a-Amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate Gobinath Rajagopalan, Chandraraj Krishnan * Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India Received 6 March 2007; received in revised form 1 June 2007; accepted 1 June 2007 Available online 17 July 2007 Abstract A catabolite derepressed Bacillus subtilis strain KCC103 was used to produce a-amylase in medium containing sugarcane bagasse hydrolysate (SBH). Addition of SBH (1% reducing sugar (w/v)) to the nutrient medium supported maximum a-amylase production of 67.4 U ml 1 . HPLC analysis of SBH showed the presence of glucose, xylose and arabinose in the ratio of 0.9:1.0:0.16 (w/w/w). In SBH-medium glucose and xylose were consumed completely while arabinose remained unutilized. Uptake rate of glucose was 2-folds higher than xylose but rate of a-amylase production with xylose was 1.5-folds higher than glucose. Arabinose had no effect on growth and a-amylase synthesis. Further, a-amylase production in SBH-medium was enhanced to 144.5 U ml 1 (2.2-fold) by response surface methodology where the levels of SBH, and other media components were varied. The modified medium consisted of (in g l 1 ) SBH: 24; peptone: 17.43; yeast extract: 1.32 and beef extract: 1.82. High level of SBH showed no significant inhibition of a-amylase synthesis. The derepressed strain KCC103 is useful to produce a-amylase economically in short time (30–36 h). Ó 2007 Elsevier Ltd. All rights reserved. Keywords: a-Amylase; Bacillus subtilis; Sugarcane bagasse hydrolysate; Catabolite repression; Response surface methodology 1. Introduction a-Amylases (EC 3.2.1.1, 1,4-a-D-glucan glucanohydroly- ase) hydrolyse starch to a range of products such as glucose and maltose or specific malto-oligosaccharide or mixed malto-oligosaccharides (Hashim et al., 2005; Messaoud et al., 2004; Takasaki, 1983; Dey et al., 2002). They are employed in industries for different purposes; glucose and maltose-forming a-amylases in alcohol fermentation and sugar syrup formulation, and malto-oligosaccharide-form- ing a-amylases in food processing (Kirk et al., 2002; Pala- cios et al., 2004). Amylases also play a significant role in starch, detergent, beverage and textile industries and its commercial production from microorganisms represent 25–33% of the world enzyme market (Nguyen et al., 2002). Industrial production of enzymes can be made eco- nomical by utilizing low cost substrates such as agricultural byproducts in the production medium. In recent years, there has been an increasing effort on efficient utilization of sugarcane bagasse which is one of the largest cellulosic agro-industrial byproducts (Pandey et al., 2000). Sugarcane bagasse can be used as a raw fiber in solid state fermenta- tion or acid hydrolysed simple sugars in submerged fer- mentation (Correa and Tengerdy, 1998; Dhillon et al., 2000; Pandey et al., 2000; Pessoa et al., 1997). The hydro- lysate of sugarcane bagasse has been employed as a carbon source for enzyme production and fermentation processes such as ethanol production (Pandey et al., 2000; Roberto et al., 1991). There are several reports on the production of lignocellulose degrading enzymes such as cellulase, xylanase and laccase from microorganisms utilising sugar- cane bagasse (Aiello et al., 1996; Adsul et al., 2004; Arorra and Gill, 2001). Bacillus sp. is well known to produce a-amylases in starch medium. A few reports describe the production of a-amylase from Bacillus sp. in media consisting of agro-byproducts 0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.06.001 * Corresponding author. Tel.: +91 44 22574111; fax: +91 44 22570509. E-mail address: kcraj@iitm.ac.in (C. Krishnan). Available online at www.sciencedirect.com Bioresource Technology 99 (2008) 3044–3050