Vol.1, No.4, 1 12-120 (2013) Advances in Enzyme Research
http://dx.doi.org/10.4236/aer.2013.14012
Optimized production and properties of thermost able
alkaline protease from Bacillus subtilis SHS-04
grown on groundnut (Arachis hypogaea) meal
Folasade M. Olajuyigbe
Department of Biochemistry, School of Sciences, Federal University of Technology, Akure, Nigeria;
Received 10 July 2013; revised 19 August 2013; accepted 5 September 2013
Copyright © 2013 Folasade M. Olajuyigbe. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Production of alkaline protease from Bacillus
subtilis SHS-04 w as investigated under different
fermentation conditions involving low-cost sub-
strates with the aim of optimizing yield of en-
zyme. Maximum enzyme production (1616.21
U/mL) was achieved using groundnut meal
(0.75%) as nitrogen source and 0.5% glucose as
carbon source at 48 h cultivation period, pH 9,
45˚C and 200 rpm. The yield was 348% increase
over comparable control samples. The alkaline
protease had optimum temperature of 60˚C and
remarkably exhibited 80% relative activity at
70˚C. It was highly thermostable showing 98.7%
residual activity at 60˚C after 60 minutes of in-
cubation at pH 9.0 and was stable in the pres-
ence of organic solvents studied. These proper-
ties indicate the viability of the protease for
biotechnological and industrial applications.
The optimized yield of enzyme achieved in this
study establishes groundnut meal as potential
low-cost substrate for alkaline protease produc-
tion by B. subtilis SHS-04.
Keywords: Alkaline Protease; Bacillus subtilis
SHS-04; Groundnut (Arachis hypogaea) Meal;
Low-Cost Substrate; Thermostable
1. INTRODUCTION
Proteases constitute one of the most important groups
of global industrial enzymes accounting for about 60%
of the total enzyme sales [1]. Among the different types
of proteases, alkaline proteases have wide applications in
the detergent, leather and pharmaceutical industries [2].
Bacterial proteases are the most significant, compared
with plants, animal and fungal proteases because they are
mostly extracellular and easily produced in large amount,
but their usefulness is limited by various factors, such as
instability at high temperatures, extreme pH, the pres-
ence of organic solvents and the need for co-factors [3].
The present known proteases are not sufficient to meet
industrial demands hence, there is a continuous search
for new proteases with novel characteristics for industrial
applications from diverse bacteria isolates.
Enzyme production by any organism is influenced by
many factors such as pH, incubation temperature, incu-
bation time, growth rate of culture and medium composi-
tion [4]. Currently, the overall cost of enzyme production
is high and the growth medium accounts for about 30% -
40% of the production cost [5]. Reducing the cost of en-
zyme production by optimization of fermentation me-
dium and process parameters is the major goal of basic
research for industrial applications [6]. To this extent, the
search for inexpensive carbon and nitrogen sources such
as agricultural residues and marine by-products for use in
media composition for protease production has been
continuous.
Maximum protease synthesis by alkalophilic Bacillus
sp. I-312 was obtained when the bacterium was grown in
a medium containing wheat flour and soybean meal as
carbon and nitrogen sources [7]. Bacillus licheniformis
RP1 was shown to produce proteases when grown in
media containing shrimp wastes powder as a sole carbon
and nitrogen source [8]. Mirabilis ja lap a tuber powder
(MJTP) was used as organic substrate for the growth and
production of fibrinolytic serine protease by Bacillus
amyloliquefaciens An6 [9].
Groundnut (Arachis hypogaea) is a species in the
legume family, Fabaceae, native to South America and
cheaply cultivated in African, Asian and Arab countries
[10]. Groundnut provides an inexpensive source of high
quality dietary protein and oil. Groundnut seed contains

F. M. Olajuyigbe / Advances in Enzyme Research 1 (2013) 112-120 113
25% to 28% protein and 46% to 51% oil on a dry seed
basis and it is a rich source of minerals (phosphorus, cal-
cium, magnesium, and potassium) and vitamins (E, K,
and B group) [11-13]. The proximate analysis and nutria-
tional value of groundnut suggest groundnut meal as
potential low-cost substrate in media composition for
microbial growth and enzyme production. In this study,
effects of low-cost substrates including groundnut meal
and process parameters were investigated on protease
production and properties of the crude enzyme were de-
termined.
