Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus
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- Gupta, A., Joseph, B., Mani, A. et al. World J Microbiol Biotechnol (2008) 24: 237. doi:10.1007/s11274-007-9462-z
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In this communication, we report the presence of a newly identified serine alkaline protease producing bacteria, Virgibacillus pantothenticus (MTCC 6729) in the fresh chicken meat samples and the factors affecting biosynthesis as well as characterization of protease. The strain produced only 14.3 U ml−1 protease in the standard medium after 72 h of incubation, while in optimized culture conditions the production of protease was increased up to 18.2 U ml−1. The strain was able to produce protease at 40°C at pH 9.0. The addition of dextrose and casein improved protease production. The protease was partially purified and characterized in terms of pH and temperature stability, effect of metal ions and inhibitors. The protease was found to be thermostable alkaline by retaining its 100% and 85% stability at pH 10.0 and at 50°C respectively. The protease was compatible with some of the commercial detergents tested, and was effective in removing protein stains from cotton fabrics. The V. pantothenticus, MTCC 6729 protease appears to be potentially useful as an additive in detergents as a stain remover and other bio-formulations.
KeywordsAlkaline proteaseDetergent compatibilityEnzymeSerine proteaseVirgibacilluspantothenticus
Among the commercially available proteases, mainly the neutral and alkaline proteases are produced by organisms belonging to the genus Bacillus (Mala et al. 1998). Alkaline proteases secreted by both neutrophilic and alkalophilic bacilli are of particular interest due to their wide applications in laundry detergents, leather processing, protein recovery or solubilization, organic synthesis, meat tenderization, detergents, food industry, photography, and pharmaceuticals etc. (Cowan 1996). Although a variety of proteolytic fungi and bacteria are available, only a few provided high enzymatic activities with commercial success. However, the great economic value of protease still gives an impetus to search for new proteases with novel properties. In the search of new proteolytic bacteria, a strain of Virgibacillus pantothenticus (MTCC 6729) was isolated from fresh chicken meat samples that produced high levels of serine alkaline protease in batch culture conditions. Of all proteases, alkaline proteases produced by Bacillus species are of great importance in detergent industry due to their high thermostability and pH stability. The stability of protease has been improved in the past few years by using protein engineering (Gupta et al. 2002) and recombinant DNA techniques (Kaneko et al. 1989; Jang et al. 1992). In place of using such modern but expensive and time consuming techniques, proper selection of wild microbial isolates can provide stable enzymes that can easily serve the purpose without any additional requirements. Moreover, such isolate could be conveniently maintained and handled during bioprocess. Following amplified rDNA restriction analysis (ARDRA) and a polyphasic study, the new genus Virgibacillus was proposed to accommodate Bacillus pantothenticus and two related organisms, which appeared to belong to an undescribed and new species (Heyndrickx et al. 1998).
The performance of protease is influenced by several factors, such as pH of industrial process, ionic strength, temperature and mechanical handling. Therefore, extensive studies were made on various nutritional and environmental factors influencing the optimum production. Newer enzymes with novel properties that can further enhance the industrial process using the current enzyme is always in demand. For production of enzyme for industrial use, isolation and characterization of new promising strains using cheap carbon and nitrogen source is a continuous process (Parekh et al. 2000). The present work was carried out to optimize the culture conditions for the alkaline protease production by Virgibacillus pantothenticus isolated from chicken meat samples and the serine alkaline proteases secreted was partially purified, characterized and studied for its compatibility with various commercially available detergents.
Materials and methods
The chemicals viz., peptone, skim milk, Tris–HCl, trichloroacetic acid, phenylmethylsulphonyl fluoride (PMSF), β-mercaptoethanol (β-ME), ethylene diamine tetra acetic acid (EDTA), sodium azide, urea, sodium dodecyl sulphate (SDS), dialysis and sampling bags used in the present study were purchased from HiMedia (Mumbai, India). All other chemicals used were of the (analytical grade) highest purity available commercially. The chicken meat samples and commercial detergents were purchased from local market.
Isolation of bacterial strains
Fresh chicken meat samples collected in sterile Nasco sampling bags were brought to the laboratory and processed for analysis within 6 h of collection. Smashed samples of meat, was serially diluted up to 10−7 fold. The diluted samples were plated onto skim milk agar plates containing peptone (0.1% w/v), NaCl (0.5% w/v), agar (2.0% w/v), and skim milk (10% v/v). Plates were incubated at 37°C for 24 h. A clear zone of skim milk hydrolysis gave an indication of protease producing organisms. Bacterial colonies exhibiting the larger zone were quantitatively determined for protease activity by spectrophotometric analysis. Depending upon the maximum proteolytic activity by qualitative and quantitative assay, the strain PR-A isolated in our laboratory was selected for further experimental studies. Bacterial isolate was characterized according to the Bergey’s Manual of Systematic Bacteriology (Holt et al. 1994) and confirmed at Institute of Microbial Technology (IMTECH), Chandigarh, India.
Optimization of culture conditions
Incubation time (12–96 h) and effects on addition of various carbon and nitrogen source were evaluated in relation to enzyme yield. The physical and chemical cultural conditions like pH (5–10), temperature (27–45°C), and mode of incubation, the static and shake conditions were optimized. The optimal temperature for growth and production of protease was investigated at a fixed substrate concentration and pH with varying temperatures. The experiments were conducted in triplicates and the results were the average of the three independent trials.
