Food Sci. Biotechnol. 23(1): 1-8 (2014) DOI 10.1007/s10068-014-0017-4 Molecular Cloning and Characterization of a Thermostable α-Amylase Exhibiting an Unusually High Activity Jong-Tae Park, Antonius Suwanto, Irawan Tan, Tommy Nuryanto, Rudy Lukman, Kan Wang, and Jay-lin Jane Received: 26 October 2012 / Revised: 25 April 2013 / Accepted: 11 June 2013 / Published Online: 28 February 2014 © KoSFoST and Springer 2014 Abstract An α-amylase gene was cloned from the thermophilic bacterium Bacillus subtilis isolated from Indonesian oil palm shell waste. The gene expressed an extracellular enzyme. Optimal hydrolysis conditions for the enzyme were 70 o C and pH 6.0. The specific activity of the enzyme was 16.0 kU per mg of protein, which was higher than for other thermostable amylases. Hydrolytic products of the enzyme using starch and glycogen were mainly maltohexaose and maltopentaose. The enzyme had a K m value of 0.099 mg/mL for amylopectin, more than 10 times lower than for amylose. The catalytic efficiency of the enzyme using amylopectin was 39,200 mL/mg·s and was 3,270 mL/mg·s using amylose. The enzyme liquefied corn starch at pH 5.0, which was successfully converted to glucose using commercial glucoamylase and pullulanase without pH adjustment. The enzyme has advantages for industrial applications. Keywords: thermostable α-amylase, kinetic parameter, starch liquefaction, molecular cloning, Geobacillus stearothermophilus Introduction α-Amylase (EC 3.2.1.1) is the representative enzyme in the glycoside hydrolase family 13 (GH 13) (1) and is an amylolytic enzyme that hydrolyzes starch and related polysaccharides by hydrolyzing internal α-1,4-glycosidic linkages. The enzyme is widely distributed in various species from bacteria to mammals. Starch is the most abundant carbon energy source on earth and consists of amylose (glucose polymers mostly linked by α-1,4-linkages) and amylopectin (glucose polymers linked by approximately 95% α-1,4-linkages, and 5% α- 1,6-branch linkages). A huge amount of starch from plants is processed for use in the textiles, paper, and food industries. Sugar conversion from corn starch is one of the most important processes in the starch industry (2). α-Amylase has been used for starch liquefaction for decades, and the properties of the enzyme have been improved for industrial applications. In the conventional method, a mixture of starch slurry and enzyme is jet-cooked at 105 o C-110 o C to partially breakdown starch molecules and reduce viscosity, and then cooled to 95 o C to continue α-amylase hydrolysis (2-4). Many commercial enzymes that are stable at a temperature of 95 o C or above have been developed by the enzyme industry from the α-amylase of Bacillus licheniformis (BLA). The wet-milling industry, however, is looking for further improvements in the properties of α-amylase, including thermostablity at low pH values with no calcium ion. Thermostable α-amylases isolated from B. stearothermophilus (BSTA), B. amylo- liquefacience (BAA), and from fungi are widely used for starch hydrolysis in the food industry (2). Jong-Tae Park, Jay-lin Jane () Department of Food Science and Human Nutrition, Iowa State University, IA 50011, USA Tel: +1-515- 294-9892; Fax: +1-515-294-8181 E-mail: jjane@iastate.edu Jong-Tae Park Department of Food Science and Technology, Chungnam National University, Daejeon 305-764, Korea Antonius Suwanto Department of Biology, Faculty of Science and Mathematics, Bogor Agricultural University, Bogor, Kediri 16680, Indonesia Irawan Tan, Tommy Nuryanto Charoen Pokphand Indonesia, Jakarta Utara 14430, Kediri, Indonesia Rudy Lukman Biotech Department of PT. BISI International, Kediri 16720, Indonesia Kan Wang Department of Agronomy, Iowa State University, Ames, IA 50011, USA RESEARCH ARTICLE