformis ALW1. The productivity was optimized by applying
one variable at a time approach followed by applying a statistical
technique in which the productivity was increased to reach 22.90302U/
mL. The partially purified enzyme was a thermophilic enzyme with
optimum activity at 70 � C and half-lives of 693.15, 231.05 and
57.76min 1at 55, 60 and 65 � C respectively. These characteristics sug­
gest it as a suitable candidate in several applications in which high
temperature is required to achieve optimal results. Additionally, the
enzyme was stable with a commercial detergent as it retained more than
80% of its activity after pre-incubation for 30min at 50 � C. Finally, the
addition of the enzyme to the detergent formulation improved its
washing performance in the removal of blood stains.
CRediT authorship contribution statement
Mohamed A. Emran: Methodology, Writing - review & editing.
Shaymaa A. Ismail: Methodology, Formal analysis, Writing - original
draft. Amal M. Hashem: Supervision.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bcab.2020.101631.
References
Abdel-Fattah, A.M., El-Gamal, M.S., Ismail, S.A., Emran, M.A., Hashem, A.M., 2018.
Biodegradation of feather waste by keratinase produced from newly isolated Bacillus
licheniformis ALW1. J. Genet. Eng. Biotechnol. 16 (2), 311–318.
Adetunji, A.I., Olaniran, A.O., 2020. Statistical modelling and optimization of protease
production by an autochthonous Bacillus aryabhattaiAb15-ES: a response surface
methodology approach. Biocatal. Agric.Biotechnol. 101528.
Ahmad, W., Tayyab, M., Aftab, M.N., Hashmi, A.S., Ahmad, M.D., Firyal, S., Wasim, M.,
Awan, A.R., 2020. Optimization of conditions for the higher level production of
protease: characterization of protease from Geobacillus SBS-4S. Waste Biomass
Valores 1–11.
Ammasi, R., Victor, J.S., Chellan, R., Chellappa, M., 2020. Alkaline protease for an
efficacious rehydration of skin matrix by a novel Bacillus crolab MTCC 5468 in
sustainable leather production: a green approach. Biotechnol. Lett. 42 (2), 249–267.
Asha, B., Palaniswamy, M., 2018. Optimization of alkaline protease production by
Bacillus cereus FT 1 isolated from soil. J. Appl. Pharmaceut. Sci. 8 (2), 119–127.
Barrett, A.J., McDonald, J.K., 1986. Nomenclature: protease, proteinase and peptidase.
Biochem. J. 237 (3), 935.
8
Biocatalysis and Agricultural Biotechnology 26 (2020) 101631
Box, G.E., Behnken, D.W., 1960. Some new three level designs for the study of
quantitative variables. Technometrics 2 (4), 455–475.
Box, G.E., Wilson, K.B., 1951. On the experimental attainment of optimum conditions.
J. Roy. Stat. Soc. B 13 (1), 1–38.
Breithaupt, H., 2001. The hunt for living gold. EMBO Rep. 2 (11), 968–971.
Delgado-García, M., Flores-Gallegos, A.C., Kirchmayr, M., Rodríguez, J.A., Mateos-
Díaz, J.C., Aguilar, C.N., Muller, M., Camacho-Ruíz, R.M., 2019. Bioprospection of
proteases from Halobacillusandaensis for bioactive peptide production from fish
muscle protein. Electron. J. Biotechnol. 39, 52–60. .
Desai, K.M., Survase, S.A., Saudagar, P.S., Lele, S.S., Singhal, R.S., 2008. Comparison of
artificial neural network (ANN) and response surface methodology (RSM) in
fermentation media optimization: case study of fermentative production of
scleroglucan. Biochem. Eng. J. 41 (3), 266–273.
Dias, D.R., Vilela, D.M., Silvestre, M.P.C., Schwan, R.F., 2008. Alkaline protease from
Bacillus sp. isolated from coffee bean grown on cheese whey. World J. Microbiol.
Biotechnol. 24, 2027–2034.
Edwards, L.J., Muller, K.E., Wolfinger, R.D., Qaqish, B.F., Schabenberger, O., 2008. An
R2 statistic for fixed effects in the linear mixed model. Stat. Med. 27 (29),
6137–6157.
Gaonkar, S.K., Furtado, I.J., 2020. Characterization of extracellular protease from the
haloarcheonHalococcus sp. strain GUGFAWS-3 (MF425611). Curr. Microbiol. 1–11.
