RESEARCH ARTICLE A novel cold-adapted pullulanase from Exiguobacterium sp. SH3: Production optimization, purification, and characterization Sarah Rajaei*, Reza Heidari*, Hossein Shahbani Zahiri, Sara Sharifzadeh, Ibrahim Torktaz and Kambiz Akbari Noghabi Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Cold-adapted enzymes, elaborated by psychrophiles and psychrotrophs, seem to have potential for current and future applications. In the current study, pullulanase production by the cold-adapted Exiguobacterium sp. SH3 was investigated. The Plackett–Burman design and the response surface methodology were applied to identify and optimize the significant variables affecting the pullulanase production of Exiguobacterium sp. SH3. The results showed that temperature, time, and CaCl 2 concentration were significant variables while shaking, starch, yeast extract, tryptone, pH, MnCl 2 , MgCl 2 , and KH 2 PO 4 were not significant. Using statistical analyses and optimizations, the pullulanase production was significantly elevated from 200 Æ 18 U/mL to 950 Æ 27U/mL (4.75 times) as compared to non-optimized conditions. A pullulanase of about 70 kDa, designated as Pul-SH3, was purified 16.2-folds from the optimized culture, and identified to be an amylopullulanase. The K m and V max of the enzyme were 0.069 mg/mL and 967 U/mL, respectively. The optimum pH and temperature for maximum activity of Pul-SH3 were 7.5 and 30°C, respectively. As a cold-adapted enzyme, Pul-SH3 retained 23% of the maximum activity at 0°C. The biochemical characteristics and N-terminal amino acid sequence of Pul-SH3 suggest that the enzyme is a novel cold-adapted amylopullulanase with remarkably high specific activity at moderate ambient temperature. Received: January 30, 2013 Revised: June 10, 2013 Accepted: June 12, 2013 Keywords: Cold-adapted / Exiguobacterium / Pullulanase 1 Introduction The starch-hydrolyzing enzymes are important because they have widespread applications in food, feed, pharmaceutical, paper, textile, and detergents industries. The enzymes account for about 25% of the global enzyme market that is anticipated to reach US $7 billion by 2013 [1]. Starch is composed of a mixture of amylose and amylopectin. Amylose is a linear polymer of glucose with a-1,4-linkages, while amylopectin is a branched polymer of glucose with main polymerizing a-1,4- and branching a-1,6-linkages. The ratio of amylopectin to amylose is variable in starch obtained of different plants. However, the amylopectin content of starch in most plants is more than 60% [2]. Among the starch- hydrolyzing enzymes are pullulanases which possess a-1,6 and/or a-1,4 hydrolysis activity on pullulan and, possibly, other carbohydrates such as starch, amylopectin, and glycogen [3–5]. According to substrate and linkage specificity as well as the end products of substrate hydrolysis, pullulanase enzymes fall into five categories: (i) pullulanase type I, hydrolyzing a-(1,6) linkages in pullulan to produce maltotriose; (ii) pullulanase type II (amylopullulanase), hydrolyzing a-(1,6) linkages in pullulan to produce malto- triose, and hydrolyzing a-(1,4) linkages in starch to produce a mixture of glucose, maltose, and maltotriose; (iii) pullulan hydrolase type I (neopullulanase), hydrolyzing a-(1,4) link- ages in pullulan to produce panose; (iv) pullulan hydrolase type II (isopullulanase), hydrolyzing a-(1,4) linkages in pullulan to produce isopanose; (v) pullulan hydrolase type III, Colour online: See the article online to view Fig. 4 in colour. Ã These authors contributed equally to this work. Correspondence: Dr. Hossein Shahbani Zahiri, National Institute of Genetic Engineering and Biotechnology (NIGEB), Pajoohesh Blvd., Hemat Highway, Tehran 14155-6343, Iran E-mail: shahbani@nigeb.ac.ir Fax: þ98-21-44580395 Abbreviations: CCD, central composite design; RSM, response surface methodology DOI 10.1002/star.201300030 Starch/Stärke 2014, 66, 225–234 225 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com