巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

Cloning, Expression and the Characterization of b-amylase from a Bacillus megaterium WS06

摘要:

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-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菌DSM319的b-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-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。

Abstract:

A b-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 b-amylase from Bacillus megaterium DSM319 and presents a normal b- 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 conserved regions of the glycosylhydrolase family 14. The amyG gene was overexpressed using the pET21a vector and Escherichia coli BL21(DE3). The recombinant enzyme was purified 7.4 fold to electrophoretic homogeneity and had a Mr of 57 kD (by SDS-PAGE). The enzyme was optimally active at pH 7.0 and 60oC and showed stability at the temperature below 60oC. This enzyme efficiently hydrolyzed starch to yield maltose from non-reducing chain ends by exo-cleavage mode.