World Journal of Microbiology and Biotechnology

, Volume 24, Issue 2, pp 237–243

Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus

Authors

  • Amit Gupta
    • National Institute of Pharmaceutical Education and Research
    • College of Biotechnology and Allied SciencesAllahabad Agricultural Institute - Deemed University
  • Abin Mani
    • College of Biotechnology and Allied SciencesAllahabad Agricultural Institute - Deemed University
  • George Thomas
    • College of Biotechnology and Allied SciencesAllahabad Agricultural Institute - Deemed University
Original Paper

DOI: 10.1007/s11274-007-9462-z

Cite this article as:
Gupta, A., Joseph, B., Mani, A. et al. World J Microbiol Biotechnol (2008) 24: 237. doi:10.1007/s11274-007-9462-z

Abstract

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.

Keywords

Alkaline proteaseDetergent compatibilityEnzymeSerine proteaseVirgibacilluspantothenticus

Introduction

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.

Enzyme assay

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

Four different meat samples were analyzed for isolation of proteolytic bacterial cultures. These samples are rich in protein and the spoilage and biodegradation of such samples are generally caused due to the microbial population present in it. Around 25 morphologically distinct bacterial colonies were isolated from each sample. Isolated cultures were separately screened for their proteolytic activity. Out of total isolates tested, approximately 50% isolates exhibited proteolytic activity at various extents. Five efficient bacterial isolates were selected for qualitative and quantitative assay at different pH (Table 1). Among the cultures tested, the laboratory isolate PR-A showed highest zone of clearance (14.5 mm) at pH 9.0 with proteolytic activity i.e., 12.5 U ml−1, followed by the laboratory isolate PR-E (9.3 U ml−1). Other isolates showed maximum activity at pH 7.0. Using morphological and biochemical characteristics based on Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994) and according to Heyndrickx et al. (1998), the bacterial isolate PR-A was identified as Virgibacillus pantothenticus. The newly identified strain is deposited at IMTECH Chandigarh, India with the Accession Number MTCC 6729. Details of the bacterial identification are given in Table 2.
Table 1

Screening of proteolytic bacterial isolates at different pH

S. no.

Isolate

Diameter of zone (mm) at different pH

Enzyme activity at pH 9.0

4.0

7.0

9.0

1

PR-A

9.5

11.5

14.5

12.5

2

PR-B

5.0

9.5

10.0

6.2

3

PR-C

4.8

9.0

08.0

7.9

4

PR-D

3.5

10.0

10.0

8.2

5

PR-E

3.2

6.0

5.5

9.3

Table 2

Morphological and biochemical characteristics of PR-A isolate

Morphological and biochemical characteristics

Results

Colony morphology

Irregular convex and opaque

Gram staining

Gram positive rods

Cells size

2–8 × 0.5–0.7 mm2

Spores

+

Aerobic growth

+

Motility

Catalase

+

Oxidase

+

Growth in NaCl

Up to 5%

Growth temperature

15–42°C

Glucose

A

Salicin

A

Fructose

A

Arabinose

Galactose

Inositol

Mannitol

Raffinose

Rhamnose

Xylose

Methyl Red

+

V-P test

Indole

Citrate

Oxidation fermentation test

Nitrate reduction

+

Identification of organism

Virgibacillus pantothenticus

+, Positive reaction; –, Negative reaction; A, acid production

Optimization of culture conditions

The maximum protease production was recorded after 72 h of incubation at 40°C (Table 3). The present results were supported by the findings of MacFarlane et al. (1992), Feng et al. (2001) and Kim et al. (2001) where the maximum protease production usually occurs at the late logarithmic to the beginning of stationary phase of growth. The results obtained in Table 3 revealed that the protease production was particularly sensitive to acidic pH. Maximum protease production was obtained at pH 9.0. Borris (1987) reported that the alkaline protease production was found to be the maximum at pH 9.0–13.0. The addition of dextrose in the production medium increased the protease production (16.4 U ml−1). However, the addition of lactose and maltose in the production medium declined the protease production. Casein was found to be the most preferred substrate (18.2 U ml−1), followed by skim milk (16.4 U ml−1) for maximum production of protease (Table 4). To test the effect of shaking conditions in the protease production, one set of culture was incubated at 40°C in a rotary shaker at 180 rpm. Whereas the other set was incubated under stationary condition. It was evident from the results that the shaking condition influenced the biomass and protease production (Table 3). Under the shaking condition, a twofold increase of protease was observed when compared to the static condition, confirming the results obtained by Wendhausen et al. (2005). Thus, the shaking might have created conditions of higher availability of the nutrients to the bacteria. Further aeration of the medium due to the enhanced mixing provided oxygen required for the microbial cell growth. However, the addition of metal ions did not show any significant increase in protease production (data not shown).
Table 3