2. MATERIALS AND METHODS
2.1. Materials
Media components were products of Sigma-Aldrich
(St Louis, MO, USA) except groundnut seeds, soybean
meal, yam (Dioscorea alata) flour and locust bean meal
which were purchased from the local market and proc-
essed using standard sieve. Groundnut meal was pre-
pared by sun drying fresh groundnut seeds for two weeks.
The dried groundnut seeds were pulverized and stored in
glass bottles at room temperature. All other chemicals
used were of analytical grade and obtained from Fisher
Scientific.
2.2. Bacterial Strain
The microorganism used in this study was an alka-
liphilic bacterium isolated from the soil of a slaughter-
house. Samples collected were plated onto skim-milk
agar plates and were incubated 24 h at 37˚C. A clear zone
of skim-milk hydrolysis gave an indication of prote-
ase-producing strains. Individual colonies were purified
through repeated streaking on fresh agar plates. Depend-
ing upon the zone of clearance, isolate SHS-04 was se-
lected for further experimental studies. This isolate was
identified as B. subtilis in the Biotechnology Unit of the
Federal Institute of Industrial Research, Lagos, Nigeria
based on methods described in Bergey’s Manual of Sys-
tematic Bacteriology [14] and maintained on nutrient
agar slants stored at 4˚C.
2.3. Submerged Fermentation
Seed inoculum was prepared by growing a loopful of
slant culture of B. subtilis SHS-04 in 20 mL of seed me-
dium containing 0.75% (w/v) peptone, 0.5% (w/v) glu-
cose, 0.05% (w/v) NaCl and 0.01% (w/v) MgSO4.7H2O
in a 200 mL conical flask with pH adjusted to 8.0. The
inoculated seed medium was incubated at 37˚C for 24 h
at 180 rpm in a shaking incubator (Stuart, UK). The 24 h
old seed culture was used as inoculum for the production
media. 2.5 mL of seed inoculum (constituting 5% v/v)
was transferred into 50 mL of production media which
had same composition as the seed medium. At the end of
48 h incubation period, cultures were harvested by cen-
trifugation at 10,000 rpm for 15 min at 4˚C. The cell free
supernatant was recovered as crude enzyme preparation
and used for further studies.
2.4. Assay of Protease Activity
Protease activity was determined by a modified pro-
cedure based on Fujiwara et al. [15] using 1.0% casein in
50 mM Glycine-NaOH buffer (pH 9.0) as substrate. The
assay mixture consisted of 2.0 mL of substrate and 0.5
mL of enzyme solution in 50 mM Glycine-NaOH buffer
(pH 9.0). The reaction mixture was incubated at 40˚C for
30 min and reaction was terminated by the addition of
2.5 mL of 10% (w/v) trichloroacetic acid. The mixture
was allowed to stand for 15 min and then centrifuged at
10,000 rpm for 10 min at 4˚C to remove the resulting
precipitate. Protease activity was determined by estimat-
ing the amount of tyrosine in the supernatant which was
done by measuring the absorbance at 280 nm. One unit
of protease activity was defined as the amount of enzyme
required to release 1 μg of tyrosine per mL per minute
under the mentioned assay conditions.
2.5. Determination of Growth Kinetics and
Protease Production of B. subtilis
SHS-04
Growth kinetics over the cultivation period and pro-
duction of protease were studied by cultivating the bac-
teria for 12, 24, 36, 48, 60, 72 and 84 h at pH 9.0 and
45˚C at 200 rpm. The growth of the microorganism was
determined by measuring the absorbance of culture at
600 nm. The cultures were centrifuged at the end of each
cultivation period and the supernatants were used for
determination of protease activity which was the measure
of protease production.
