巨大芽孢杆菌_淀粉酶基因克隆_表达和酶学性质分析.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, October 25; 24(10): 1740-1746 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 - β 吴襟, 张树政 , 100101 : 从巨大芽孢杆菌(Bacillus megaterium) 的全基因组DNA 文库中筛选出一个β-淀粉酶基因 amyG, 分析测定了其 核苷酸序列并进行了诱导表达; 其中 amyG 编码的蛋白有 545 个氨基酸、分子量为 60.194 kD, 与已报道的巨大芽孢杆 β 菌DSM319 的 -淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG 从N 末端到C 末端依次由信号肽 域、糖基水解酶催化功能域和淀粉结合域3 个功能域组成。其中催化功能域里含有第 14 家族糖基水解酶常见的几个高 度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4 倍, 获得凝胶电泳均一的蛋白样品; 经SDS电 o o 泳测定, 酶AmyG 的分子量为57 kD。该酶的最适反应温度为60 C, 最适反应pH 为7.0; 在温度不超过60 C 时, 酶活 较稳定; AmyG 能迅速降解淀粉生成麦芽糖, 属于外切β-糖苷酶。 : 巨大芽孢杆菌, β-淀粉酶, 基因克隆和表达, 酶学性质 Cloning, Expression and the Characterization of β-amylase from a Bacillus megaterium WS06 Jin Wu, and Shuzheng Zhang Institute of Microbiology , Chinese Academy of Sciences, Beijing 100101, China Abstract: A β-amylase gene (amyG) was cloned from a Bacillus megaterium WS06 and expressed in the Escherichia coli. Nucleotide sequence anlysis showed the amyG gene is composed of 1638 bp (545 amino acid residues with a Mr of 60.194 kD). The AmyG shows 94.5% sequence homologies with β-amylase from Bacillus megaterium DSM319 and presents a normal β-amylase primary structure, constituted by three parts: the N-terminal signal sequence, the catalytic domain and the C-terminal starch binding domains. The deduced amino acid sequence revealed that several highly