Effect of various parameters on protease production of Virgibacillus pantothenticus

Parameters

Protease activity (U ml−1)

(a) Incubation time (h)

24

6.8

36

10.2

48

12.4

60

13.6

72

14.3

84

11.5

96

5.4

(b) Temperature (°C)

27

12.4

32

14.2

37

14.6

40

15.2

45

8.9

(c) Aeration

Static

6.4

Shaking

15.9

(d) pH

5

6.2

6

8.4

7

10.8

8

12.4

9

15.8

10

12.0

Table 4

Effect of carbon sources and substrates (0.5% w/v) on protease production of Virgibacillus pantothenticus

Parameters

Carbon source

Protease activity (U ml−1)

(a) Carbon source

Glucose

15.8

Dextrose

16.4

Lactose

6.8

Maltose

5.0

Wheat bran

11.5

(b) Substrates

Casein

18.2

Skim milk

16.4

Purification and characterization of protease

Purification of protease

The results of purification of protease from V. pantothenticus are summarized in Table 5. Approximately 4.62-fold purification from the initial culture broth was achieved during ammonium sulphate precipitation (80%) with a recovery of 36.90% enzyme. The specific activity of the final partially purified enzyme was 5.6 U mg−1 protein. Most of commercial applications like detergent do not require homogenous enzyme preparation; a certain degree of purity, however, enables efficient and successful usage (Sharma et al. 2001).
Table 5

Summary of partial purification procedure of alkaline protease from Virgibacillus pantothenticus

Purification step

Total activity (Units)

Total protein (mg)

Sp. Activity (U mg−1)

Purified (fold)

Yield (%)

Crude enzyme

27,300

22,500

1.21

1.00

100

(NH4)2SO4 precipitation (dialyzed)

10,080

1,800

5.60

4.62

36.90

Effect of pH

Although the enzyme was active over a broad range of pH, optimum activity was found at pH 10.0 indicating to an alkaline protease (Fig. 1). This enzyme was active in alkaline conditions; indicating its potential use in detergent formulations as reported (Towatana et al. 1999; Gupta et al. 2002). Sana et al. (2006) reported that the protease from gamma Proteobacterium showed an optimum activity at pH 9.0 and maintained activity in the range 30–70°C which makes it suitable for use in the detergent industry. Gupta et al. (2002) mentioned that alkaline protease which was useful for detergent applications are mostly active in the pH range 8.0–12.0. The enzyme activity was reduced to about 27% at pH 6.0 and lost 20% of its activity at pH 12.0.
https://static-content.springer.com/image/art%3A10.1007%2Fs11274-007-9462-z/MediaObjects/11274_2007_9462_Fig1_HTML.gif
Fig. 1

Enzyme activity (■) and stability (○) of purified protease from Virgibacillus pantothenticus, at various pH. The relative activity (%) was calculated relative to the case of reaction at which maximum activity was taken as 100%

Effect of temperature

Optimum temperature for protease activity was found to be at 50°C and it was 82% stable at this temperature for 1 h (Fig. 2). Comparing the present results with those of El-Hawary and Ibrahim (1992) and Nilegaonkar et al. (2002) it could be concluded that the optimum temperature of proteolytic activity frequently exceeded the optimum temperature for enzyme production. It was also suggested that the stability of protease enzyme could be due to their genetic adaptability to carry out their biological activity at a higher temperature (Gaure et al. 1989; Whittle and Bloomfield 1999; Kanekar et al. 2002).
https://static-content.springer.com/image/art%3A10.1007%2Fs11274-007-9462-z/MediaObjects/11274_2007_9462_Fig2_HTML.gif
Fig. 2