2.6. Effect of Temperature on Growth and
Protease Production
Optimal temperature for growth and protease produc-
tion by B. subtilis SHS-04 was determined by investi-
gating growth kinetics and protease production at fixed
media concentration and pH with varying temperatures.
Cultures were grown at 20˚C, 30˚C, 37˚C, 40˚C, 45˚C,
50˚C, and 60˚C at 200 rpm, pH 9.0 for 48 h which was
the cultivation period for optimal growth of microorgan-
ism and protease production obtained earlier in this study.
2.7. Effect of pH on Growth and Protease
Production
Cultures of B. subtilis SHS-04 were grown at fixed
media concentration and temperature, 45˚C and varying

F. M. Olajuyigbe / Advances in Enzyme Research 1 (2013) 112-120
114
pH of 6.0 to 11.0 at 200 rpm for 48 h using 50 mM of the
following buffer solutions: sodium citrate (pH 6.0),
Tris-HCl (pH 7.0 to 8.0) and glycine-NaOH (pH 9.0 to
11.0) for media preparation. Optimal pH for growth and
protease production by B. subtilis SHS-04 was deter-
mined at the end of the cultivation period by measuring
the absorbance of culture at 600 nm and determining
protease activity in the cell free supernatant obtained
after centrifugation at 10,000 rpm and 4˚C for 15 min.
2.8. Effect of Nitrogen Sources on Growth
Kinetics and Protease Production
Protease production from B. subtilis SHS-04 was in-
vestigated using various nitrogen sources which included
inexpensive and readily available groundnut meal, locust
bean meal and soybean meal. Other nitrogen sources
tested were gelatin, beef extract and peptone. Effects of
various nitrogen sources (0.75%, w/v) on growth and
protease production were studied at pH 9.0 and tempera-
ture, 45˚C. Groundnut meal, beef extract, locust bean
meal, gelatin, yeast extract and soybean meal were used
to substitute peptone in different production media. Cul-
tures were grown for 48 h at 200 rpm; growth and prote-
ase production were measured at the end of the cultiva-
tion period to determine the best nitrogen sources for
optimal enzyme production.
2.9. Effect of Carbon Sources on Growth
Kinetics and Protease Production
Various carbon sources were investigated for their ef-
fects on protease production by B. subtilis SHS-04 at pH
9.0 and temperature, 45˚C. The carbon sources (0.5%,
w/v) tested are fructose, maltose, lactose, yam flour, su-
crose and soluble starch. These were used to replace
glucose in different production media. Cultures were
grown for 48 h at 200 rpm. Growth and protease produc-
tion were measured at the end of the cultivation period to
determine the best carbon sources for optimal enzyme
production.
2.10. Effect of Temperature on Activity and
Stability of Protease
Effect of temperature on activity of crude enzyme was
determined by incubating the reaction mixture at tem-
peratures ranging from 30˚C to 80˚C for 30 min in the
presence and absence of 5 mM CaCl2 then the activity of
the protease was measured. The thermal stability was
determined by incubating the crude protease at tempera-
tures ranging from 50˚C to 80˚C in the presence and ab-
sence of 5 mM CaCl2 for 30, 60, 90 and 120 min, respec-
tively and the residual protease activity was determined
according to the standard assay procedure.
2.11. Effect of pH on Activity and Stability of
Protease
The effect of pH on activity of protease was deter-
mined by assaying for enzyme activity at different pH
values ranging from 4.0 to 12.0. The pH was adjusted
using 50 mM of the following buffer solutions: sodium
acetate (pH 4.0 to 5.0), sodium citrate (6.0), Tris-HCl
(pH 7.0 to 8.0) and glycine-NaOH (pH 9.0 to 12.0). Re-
action mixtures were incubated at 40˚C for 30 min and
the activity of the protease was measured. To determine
the effect of pH on stability of protease, the protease was
incubated in relevant buffers of varying pH (4.0 to 12.0)
without substrate for 60 min at 40˚C. The residual prote-
ase activity was determined as described earlier.