Gulmus, E.O., Gormez, A., 2020. Characterization and biotechnological application of
protease from thermophilic Thermomonashaemolytica. Arch. Microbiol. 202 (1),
153–159.
Hakim, A., Bhuiyan, F.R., Iqbal, A., Emon, T.H., Ahmed, J., Azad, A.K., 2018. Production
and partial characterization of dehairing alkaline protease from Bacillus subtilis
AKAL7 and ExiguobacteriumindicumAKAL11 by using organic municipal solid wastes.
Heliyon 4 (6), e00646.
Hammami, A., Bayoudh, A., Hadrich, B., Abdelhedi, O., Jridi, M., Nasri, M., 2020.
Response-Surface Methodology for the production and the purification of a new
H2O2-tolerant alkaline protease from Bacillus invictae AH1 strain. Biotechnol. Prog.,
e2965
Harer, S.L., Bhatia, M.S., Bhatia, N.M., 2018. Isolation, purification and partial
characterization of thermostable serine alkaline protease from a newly isolated
Bacillus thuringinsis-SH-II-1A. Afr. J. Biotechnol. 17, 178–188.
Hashem, A.M., Abdel-Fattah, A., Ismail, S., El-Gamal, M., Esawy, M., Emran, M.A.,
2018a. Optimization, characterization and thermodynamic studies on B.licheniformis
ALW1 keratinase. Egypt. J. Chem. 61 (4), 591–607.
Hashem, A.M., Ismail, S., Hosny, A.E.D., Awad, G., Ismail, S., 2018b. Optimization of
Dothideomycetes sp. NrC-SSW chitosanase productivity and activity using response
surface methodology. Egypt. J. Chem. 61 (6), 973–987.
Horn, S.J., Sørlie, M., Vaaje-Kolstad, G., Norberg, A.L., Synstad, B., Vårum, K.M.,
Eijsink, V.G.H., 2006. Comparative studies of chitinases A, B and C from
Serratiamarcescens. Biocatal. Biotransform. 24 (1–2), 39–53.
Iqbalsyah, T.M., Malahayati, M., Atikah, A., Febriani, F., 2019. Cultivation conditions for
protease production by a thermo-halostable bacterial isolate PLS A. J. Natah 19 (1),
18–23.
Ismail, S.A., 2019. Microbial valorization of shrimp byproducts via the production of
thermostable chitosanase and antioxidant chitooligosaccharides. Biocatal. Agric.
Biotechnol. 20, 101269.
Ismail, S.A., Hassan, A.A., Emran, M.A., 2019a. Economic production of thermo-active
endo β-mannanase for the removal of food stain and production of antioxidant
manno-oligosaccharides. Biocatal. Agric. Biotechnol. 22, 101387.
Ismail, S.A., Hassan, M.E., Hashem, A.M., 2019b. Single step hydrolysis of chitin using
thermophilic immobilized exochitinase on carrageenan-guar gum gel beads.
Biocatal. Agric. Biotechnol. 21, 101281.
Ismail, S.A., Serwa, A., Abood, A., Fayed, B., Ismail, S.A., Hashem, A.M., 2019c. A study
of the use of deep artificial neural network in the optimization of the production of
antifungal exochitinase compared with the response surface methodology. JJBS 12
(5), 543–551.
Jadhav, H.P., Sonawane, M.S., Khairnar, M.H., Sayyed, R.Z., 2020. Production of
alkaline protease by rhizosphericBacillus cereus HP_RZ17 and
Paenibacillusxylanilyticus HP_RZ19. Environ. Sustain 1–9.
Jana, A., Maity, C., Halder, S.K., Das, A., Pati, B.R., Mondal, K.C., Mohapatra, P.K.D.,
2013. Structural characterization of thermostable, solvent tolerant, cytosafetannase
from Bacillus subtilis PAB2. Biochem. Eng. J. 77, 161–170.
Joo, H.S., Kumar, C.G., Park, G.C., Kim, K.T., Paik, S.R., Chang, C.S., 2002. Optimization
of the production of an extracellular alkaline protease from Bacillus horikoshii.
Process Biochem. 38 (2), 155–159.
Lakshmi, B.K.M., Sri, P.R., Devi, K.A., Hemalatha, K.P.J., 2014. Media optimization of
protease production by Bacillus licheniformis and partial characterization of alkaline
protease. Int. J. Curr. Microbiol. App. Sci. 3 (5), 650–659.
