618 Research Article Received: 21 August 2008 Revised: 14 October 2008 Accepted: 14 October 2008 Published online in Wiley Interscience: 29 December 2008 (www.interscience.wiley.com) DOI 10.1002/jctb.2090 Optimization of agro-residual medium for α -amylase production from a hyper-producing Bacillus subtilis KCC103 in submerged fermentation Gobinath Rajagopalan and Chandraraj Krishnan ∗ Abstract BACKGROUND: Although submerged fermentation (SmF) is the conventional method in industry, use of low-cost agro-residues for α-amylase production in SmF has not been well established. Here we optimized agro-residue-based medium and culture conditions for α-amylase production in SmF using a hyper-producing Bacillus subtilis KCC103. RESULTS: B. subtilis KCC103 produced α-amylase in SmF by utilizing agro-residues. Wheat bran (WB) and sunflower oil cake (SFOC) were selected as the best substrates using shake flasks. Medium containing WB (carbohydrate rich) and SFOC (rich in protein and free amino acids) at 1 : 1 (w/w) ratio produced high levels (90 IU mL −1 ) of α-amylase at 30 – 36 h in a shake flask. The α-amylase yield was 14-fold enhanced (1258 IU mL −1 ) by optimizing process parameters and medium composition following response surface methodology in a bioreactor. The optimal conditions were: WB 1.27%, SFOC 1.42%, pH 7, 37 ◦ C and 10 – 12 h. Both in shake flask and bioreactor α-amylase synthesis was not repressed by the release of simple sugars into the medium. CONCLUSION: KCC103 with catabolite derepression and hyperproducing ability is useful for economic α-amylase production using low-cost agro-residual substrates in conventional SmF. Since the production time (10– 12 h) is much shorter than other strains this would improve productivity and further reduce the cost of α-amylase production. c  2008 Society of Chemical Industry Keywords: α-amylase production; Bacillus subtilis; catabolite repression; wheat bran; oil cake; media optimization INTRODUCTION α-Amylases are among the most important enzymes in present- day biotechnology. Although α-amylases can be derived from several sources, including plants, animals and microorganisms, microbial enzymes generally meet industrial demands. Currently, several microbial α-amylases are available commercially and they have almost completely replaced the chemical hydrolysis of starch in the starch-processing industries. 1 α-Amylases find potential application in a number of industrial processes such as the food, fermentation, textile and paper industries. α-Amylase can be produced by submerged fermentation (SmF) or solid-state fermentation (SSF). 2,3 Industrial-scale production of α-amylase is traditionally obtained from SmF because of the ease of handling, high product recovery and greater control of environmental factors such as temperature and pH. 4,5 Production of α-amylase by SmF using synthetic media has been reported for Bacillus spp. 1 The main drawback of SmF in α-amylase production is cost of raw materials in the synthetic media, which include expensive components such as soluble starch, glycerol and/or maltose as carbon source, and peptone, tryptone, meat extract, yeast extract and/or soy peptone as nitrogen source. 2,3 Replacement of expensive components of the synthetic media with low-cost agro-residues would lead to reduced cost of α-amylase production by SmF for application in industries. B. subtilis KCC103 is a catabolite derepressed for α-amylase synthesis and is able to hyper-produce α-amylase using diverse carbon sources. Because of catabolite derepression in KCC103, α-amylase production begins in early growth phase and the efficiency of production increases due to shorter production time and hyper-production of α-amylase. This strain is capable of producing α-amylase by utilizing various cellulosic biomass sources and this property allows economic production of α-amylase. It synthesizes a single α-amylase with a molecular mass of 53 kDa. 6 The α-amylase can form malto-oligosaccharides, which are useful in the food industry. Hence formulation of a cheap medium for this enzyme would be a step towards process development for production of low-cost α-amylase, which in turn would minimize the cost of malto-oligosaccharide. Here we describe the optimization of process parameters and low-cost agro-residue-based medium for α-amylase production in SmF using KCC103. ∗ Correspondence to: Chandraraj Krishnan, Department of Biotechnology, Indian Institute of Technology Madras, Chennai-600036, India. E-mail: kcraj@iitm.ac.in Department of Biotechnology, Indian Institute of Technology Madras, Chennai- 600036, India J Chem Technol Biotechnol 2009; 84: 618–625 www.soci.org c  2008 Society of Chemical Industry