A highly thermostable, alkaline CMCase produced by a newly isolated Bacillus sp. VG1 J. Singh, N. Batra and R.C. Sobti* Department of Biotechnology, Punjab University, Chandigarh – 160014, India *Author for correspondence: Tel.: þ91-172-534087, Fax: þ91-172-541409, E-mail: rcsobti@punjabuniv.chd.nic.in Received 30 March 2001; accepted 28 August 2001 Keywords: Alkaline, Bacillus, carboxymethyl cellulase, detergents, thermostable Summary An extracellular, highly thermostable and alkaline CMCase was purified from Bacillus sp. VG1 using ion exchange and gel filtration chromatography. Enzyme was optimally produced in a medium containing 1.0% CMC and 0.5% tryptone. The purified CMCase had a pH optimum of 9–10 and a half life of 12 min even at 100 °C. The enzyme activity was reduced by Hg 2þ and stimulated by Co 2þ ,Na þ and K þ . Various detergents and proteinases moderately inhibited the CMCase activity. The molecular weight studies showed a single band on SDS–PAGE. Introduction Cellulose is the most abundant and renewable polymer on earth. Its enormous potential as renewable energy source was recognized after World War II, when ‘Cellulase’ was introduced to degrade clothing, tents and sand bags in the South Pacific (Bhat & Bhat 1997). Recently the potential of cellulases was revealed in various industries, including food, textiles and laundry, pulp and paper and agriculture as well as in research and development (Wong & Saddller 1993; Poutanen 1997; Cavaco-Paulo 1998; Screws & Pedersen 1998; Uhlig 1998; Bajpai 1999; Bhat 2000). During the washing process, interaction between the ingredients of the detergent and the surface of the fabric lowers surface tension and enhances the repulsive force between the soil and fabric (Murata et al. 1991). Modern types of heavy duty detergents contain one or more enzymes such as protease, amylase, cellulase, lipase and lactase (Ito et al. 1998; Foley & Jones 1999) to increase their effectiveness as detergents. Cellulases (including CMCases) are added to laundry detergents to improve softness and colour brightness and to remove dirt (soil) from cotton blended garments (Ito 1997; Cavaco-Paulo 1998; Bhat 2000). The world market for industrial enzymes has grown sharply to US$ 1.6 billion (Demain 2000) and is expected to be US$ 1.7–2.0 billion by the year 2005 (Godfrey & West 1996a). Bacillus sp. VG1, that grows at pH 7.0–11.0, produces a highly thermostable, alkaline and extracellular CMCase. The production of enzyme has been optimized. It has been purified and characterized to exploit its potential as an effective additive for laundry detergents. Material and methods Chemicals All the analytical reagents and media components were purchased from Hi-Media (Bombay, India) and Sigma Chemicals (St. Louis, USA). Bacterial strain, medium and culture conditions Alkaline CMCase-producing bacteria were screened from soil samples near the hot water springs, Manikaran (Distt. Kullu, HP, India) on CMC–Trypan blue plates (Shikata et al. 1990). The medium contained per litre: CMC 10 g; yeast extract 5 g; NaCl 5 g; KH 2 PO 4 1g and agar 20 g. Its pH was adjusted to 9.5 with sodium carbonate (10% w/v). The culture plates were incubated at 45 °C. Six strains producing CMCases were isolated and strain VG1 identified as Bacillus sp. was selected for further work. It was grown in the liquid medium at 45 °C for 72 h at 150 rev/min. The centrifuged culture supernatant was used as a source of crude enzyme. Assay for CMCase The diluted enzyme (250 ll) was mixed with an equal amount of CMC (4.0% w/v in Tris/HCl buffer, pH 8.5) and after an incubation of 30 min at 60 °C, 750 ll solution of dinitrosalicylic acid reagent was added and the mixture was heated in a boiling water bath for 10 min (Miller 1959). This was followed by the addition of 1.25 ml buffer and the absorbance was measured at 600 nm (Beckman DU 640B). World Journal of Microbiology & Biotechnology 17: 761–765, 2001. 761 Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.