Stabilization of Protease from Bacillus licheniformis MZK05M9

Abstract

In this study, the effects of temperature, lyophilization and various additives on the storage stability of extracellular protease enzyme from Bacillus licheniformis MZK05M9 (BlM9) were investigated. At 4°C, 100% activity of the enzyme was retained for 30 days whereas at 30°C, the enzyme showed instability. The lyophilized enzyme retained 100% of its activity for 35 days and 90% of its activity for 120 days. Among different salts, sugars, surfactants and polymers, the Polyethylene glycol 4000 (PEG 4000) stabilized the enzyme most. The addition of 0.5% (w/v) PEG stabilized the enzyme to retain 100% of its activity for 18 days and 90% level for up to 30 days. Thus, the PEG 4000 at 0.5% level can be used as additive to increase the storage stability of BlM9 enzyme in an industrial level.

Full text

Bangladesh Journal of Industrial Microbiology and Biotechnology 2017; 1(1): 39-45
ISSN: 2521-0386
www.bjimb.org
Stabilization of Protease from Bacillus licheniformis MZK05M9
Md. Arafat Al Mamun1, Md. Mahmuduzzaman Mian2, Shamima Begum3, Amika Ahmed Manzum1,
Shakila Nargis Khan2, Md. Mozammel Hoq2*
1Centre for Advanced Research in Sciences (CARS).
2Department of Microbiology, University of Dhaka, Dhaka-1000.
3BRAC University, Dhaka-1212.
(*Corresponding author’s e-mail: mhoq@du.ac.bd)
Received: 15 April, 2017; Revised: 01 May, 2017; Accepted: 12 May, 2017
Cite as: Mamun MAA, Mian MM, Begum S, Manzum AA, Khan SN, Hoq MM. Stabilization of protease from Bacillus
licheniformis MZK05M9. Bangladesh J Ind Microbiol Biotechnol. 2017; 1(1): 39-45.
Introduction
Microbial proteases have tremendous uses in food, pharmaceuticals, detergent and leather industries.
A mutant strain Bacillus licheniformis MZK05M9 (BlM9) can produce alkaline protease (BlM9
enzyme) with bating potentiality in leather processing [1]. The instability of the enzymes is a
limiting factor that causes difficulty in storage and subsequently leads to high unit costs of the
enzyme [2]. Therefore, the storage of BlM9 enzyme in room temperature is challenging while using
bulk amounts in an industrial scale. Effects of the aqueous environment and microbial
contamination may cause the limited stability of the enzyme [3,4]. The enzyme may also be
denatured by changes in temperature, pH, pressure and ionic strength [5].
Generally, the addition of excipients to the enzymes is the most frequent method of stabilizing the
enzymes. Various classes of additives such as ligands, substrates, salts, polyols, sugars, DMSO,
Abstract
In this study, the effects of temperature, lyophilization and various additives on the storage stability of
extracellular protease enzyme from Bacillus licheniformis MZK05M9 (BlM9) were investigated. At 4°C, 100%
activity of the enzyme was retained for 30 days whereas at 30°C, the enzyme showed instability. The lyophilized
enzyme retained 100% of its activity for 35 days and 90% of its activity for 120 days. Among different salts,
sugars, surfactants and polymers, the Polyethylene glycol 4000 (PEG 4000) stabilized the enzyme most. The
addition of 0.5% (w/v) PEG stabilized the enzyme to retain 100% of its activity for 18 days and 90% level for up
to 30 days. Thus, the PEG 4000 at 0.5% level can be used as additive to increase the storage stability of BlM9
enzyme in an industrial level.
Keywords: Stabilization, protease, Bacillus, lyophilization, excipient
Research Article Open Access