2.12. Effects of Organic Solvents on Activity
and Stability of Protease
Effects of glycerol, methanol, isopropanol, dimethyl
sulfoxide (DMSO), benzene, and acetone on crude pro-
tease activity were studied by introducing the selected
organic solvent into the reaction mixture at a final con-
centration of 25% v/v and protease activity was deter-
mined according to the standard assay procedure. Or-
ganic solvent stability of protease was investigated by
pre-incubating 0.75 mL of crude protease with 0.25 mL
of organic solvent at 40˚C for 30 min with shaking. The
residual protease activity was determined according to
the standard assay procedure and compared with the
control. Distilled water was used to replace organic sol-
vent in the control.
3. RESULTS AND DISCUSSION
3.1. Growth Kinetics of B. subtilis SHS-04
and Protease Production
Growth kinetics of B. subtilis SHS-04 and protease
production were studied to determine the cultivation pe-
riod that favours maximum yield of enzyme and evaluate
the effect of growth of organism on protease production.
Growth of organism was exponential up to 48 h, fol-
lowed by a stationary phase (Figure 1). Protease produc-
tion started in the exponential phase corresponding with
growth of organism and optimum production of 464.5 U/
mL was evident at 48 hour which was the late exponen-
tial phase of B. subtilis SHS-04. This suggests that pro-
duction of protease by this Bacillus sp. is dependent on
cell growth. Incubation time has been reported to play
substantial role in enzyme production [4,16,17]. There
was decline in protease production after 48 h (Figure 1)
which could be due to autolysis of the protease which
was consequent upon accumulation of the enzyme in the
production media.

F. M. Olajuyigbe / Advances in Enzyme Research 1 (2013) 112-120 115
Figure 1. Growth kinetics of B. subtilis SHS-04 and protease
production over 84 h cultivation period. Protease activity
(U/mL) was used to measure protease production. Symbols and
bars represent mean values and standard deviations of triplicate
determinations.
3.2. Effects of Temperature on Growth and
Production of Protease
In bioprocesses, specific temperature requirement and
its regulation is one of the most critical parameters [18].
B. subtilis SHS-04 exhibited cell growth and protease
production at all temperatures studied (20˚C to 60˚C).
Optimum temperature for cell growth was 37˚C but op-
timum protease production was recorded at 45˚C (Figure
2). Sepahy and Jabalameli [19] reported a similar finding
on a Bacillus sp. with maximum protease production at
45˚C. Gouda [20] reported a lower optimum temperature
of 30˚C for protease production by Bacillus sp. MIG and
Nascimento [21] reported a higher optimum tempera-
ture of 60˚C for Bacillus sp. SMIA-2, respectively. Cul-
tivation temperature affects protein synthesis by influ-
encing rate of biochemical reactions within the cell and
consequently inducing or repressing enzyme production
[22].
3.3. Effects of pH on Growth and Production
of Protease
B. subtilis SHS-04 grew over the entire pH 6.0 - 11.0
studied with optimum growth recorded at pH 9.0. Prote-
ase production followed same trend as cell growth with
optimum enzyme production of 462.6 U/mL at pH 9.0
(Figure 3). Cell growth and protease production declined
after pH 9.0. Bacillus sp. strain APP1 also grew well in
pH range 5.0 - 12.0 but demonstrated optimum protease
production at pH 9.0 [23]. Similar finding was reported
by Joshi et al. [24] on Bacillus cereus which showed
Figure 2. Effects of temperature on growth and protease pro-
duction over 48 h cultivation period. Symbols and bars repre-
sent mean values and standard deviations of triplicate determi-
nations.
Figure 3. Effects of pH on growth and protease production
over 48 h cultivation period. Symbols and bars represent mean
values and standard deviations of triplicate determinations.
maximum protease production at pH 9.0. Some have
reported optimum protease production at pH 7.0 and 8.0
[21,25]. Further studies to optimize protease production
from B. subtilis SHS-04 were carried out at pH 9.0. The
pH of culture medium has earlier been reported to have
strong influence on metabolic processes within the cell
[26].
