The production of alkaline protease by a Bacillus species isolate

ELSEVIER

TI (IIlIOI.OGY Bioresource Technology 67 (1999) 201-203

Short Communication

The production of alkaline protease by a Bacillus species isolate S. Mehrotra, P.K. Pandey, R. Gaur, N.S. Darmwal* Department of Microbiology, Dr R. M. L. Avadh University, Faizabad 224 001, U.P., India Received 23 April 1998; revised 16 May 1998; accepted 27 May 1998

Abstract Alkalophilic bacterial strains (52) isolated from saline-alkali soils were screened on milk agar medium for their ability to produce alkaline protease. Fifteen positive isolates were further examined for their extent of alkaline protease production. The most potent producer was identified as Bacillus sp. Maximum enzyme activity was achieved in the presence of glucose (1% w/v) and ammonium chloride (1% w/v) at pH 10.5 and 40°C over a 20 h incubation. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Alkaline.protease;Bacillus sp.; Alkalophiles

1. Introduction

Proteases are the most important industrial enzymes, accounting for about 60% of the total enzyme market (Ng & Kenealy, 1986). Alkaline proteases are of particular interest due to their wide applications in detergent, tanning and dairy industries (Grebeskova et al., 1988; Wilson et al., 1992; Phadatara et al., 1993). Of late, some newer applications, for instance recovery of silver from photographic plates (Fujiwara et al., 1991) and peptide synthesis (Cerovsky, 1992), have also been explored. In view of the commercial interest, microbes from varied habitats have been examined by many researchers for industrially-suitable alkaline proteases (Steele et al., 1992; Sen & Satyanarayana, 1993; Paliwal et al., 1994; Gajju et al,, 1996). In the present work the authors attempted to isolate bacteria from saline-alkali soils and optimized condi-

*Corresponding author.

tions for maximum alkaline protease production by a promising strain.

2. Methods

2.1. Isolation and screening

Fifty-two alkalotrophic bacterial strains were isolated, employing an enrichment culture technique (Boyer et al., 1973); from saline-alkali soils of the Avadh region in Uttar Pradesh, India. All the strains were screened for their alkaline protease production on milk agar medium containing (g/l): skimmed milk, 100; yeast extract, 10; agar, 20; pH 8.0 (adjusted after autoclaving). Fifteen isolates found positive for alkaline protease were again tested for their enzyme production in the growth medium containing (g/l): glucose, 10; peptone, 5; yeast extract, 5; KH2PO4, 1; MgSO4.7H20, 0.2. The pH of the broth was adjusted to 10.0 after autoclaving by adding sterilized NaeCO3 solution (20% w/v) at 10% (v/v). The above medium (50ml) was inoculated at 1.0% (v/v) with a 24 h old (O.D. 0.6) culture and incubated at 37°C in a shaker (120 rev/min) for 18 h. The grown cultures were then centrifuged at

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S. Mehrotara et al.IBioresource Technology 67 (1999) 201-203

10000g for 25 min and the supernatants used for alkaline protease assay. The promising strain, identified as Bacillus sp. (Institute of Microbial Technology, Chandigarh, India), was selected for optimization of protease production. The culture was maintained on slants containing the yeast extract peptone growth medium with agar and stored at 4 ° + I°C.

2.2 Enzyme production optimization The effect of incubation time (0-36 h), temperature of incubation (35-50°C) and pH (8.0-11.5) of the growth medium were studied. The production of alkaline protease after substituting glucose (1.0% w/v) with sucrose, fructose, mannitol, maltose, starch, lactose at 1.0% (w/v) and yeast extract (0.5% w/v) +peptone (0.5% w/v) with NH4NO3, (NH4)2504, NaNO3, NH4H2PO4, NH4CI, peptone and casein at 1.0% (w/v) was studied.