Stabilization of protease from Bacillus licheniformis MZK05M9
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Bangladesh Journal of Industrial Microbiology and Biotechnology 2017; 1(1): 39-45
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glycerol, polyethylene glycols and synthetic polymers can be employed for enzyme stabilization [2].
Lyophilization may also be another process of stabilizing protease enzyme [5].
In this study, various additives such as salts, sugars, surfactants and polymers were used to check
their effects on storage stability of BlM9 enzyme.
Methods
Microorganism
A mutant B. licheniformis MZK05M9 (BlM9) developed through classical mutation [6] was used in
this experiment. This organism was preserved in the Enzyme and Fermentation Biotechnology
laboratory, Department of Microbiology, University of Dhaka. Stock culture of the organism was
maintained on nutrient agar medium at 4°C in refrigerator for routine laboratory use and 15%
glycerol broth at −70°C for long term preservation.
Production of protease
Production of protease from the mutant strain BlM9 was carried out in a medium containing
molasses, soybean meal, K2HPO4, NaCl and MgSO4 (% w/v is not shown here). The fermentation
was carried out in 7 L bench-top bioreactor (Bioflo 110, New Brunswick Scientific, USA) with a
working volume of 3.5 L. The vegetative bacterial inoculum was transferred to the fermentation
medium at a level of 5 % (v/v). The batch was carried out at temperature of 37°C and initial pH 7.5.
The dissolved oxygen level in the culture were controlled by cascading mode maintained by both
agitation and aeration where high and low limits of agitation were 300 rpm and 150 rpm respectively
and high and low limits of aeration were 3.5 SLPM and 1 SLPM respectively.
Excipients used
Various additives viz. Salts (MnSO4 and NH4SO4), sugars (sucrose, fructose, and glucose), polymer
(Polyethylene glycol, PEG 4000) and Surfactants (Tween-80 and Triton X-100) were used to study
their effects on enzyme storage stability. After centrifugation of the fermentation broth, the cell free
supernatant (protease enzyme) was taken into sterile Duran bottles. The various additives were added
to the cell free enzyme at 0.5% (w/v) level except PEG 4000. PEG 4000 was added to the enzyme at
0.25, 0.5 and 1 % (w/v) level. After addition of the additives the bottles containing enzymes were
incubated at 30°C for 90 days.
Lyophilization of the protease
After centrifugation of fermentation broth, the cell free supernatant (120 ml) was taken in petri-
dishes and was subjected to freezing at -20°C for one hr. The petri-dishes were then placed carefully
in the lyophilization unit (FD5512, Ilshin lab Co. Ltd., Korea). The operation was carried out for 5 to
6 hr while monitoring at regular intervals. Finally, the petri-dishes were removed from the
lyophilization unit, the powdered product was transferred to a Duran bottle and the bottle was tightly
sealed. To assay the activity of the lyophilized product, it was re-suspended in distilled water and
assay was performed to determine the protease activity. The product was stored at 30°C and the

Stabilization of protease from Bacillus licheniformis MZK05M9
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Bangladesh Journal of Industrial Microbiology and Biotechnology 2017; 1(1): 39-45
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relative activity of the lyophilized enzyme was checked every 7 days. The recovery of lyophilization
was calculated by the following formula [5].
Recovery (%) = Activity of the sample after lyophilization
Activity of the sample before lyophilization X100
Determination of enzyme activity
Enzyme activity was determined with azo-casein (Sigma-Aldrich) as substrate by a modified
procedure described by Krieger and Lockwood (1981) [7]. 400 l of 1% Azo-casein solution in
0.05M Tris -HCI buffer (PH 8.5) was added to 400 l of culture supernatant and kept for 1 hr at 37°C
in a water bath. The reaction was stopped by the addition of 135 l of 35% trichloroacetic acid
(TCA) and the mixture was kept at 4°C for at least 10 min. After centrifugation at 13,000 rpm for 10
min, 0.75ml of supernatant was mixed with 0.75 ml of 1.0M NaOH and the absorbance was taken at
440 nm within one minute in a spectrophotometer (GENESYSTM 5, Thermospectronic, USA). The
control was prepared by adding TCA before mixing the culture supernatant with azo-casein solution.
One unit of protease activity was determined as the amount of enzyme that produces an increase in
absorbance of 0.01 under the above assay condition. The value obtained is expressed in U/ml.
Relative activity of a test sample was determined as the percentage of the initial activity of that
sample.
Results and Discussion
Recovery of the enzyme activity upon lyophilization of the alkaline protease
After complete lyophilization solid powdered enzyme was kept in Duran bottle and was tightly
sealed (figure 1)
Figure 1:Lyophilized BlM9 enzyme
By determining the enzyme activity before and after lyophilization it was observed that this process
recovered the enzyme activity at 98.5 % level (Table 1). Previously Anjum et al. [5] reported that
after lyophilization of protease from Bacillus subtilis, the recovery of 82% was achieved.

