Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04

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;

folajuyi@futa.edu.ng

Received 10 July 2013; revised 19 August 2013; accepted 5 September 2013

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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