3.4. Effects of Nitrogen Sources on Growth
and Production of Protease
All the nitrogen sources studied supported cell growth
and protease production from B. subtilis SHS-04 at pH

F. M. Olajuyigbe / Advances in Enzyme Research 1 (2013) 112-120
116
9.0 and temperature 45˚C. However, maximum enzyme
production of 1616.2 U/mL was achieved with ground-
nut meal as nitrogen source (Figure 4). This was 348%
increase in protease production over what was obtained
in the basal media which contained peptone as nitrogen
source. Groundnut meal served as complex organic ni-
trogen source and also provided vitamins and minerals
[13] which in this study promoted growth and enzyme
production in B. subtilis SHS-04. The stimulative role of
oil seed cakes on alkaline protease production has been
reported in some studies [27]. Beef extract and soybean
meal supported 234% and 145% increase in protease
yield over peptone. These findings show that organic
nitrogen sources are effectively utilized for protease
production [4]. Joo and Chang [7] reported optimum
protease production from Bacillus sp. I-312 in a medium
containing wheat flour and soybean meal as the carbon
and nitrogen sources. Furthermore, protease production
from Bacillus cereus MCM B-326 using media contain-
ing deoiled groundnut cakes have been reported but with
enzyme yield below 200 U/mL [28]. The protease pro-
duction by B. subtilis SHS-04 using groundnut meal as
nitrogen source in the present study has greatly enhanced
protease production more than other nitrogen sources
studied. These results indicate that groundnut meal is an
excellent low-cost and readily available substrate for
protease production by B. subtilis SHS-04.
3.5. Effects of Carbon Sources on Growth
and Production of Protease
B. subtilis SHS-04 grew and produced protease in the
presence of various carbon sources tested (Figure 5).
Figure 4. Effects of nitrogen sources on growth and protease
production. Symbols and bars represent mean values and stan-
dard deviations of triplicate determinations.
Figure 5. Effects of carbon sources on growth and protease
production. Symbols and bars represent mean values and stan-
dard deviations of triplicate determinations.
However, only glucose and lactose were most effectively
utilized by the organism for protease production with
optimum enzyme production of 465.2 U/mL in the pres-
ence of 0.5% glucose and 415.0 U/mL with 0.5% lactose.
Protease production was repressed in the presence of
fructose, maltose and soluble starch (Figure 5). Our re-
sults agree with some earlier reports where production of
extracellular protease was enhanced in the presence of
glucose as carbon sources [7,29]. However, other previ-
ous studies have reported the repression of protease pro-
duction by glucose [24,30].
3.6. Thermostability of Crude Protease
It has been well reported that the stability of alkaline
proteases is dependent on calcium ion [31-33]. It is
highly remarkable that alkaline protease from B. subtilis
SHS-04 demonstrated unique stability both in the pres-
ence and absence of Ca2+ with optimum temperature of
60˚C as shown in Figure 6. The protease had relative
activity of 80% at 70˚C in the absence of Ca2+ and 89 %
relative activity in the presence of Ca2+. The enzyme
retained 76% of its original activity at 80˚C in the pres-
ence of 5 mM Ca2+ and exhibited 46% relative activity in
the absence of calcium ion at 80˚C (Figure 6). Some
reports on crude and purified thermo tolerant protease
show considerable activity over 40˚C to 65˚C but are
completely inactivated at 80˚C [25,34,35]. The protease
under study showed very high thermostability retaining
99% of its original activity at 60˚C after 60 min of incu-
bation at pH 9.0 in the presence of 5 mM Ca2+ as shown
in Figure 7 and 78% in the absence of Ca2+. The thermo-
stability decreased sharply on increase of temperature to
70˚C exhibiting 46% residual activity in the presence of
Ca2+ and 35% residual activity in the absence of this

F. M. Olajuyigbe / Advances in Enzyme Research 1 (2013) 112-120 117
Figure 6. Effect of temperature on activity of protease from
Bacillus subtilis SHS-04 in the presence (bold line) and ab-
sence of CaCl2 (dashed line).