& Horikoshi, 1976; Manachini et al., 1988; Sen & Satyanarayan, 1993). However, an optimum temperature of 45°C for alkaline protease production by Bacillus licheniformis S-40 has been reported by Sen & Satyanarayana (1993). The Bacillus sp. was capable of utilizing a wide range of carbon sources. However, the best carbon source in the present study for alkaline protease production was glucose followed by fructose (Table 1). Glucose has been reported to suppress protease production (Sonnleitner, 1983; Sen & Satyanarayana, 1993), but in the present study it was found to be a relatively good carbon source for enzyme production. Other workers have also reported better alkaline protease production in the presence of glucose as a carbon source (Sinha & Satyanarayana, 1991; Gajju et al., 1996). In general, both inorganic and organic nitrogen sources were utilized efficiently by the Bacillus sp. for protease production. However, an inorganic source (ammonium

2.3. Enzyme assay The method of Auson-Hagihara was followed (Hagihara et al., 1958) in which enzyme (1 ml) was added to 1 ml casein solution (1% w/v casein solution prepared in 10 mM carbonate-bicarbonate buffer of pH 10.5) and incubated at 37°C for 10min. The reaction was terminated by 3 ml of 10% trichloroacetic acid and the contents filtered through a Whatman No. 1 filter paper. The filtrate absorbance was read at 275 nm and extrapolated against a tyrosine standard curve. A unit of alkaline protease activity was defined as 1/zg of tyrosine liberated m1-1 min -1 under the experimental conditions.

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3. Resultsanddiscussion Only 15 from 52 alkalophilic bacteria isolated from saline-alkali soil exhibited prominent clear zones around their colonies on milk agar medium. Based on this, a bacterial isolate identified as Bacillus sp. w a s selected for further studies. Although alkaline protease was produced throughout the course of fermentation (4-36 h), the enzyme production was high at 20 h and corresponded with the late exponential phase of growth (Fig. l(a)). Ward (1986) has also reported that Bacillus sp. usually produce more alkaline protease during the late exponential phase. The function of this enzyme is obscure, but its production is correlated with the onset of a high rate of protein turnover during sporulation in certain bacilli. Figure l(b) depicts that maximum protease production by Bacillus sp. was at initial pH 10.5 and 40°C. Other workers have also reported greater enzyme production by Bacillus sp. in the alkaline range of 7-12

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pH Fig. 1. (a) Effect of incubation period on growth (o) in terms of absorbance at 665 nm and alkaline protease (e) production in terms of U/ml/min by Bacillus sp. (b) Effect of pH and temperature (QC) 35 (o), 40 (e), 45 (1), 50 (D) on alkaline protease production by Bacillus sp. The values of enzyme activity represent averages of at least two experiments.

S. Mehrotara et aL/Bioresource Technology 67 (1999) 201-203 Table 1 Effect of carbon and nitrogen sources on alkaline protease production by Bacillus sp. at pH 10.5 and 40"C during 20 h incubation Carbon (1% w/v)

Enzyme (U/ml/min)

Nitrogen (1% w/v)

Enzyme (U/ml/min)

Sucrose Fructose Mannitol Glucose Maltose Starch Lactose

18.5 26.0 19.1 28.1 18.2 17.5 18.5

NH4NO3 (NH4)2SO4 NaNO3 NH4H2PO4 NHaCI Peptone Casein Yeast extract (0.5% w/v) + peptone (0.5% w/v)

24.0 24.3 25.3 23.8 30.8 24.8 24.4 28.5

These values of enznyme activity represent averages of at least two experiments.

chloride) gave relatively better results, followed by yeast extract+peptone, for enzyme production in the present study (Table 1). Supplementation of media containing NI-LCI (1% w/v) with various concentrations of yeast extract and casein did not yield better results (not shown). Conflicting reports regarding the effect of organic and inorganic nitrogen sources on alkaline protease production by Bacillus sp. are available. Sinha & Satyanarayana (1991) reported higher protease production with inorganic nitrogen sources such as ammonium sulphate and ammonium nitrate, while many others have found organic sources better suited to Bacillus sp. for enzyme production (Fujiwara & Yamamoto, 1987; Sen & Satyanarayana, 1993; Gajju et al., 1996). Thus, it may be concluded that our Bacillus isolate could be thought of as a candidate for the production of alkaline protease, at 40°C and pH 10.5 during 20 h fermentation in a medium containing glucose (1% w/v) and ammonium chloride (1% w/v) as carbon and nitrogen sources. Further study could reveal its possible exploitation for commercialization.

Acknowledgements Research grant from All India Council for Technical Education, New Delhi to one of the authors (NSD) is gratefully acknowledged. Authors also wish to thank Professor S. K. Garg and Dr Rajat Sandhir for valuable suggestions and help in computation of results, respectively.

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