Preparation of protease enzyme
The liquid medium used for the production of protease was composed (g/l) of; sucrose, 5.0; citric acid, 5.0; yeast extract, 10.0; K2HPO4, 1.0; MgSO4·7H2O, 0.1 and CaCl2·2H2O, 0.1. The pH of medium was adjusted to 9.0 with 10% (w/v) Na2CO3 solution. The medium was inoculated at 5% (v/v) with a 20 h old culture and incubated at 37 °C in a shaker (180 rpm) for 48 h. The culture medium was centrifuged at 7,500 rpm for 10 min at 4°C. The cell free supernatant was precipitated with 80% ammonium sulphate at 4°C. After centrifugation at 15,000 g for 20 min at 4°C, the pellet was dissolved in a small amount of 5 mM Tris–HCl buffer, pH 7.0 and dialyzed overnight against the same buffer. This partially purified enzyme was used for further studies.
For measuring protease activity, the crude enzyme (0.2 ml) was mixed with 2.5 ml of 1% casein in phosphate buffer (pH 7) and incubated for 10 min at 37°C. The reaction was terminated by adding 5 ml of 0.19 M trichloroacetic acid (TCA). The reaction mixture was centrifuged and the soluble peptide in the supernatant fraction was measured with tyrosine as the reference compound (Liang et al. 2006). One unit of protease activity is defined as the activity that releases 1 μmol tyrosine ml−1 in 1 min at 37°C. Protein concentration was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard.
Effect of pH on protease activity and stability
The effect of pH on enzyme activity was determined by incubating the reaction mixture at various pH ranging from 4.0 to 12.0 using different buffer systems (Sana et al. 2006). The buffers used for the purpose were 0.1 M citrate (pH 4.0–5.0), 0.2 M sodium phosphate (pH 6.0–8.0), 0.2 M glycine–NaOH (pH 9.0–10.0) and 0.1 M glycine–NaOH (pH 11.0–12.0). The pH stability was obtained by measuring the relative activity of enzyme after 1 h of preincubation in buffers of various pH values (4.0–12.0) at 30°C.
Effect of temperature on enzyme activity and stability
The optimum temperature for enzyme activity was determined by assaying relative enzyme activity at various temperatures from 20 to 80°C. The thermostability of enzyme was measured after preincubation of enzyme in the same buffer for 30 min at various temperatures (20–80°C).
Effect of protease inhibitors and chelators on enzyme activity
The effect of various protease inhibitors (5 mM and 10 mM) such as PMSF, β-ME, and a chelator of divalent cations (EDTA), other chelators like sodium azide, urea, SDS were determined by preincubation with the enzyme solution for 30 min at 40°C before the addition of substrate. The residual protease activity was measured.
Effect of various metal ions on protease activity
The effects of metal ions like Fe3+, Mg2+, Co2+; Zn2+, Hg2+, and Cu2+ (5 mM) were investigated by adding them to the reaction mixture. Residual protease activities were measured.
Compatibility with detergents
The compatibility of V. pantothenticus protease with local laundry detergents was studied. Detergents used were Ariel (Procter and Gamble, India), Ghari (Rohit Surfactants Pvt. Ltd., India), Surf Excel (Hindustan Lever Ltd., India), Wheel (Hindustan Lever Ltd., India) and UltraVim (Hindustan Lever Ltd., India). The detergents were diluted in distilled water (0.7% w/v) and boiled for 10 min to denature the enzymes present in the solution. The detergent solution was then incubated with protease (1:1) for 3 h at 60°C, and the residual activity was determined. The enzyme activity of control sample (without any detergent) was taken as100%.
Results and discussion
Isolation and identification of bacterial strains
Screening of proteolytic bacterial isolates at different pH
Diameter of zone (mm) at different pH
Enzyme activity at pH 9.0
Morphological and biochemical characteristics of PR-A isolate
Morphological and biochemical characteristics
Irregular convex and opaque
Gram positive rods
2–8 × 0.5–0.7 mm2
Growth in NaCl
Up to 5%
Oxidation fermentation test
Identification of organism
Optimization of culture conditions
Effect of various parameters on protease production of Virgibacillus pantothenticus
Protease activity (U ml−1)
(a) Incubation time (h)
(b) Temperature (°C)
Effect of carbon sources and substrates (0.5% w/v) on protease production of Virgibacillus pantothenticus
Protease activity (U ml−1)
(a) Carbon source
Purification and characterization of protease
Purification of protease
Summary of partial purification procedure of alkaline protease from Virgibacillus pantothenticus
Total activity (Units)
Total protein (mg)
Sp. Activity (U mg−1)
(NH4)2SO4 precipitation (dialyzed)
Effect of pH
Effect of temperature
Effect of metal ions
Effect of protease inhibitors
Compatibility and stability of protease with detergents
In conclusion, we have partially purified and characterized an alkaline serine protease from V. pantothenticus. Although many alkaline serine proteases have been reported from microbial origins, to our knowledge this is the first report on purification and characterization of a serine alkaline protease from this bacterium. The desirable properties of our protease, such as stability at high alkaline pH and high temperature permit its potential to be exploited as a detergent additive. From an industrial perspective, present investigation might be fruitful in identifying one such enzyme, which can be exploited commercially.
The authors are grateful to Prof. P. W. Ramteke, Director-Research, AAI-DU, Allahabad, India for providing laboratory facilities to carry out the present work.