Stabilization of protease from Bacillus licheniformis MZK05M9
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Bangladesh Journal of Industrial Microbiology and Biotechnology 2017; 1(1): 39-45
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Table 1: Recovery of the lyophilized enzyme
Initial activity of the enzyme
1020 U/ml
Total activity of 120 ml enzyme
122400 U
Total activity of lyophilized enzyme
120560 U
Total loss of activities during lyophilization
1.5%
Recovery after lyophilization
98.5 %
Effect of temperature on enzyme stability
The enzyme retained 100% activity for up to 30 days at 4°C whereas it showed instability at
30°C.The enzyme retained 98.5 % of its activity after lyophilization (Table 1). The lyophilized
enzyme was then stored in a Duran bottle (figure 1) at 30°C. It was found that the lyophilized
enzyme retained 100% of its initial activity even after 35 days (figure 2) and 90 % after 120 days.
This suggests that lyophilization stabilizes the enzyme significantly. Lyophilization is an effective
method for storing of enzyme as this method dries up the product and protects the enzyme against
denaturation due to chemical reactions occurring in aqueous environment. During the process of
freezing and drying, changes occur in product that may denature the enzyme [8]. But in case of BlM9
enzyme, during lyophilization only 1.5% activity was denatured.
Figure 2: Stability profile of BlM9 enzyme
Effect of polymer (PEG-4000) and surfactants (Triton X-100 and Tween-80) on storage stability of
the BlM9 enzyme
Effect of PEG on the stability of the protease was investigated by incubating the enzyme with
various amounts (% w/v) of PEG such as 0.25%, 0.5% and 1%. The stabilities of the enzyme were
then compared by taking activities for up to 90 days. At 30°C, the enzyme was significantly
stabilized by 0.5% PEG with 100 % activity for up to 18 days and 90% activity for up to 30 days.
PEG 0.25%, and PEG 1%, retained 100% activity for up to 8 and 10 days, respectively (figure 3).

Stabilization of protease from Bacillus licheniformis MZK05M9
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Figure 3: Effect of polymer (PEG-4000) on storage stability of the BlM9 enzyme
Triton X-100 stabilized the enzyme at 100 % level for up to 8 days with the half shelf life of 50 days
where as the Tween -80 partially stabilized the enzyme with half shelf life of 40 days (figure 4).
Figure 4: Effect of surfactants (Triton X-100 and Tween-80) on storage stability of the BlM9 enzyme
Effect of salts and sugars on storage stability of the BlM9 enzyme
Effect of salts and sugars on the stability of alkaline protease was investigated by incubating the
enzyme with sugars such as fructose, glucose and sucrose and salts such as MnSO4 and NH4SO4 at
30°C. The stabilities of the samples were then compared by taking protease activity at 03 days
intervals. Sugars such as fructose, glucose and sucrose did not show any noticeable stabilization
effect on the alkaline protease rather enzyme incubated with glucose lost its activity more rapidly.
Salts stabilize the enzyme by salting out effect of hydrophobic residues from surface into the interior
of enzyme molecule thereby compressing the enzyme leading to higher stability of the enzyme [9].
However in this study, ions such as NH4+ and Mn2+provided some protective effect (figure 5).

Stabilization of protease from Bacillus licheniformis MZK05M9
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Bangladesh Journal of Industrial Microbiology and Biotechnology 2017; 1(1): 39-45
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Figure 5: Effect of salts and sugars on storage stability of the BlM9 enzyme
Conclusions
From the study it was found that lyophilization method stabilized the enzyme most. But sometimes
in large scale, using of liquid enzyme is more cost effective. Therefore, the PEG 4000 at 0.5% level
may be used to stabilize the liquid enzyme at room temperature for using at industrial scale.
Author contribution
MMH and MAAM designed the study. MAAM, MMM, SB and AAM performed the experiments.
SNK analyzed the data and MAAM wrote the manuscript. MMH and SNK reviewed the data. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests
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