芽孢杆菌α-淀粉酶基因的克隆、表达和酶学性质分析

Cloning and expression of the α-amylase gene from a Bacillus sp. WS06, and characterization of the enzyme

在仔猪结肠内容物中分离出一株能利用淀粉的芽孢杆菌Bacillussp.WS06,构建了全基因组DNA文库,从中筛选出α_淀粉酶基因amyF,分析测定了其核苷酸序列并进行了表达;其中amyF编码的蛋白有526个氨基酸、分子量为58.6kD;它与已报道的Bacillusmegaterium的α_淀粉酶序列有93%的同源性。经过氨基酸序列比较分析还发现,AmyF含有淀粉酶家族中4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22.2倍,获得凝胶电泳均一的蛋白样品;经SDS_PAGE检测,AmyF酶分子量为57kD。该酶的最适反应温度为55℃～60℃,酶的最适反应pH为7.0,在温度不超过55℃时,酶活较稳定;AmyF能迅速降解淀粉生成麦芽寡糖,属于内切糖苷酶。

A Bacillus sp. WS06, An amyF gene from this Bacillus composed which produces an extracellular a-amylase, was isolated from the cecum in a piglet strain was cloned and its nucleotide sequence was determined. An open reading frame of 1581 bases, which encodes 526 amino acid residues was found. The amyF gene shows high sequence homologies with other microbial amylase genes, such as Bacillus megaterium and Bacillus polymyxa （93% and 53% identity）. The deduced amino acid sequence revealed that four highly conserved regions of the a-amylase family. The amyF gene was overepressed using the pET21a vector and Escherichia coli BI21 （DE3）. The recombinant enzyme was purified 22.2 fold to electrophoretic homogeneity and had a molecular mass of 57kD （by SDS-PAGE）. The enzyme was optimally active at pH 7 and 55 - 60℃ and showed stability at the temperature below 55℃. This enzyme efficiently hydrolyzed various types of starch to yield a series of malto-oligosaccharides by endo-cleavage mode.