Enzyme activity (■) and stability (○) of purified protease from Virgibacillus pantothenticus at various temperatures. The relative activity (%) was calculated relative to the case of reaction at which maximum activity was taken as 100%

Effect of metal ions

The heavy metal, Co2+ showed a stimulatory effect on protease activity whereas other metal ions had slight inhibitory effect (Fe3+, Mg2+, Zn2+, Hg2+ and Cu2+) on enzyme activity (Fig. 3). Similar results were reported with metal ions viz., Hg2+ and Cu2+ which inhibited the alkaline protease activity of Bacillus thermoruber (Manachini et al. 1988) and Alcligenes faecalis (Thangam and Rajkumar 2002).
https://static-content.springer.com/image/art%3A10.1007%2Fs11274-007-9462-z/MediaObjects/11274_2007_9462_Fig3_HTML.gif
Fig. 3

Effect of different concentration of metal ions (2 mM) on Virgibacillus pantothenticus alkaline protease activity. The residual activity (%) was calculated relative to the case of reaction without the addition of any heavy metals ions, which was taken as 100%

Effect of protease inhibitors

Inhibition studies primarily give an insight into the nature of an enzyme, its cofactor requirements, and the nature of the active reaction center (Sigma and Mooser 1975). The effect of various protease inhibitors on activity of the newly isolated protease is given in Fig. 4. The protease activity was partially inhibited by 5 mM PMSF and SDS and completely inhibited at 10 mM of both the inhibitors, thereby suggesting that the enzyme was serine protease (Damare et al. 2006). In the case of other inhibitors, the protease was not inhibited by EDTA, while a slight inhibition was observed with DTT and β-ME. EDTA had no inhibitory effect on the protease activity; hence, this enzyme is not a metalloprotease. PMSF sulphonates, the essential serine residue in the active site of the protease and has been reported to result in the complete loss of enzyme activity (Gold and Fahrney 1964). Our findings were also similar to those of Tsuchida et al. (1986) and Yamagata and Ichishima (1989) where the protease was completely inhibited by PMSF. This indicated that the enzyme isolated in the present study was serine alkaline protease.
https://static-content.springer.com/image/art%3A10.1007%2Fs11274-007-9462-z/MediaObjects/11274_2007_9462_Fig4_HTML.gif
Fig. 4

Effect of inhibitors on activity of protease. The residual activity (%) was calculated relative to the case of reaction without the addition of any inhibitors, which was taken as 100%

Compatibility and stability of protease with detergents

Besides pH, a good detergent protease is expected to be stable in the presence of commercial detergents. The V. pantothenticus protease showed excellent stability and compatibility in presence of the locally available detergents (Ariel, Ghari, Surf Excel, Wheel and UltraVim). Our protease showed stability and compatibility with a wide range of commercial detergents at 40°C especially in the presence of Ultra Vim followed by Ghari and Wheel (Fig. 5). The enzyme retained more than 50% activity with most of the detergents tested even after 3 h of incubation at 40°C. As the protease produced by our isolate V. pantothenticus was stable over a wide range of pH values, temperatures and also showed compatibility with various commercial detergents tested. It can be used as an additive component for the detergent to improve the washing performance. The supplementation of the enzyme preparation in detergent (i.e., Ultra Vim, Ghari and Wheel) could significantly improve the cleansing of the bloodstains from cotton fabrics. The data obtained from the experiments carried out in presence of commercial detergents, the broader substrate specificity of protease in cleaving most of the proteins suggest strongly that the proteolytic enzyme from V. pantothenticus has all the potential to be used as a laundry detergent additive, in order to improve the performance of heavy-duty laundry detergents. It is now well established that proteases exhibiting activity in the high-alkaline range have potential in detergent and stain removing formulations. Their utility can be significant only if they also exhibit compatibility with various detergents (Phadtare et al. 1993; Anwar and Saleemuddin 1997). Taking this into consideration the compatibility with commercial detergents containing various additives was investigated.
https://static-content.springer.com/image/art%3A10.1007%2Fs11274-007-9462-z/MediaObjects/11274_2007_9462_Fig5_HTML.gif
Fig. 5

Stability and compatibility of alkaline protease of Virgibacillus pantothenticus in the presence of various commercial 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.

Acknowledgement

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.

Copyright information

© Springer Science+Business Media B.V. 2007