Figure 7. Thermostability of protease in the absence (dashed
lines) and presence (bold lines) of 5 mM CaCl2. Stability at
50˚C (♦), 60˚C (●), 70˚C (▲) and 80˚C (■) were determined by
assaying for activity of enzyme after incubation at specified
temperatures for 30, 60, 90 and 120 minutes, respectively.
metal ion at 70˚C after 60 min of incubation at pH 9.0
(Figure 7). These results are similar to earlier reports
which show that calcium was required for the stability of
proteases [31,36]. This is further established by Alexan-
der et al. [33] who reported that there are two calcium-
binding sites in the crystal structure of subtilisin which
play very important role in maintaining the thermostabil-
ity.
3.7. pH Stability of Crude Protease
The optimum pH for protease activity was 9.0. The
protease activity rapidly declined above pH 9.0 with the
enzyme having relative activity of 53% at pH 10.0 (Fig-
ure 8), the activity decreased significantly after pH 11.0
showing only 11% relative activity at pH 12.0. The en-
zyme was very stable over a broad range of pH (7.0 -
11.0). The important detergent enzymes, subtilisin Carl-
berg and subtilisin Novo showed maximum activity at
pH values of 8.0 - 10.0 [30].
3.8. Organic Solvent Stability of Protease
Microbial proteases have been successfully applied to
the synthesis of several small peptides of pharmaceutical
and nutritional interests [37]. However, the use of prote-
ases in peptide synthesis is limited by the specificity and
the instability of the enzymes in the presence of organic
solvents [36]. Protease from B. subtilis SHS-04 had
shown remarkable activity and stability in the presence
of the organic solvents studied (Figure 9). Protease
showed above 80% relative activity in the presence of
acetone, methanol and glycerol when compared with
control. The enzyme had relative activity of 78% and
66% in the presence of isopropanol and DMSO. The ac-
tivity of the protease was inhibited by almost 50% in the
presence of benzene (Figure 9). The effect of organic
solvents on stability of protease was determined by pre-
incubating the enzyme with selected organic solvent
(25% v/v) for 30 min with shaking. Figure 9 shows that
protease from B. subtilis SHS-04 retained above 90% of
its original activity in the presence of DMSO, acetone
Figure 8. Effect of pH on activity and stability of protease from
Bacillus subtilis SHS-04.

F. M. Olajuyigbe / Advances in Enzyme Research 1 (2013) 112-120
118
Figure 9. Effect of organic solvents (25% v/v) on activity and
stability of protease from B. subtilis SHS-04. Symbols and bars
represent mean values and standard deviations of triplicate
determinations. Dimethyl sulfoxide is abbreviated DMSO.
and methanol with 75%, 65% and 64% residual activity
in the presence of glycerol, isopropanol and benzene.
This property is highly remarkable when compared with
the alkaline protease reported by Hadj-Ali et al. [36]
from Bacillus licheniformis NH1which exhibited only
12% residual activity in the presence of isopropanol.
Also, a purified protease from Pseudomonas aeruginosa
PseA strain was stable in the presence of some organic
solvents but unstable in benzene [38]. However, the pro-
tease under study from B. subtilis SHS-04 was stable in
the presence of isopropanol, benzene, DMSO, acetone,
methanol and glycerol.
4. CONCLUSION
This study ascertains groundnut meal as the most ef-
fective low-cost substrate for protease production from B.
subtilis SHS-04. The optimized condition for maximum
enzyme production (1616.21 U/mL) using groundnut
meal (0.75%) was achieved at 48 h cultivation period,
pH 9, 45˚C and 200 rpm. The enzyme produced was sta-
ble over a wide range of pH and temperature, and in the
presence of organic solvents. The use of low-cost growth
medium for protease production would significantly re-
duce the cost of enzyme production.
5. ACKNOWLEDGEMENTS
The author is very grateful to the International Foundation for Sci-
ence (IFS), Sweden for supporting this research through the awarded
IFS grant (F/3775-2). Author also acknowledges with thanks the sup-
port received from Mr. Olatope and staff of the Biotechnology Unit of
Federal Institute of Industrial Research, Lagos in identification of bac-
terial isolates.
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