Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

文献类型： 外文期刊

第一作者： Guan, Qingtian

作者： Guan, Qingtian;Guo, Xiaohan;Han, Ting;Wei, Mengwei;Zeng, Fan;Liu, Lin;Li, Zhe;Wang, Yuhan;Zhang, Shihong;Jia, Baolei;Jin, Meiling;Cheong, Gang-Won

作者机构：

关键词： Amylopullulanase;GH57 family;Thermophilic archaeon;Extremely stable;Industrial application

期刊名称：PROCESS BIOCHEMISTRY （ 影响因子：3.757； 五年影响因子：3.665 ）

ISSN： 1359-5113

年卷期： 2013 年 48 卷 5-6 期

页码：

收录情况： SCI

摘要： Thermostable amylopullulanases can catalyse the hydrolysis of both alpha-1,4 and alpha-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 degrees C. Apu-Tk was also active at a broad range of pH (4-7), with an optimum pH of similar to 5.0-5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% beta-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications. (C) 2013 Elsevier Ltd. All rights reserved.

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