Lakshmi, B.K.M., Kumar, D.M., Hemalatha, K.P.J., 2018. Purification and
characterization of alkaline protease with novel properties from Bacillus cereus strain
S8. J. Genet. Eng. Biotechnol. 16 (2), 295–304.
Lakshmi, B.K.M., Hemalatha, K.P.J., 2016. Production of alkaline protease from Bacillus
licheniformis through statistical optimization of growth media by response surface
methodology. Ferment. Technol. 5 (2), 130–137. .
Lemes, A.C., Pav� on, Y., Lazzaroni, S., Rozycki, S., Brandelli, A., Kalil, S.J., 2016. A new
milk-clotting enzyme produced by Bacillus sp. P45 applied in cream cheese
development. LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 66,
217–224. .
Limkar, M.B., Pawar, S.V., Rathod, V.K., 2019. Statistical optimization of xylanase and
alkaline protease co-production by Bacillusspp using Box-Behnken Design under
submerged fermentation using wheat bran as a substrate. Biocatal. Agric. Biotechnol.
17, 455–464.
M.A. Emran et al.
Lineweaver, H., Burk, D., 1934. The determination of enzyme dissociation constants.
J. Am. Chem. Soc. 56 (3), 658–666.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with
folin phenol reagent. J. Boil. Chem. 193, 265–275.
McDonald, C.E., Chen, L.L., 1965. The Lowry modification of the Folin reagent for
determination of proteinase activity. Anal. Biochem. 10 (1), 175–177.
Mhamdi, S., Ktari, N., Hajji, S., Nasri, M., Kamoun, A.S., 2017. Alkaline proteases from a
newly isolated Micromonosporachaiyaphumensis S103: characterization and
application as a detergent additive and for chitin extraction from shrimp shell waste.
Int. J. Biol. Macromol. 94, 415–422.
Mian, M.M., Mamun, M.A., Khan, S.N., Hoq, M.M., 2018. Efficient medium for protease
production by Bacillus licheniformis mzk05m9 optimized through response surface
methodology. Microb. Bioact. 1 (1), 22–28.
Niyonzima, F.N., More, S.S., 2015. Microbial detergent compatible lipases. J. Sci. Ind.
Res. 74 (2), 105–113.
Olajuyigbe, F.M., Falade, A.M., 2014. Purification and partial characterization of serine
alkaline metalloprotease from Bacillus brevis MWB-01. Bioresour. Bioprocess. 1 (1),
8.
Plackett, R.L., Burman, J.P., 1946. The design of optimum multifactorial experiments.
Biometrika 33 (4), 305–325.
Biocatalysis and Agricultural Biotechnology 26 (2020) 101631
Priya, K., Jayanthi, J., Gayathiri, E., Ragunathan, M., 2019. Screening and optimization
of protease producing bacterial isolate from the gut of Portunuspelagicus and its
prospecting potential in detergent industry. Uttar Pradesh J. Zool. 40 (4), 187–199.
Qureshi, A.S., Bhutto, M.A., Khushk, I., Dahot, M.U., 2011. Optimization of cultural
conditions for protease production by Bacillus subtilis EFRL 01. Afr. J. Biotechnol. 10
(26), 5173–5181.
Qureshi, A.S., Simair, A.A., Ali, C.H., Khushk, I., Khokhar, J.A., Ahmad, A., Danish, M.,
Lu, C., 2018. Production, purification and partial characterization of organo-solvent
tolerant protease from newly isolated Bacillus sp. BBXS-2. Ferment. Technol. 7 (1),
151.
Ramkumar, A., Sivakumar, N., Gujarathi, A.M., Victor, R., 2018. Production of
thermotolerant, detergent stable alkaline protease using the gut waste of
Sardinellalongicepsas a substrate: optimization and characterization. Sci. Rep. 8 (1),
1–15.
Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., Ashraf, M., 2019. Microbial
proteases applications. Front.Bioeng. Biotech. 7, 110.
Ward, N.E., 1995. With dietary modifications, wheat can be used for poultry. Feedstuffs
67 (33), 1–4.
Zheng, L., Yu, X., Wei, C., Qiu, L., Yu, C., Xing, Q., Fan, Y., Deng, Z., 2020. Production
and characterization of a novel alkaline protease from a newly isolated
Neurosporacrassa through solid-state fermentation. Lebensm. Wiss. Technol. 122,
108990. .