Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India P.P. Kanekar * , S.S. Nilegaonkar, S.S. Sarnaik, A.S. Kelkar Division of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, Maharashtra, India Received 20 April 2000; received in revised form 11 January 2002; accepted 11 January 2002 Abstract Alkaliphilicbacteriabelongingtothegenera Bacillus, Staphylococcus, Micrococcus, Pseudomonas and Arthrobacter isolatedfrom sediment samples of the alkaline Lonar lake situated in the Buldhana District of Maharashtra State, India, were studied for the production of protease activity. Among the 54 isolates obtained, Arthrobacter ramosus and Bacillus alcalophilus exhibited high protease activity using soyacake as a sole source of carbon and nitrogen. Protease activity was optimum at 1% initial substrate concentration, at 30 °Candundershakecultureconditionforbothorganisms.Theenzymewasthermostable(65 °C),stableatpH 12 and also active in the presence of commercial detergent. This enzyme removed blood stains from cotton fabric indicating its potential use in detergent formulations. Ó 2002 Published by Elsevier Science Ltd. Keywords: Lonar lake; Alkaliphilic bacteria; Protease activity 1. Introduction Alkaliphilic microorganisms are considered as ex- tremophilesbecausetheyhavepHoptimaforgrowthin an alkaline range 9.0–10.0. The environments fre- quentedbythesealkaliphilesaredifferentfromstandard laboratory conditions. Naturally occurring highly alka- line environments are minimal with the most notable being soda lakes, alkaline springs, desert soils and soils containing decaying proteins. Alkaliphilic bacteria have been isolated from different ecological systems such as alkaline soil, CaCO 3 kilns and potato processing waste and identified as belonging to the genera Pseudomonas, Micrococcus, Arthrobacter, Corynebacterium, Bacillus and Staphylococcus along with Exiguobacterium auran- tiacum, Clostridium putrificium and Proteus mirabilis (Horikoshi and Akiba, 1982; Kroll, 1990; Nihalani and Satyanarayana, 1992; Joshi and Ball, 1993). Alkaliphiles are reported to be a rich source of al- kaline active enzymes e.g. amylase, protease, cellulase, xylanase, and other enzymes which have numerous ap- plications in industrial processes (Hayashi et al., 1988; Horikoshi, 1991) including detergent, food, pharma- ceutical,leathertanning,(TaylorandRichardson,1979) peptide synthesis, recovery of silver from X-ray films and production of useful biomass from waste such as horn,hair,feathers(AnwarandSaleemuddin,1998)and hepatopancreas (Obara et al., 1999). Proteases from particular alkaliphilic bacteria have been described previously (Horikoshi, 1971; Manachini et al., 1988; Takami et al., 1989, 1990; Kwon et al., 1994; Chaphal- kar and Dey, 1994, 1998). The alkaline soda lake of Lonar, District Buldhana, Maharashtra State, India, is unique in Asia. Geological and chemical reports are available on Lonar lake which issituated400kmawayfromPune(JhingramandRao, 1954; Nandy and Deo, 1961). However minimal data is availableonthisLakeasasourceofbiologicalmaterial. This paper describes production of protease activity by alkaliphilic strains of Arthrobacter ramosus and Bacillus alcalophilus isolated from sediment samples of alkaline Lonar Lake. 2. Methods 2.1. Qualitative detection of protease The 54 isolates of alkaliphilic bacteria from sediment samples of Lonar Lake (Kanekar et al., 1997) were Bioresource Technology 85 (2002) 87–93 * Corresponding author. Tel.: +91-020-5653680; fax: +91-020- 5651542. E-mail address: kanekarp@rediffmail.com (P.P. Kanekar). 0960-8524/02/$ - see front matter Ó 2002 Published by Elsevier Science Ltd. PII:S0960-8524(02)00018-4