巨大芽孢杆菌淀粉酶基因的克隆及其在枯草杆菌中的表达

Molecular Cloning of α-amylase Gene from Bacillus megaterium and Its Expression in Bacillus subtilis

以λ噬菌体为载体,采用鸟枪法由B.megaterium基因组克隆得到了1个淀粉酶基因,并已被亚克隆到E.coli和B.subtilis中,其表达水平较B.megaterium高250倍。克隆株产生的淀粉酶对直链淀粉的早期水解产物主要为麦芽三糖和麦芽糖,随着水解时间的延长,又将它们转变为葡萄糖。同时能以麦芽三糖为底物水解为麦芽糖和葡萄糖。受体菌的平行提取物无上述水解活性。因而该酶被确定为糖化型α-淀粉酶。SDS-凝胶电泳法确定酶分子量为58000道尔顿。

Using Bacteriophage X and plasmid pAT153 and pNQ122 as vectors, α-Amylase gene from B. megaterium has been cloned into both hosts of E. colt and B. subtilis. Expression level of the gene is 250 times higher than B. megaterium when it resides in B. subtilis.The enzyme produced by B. subtilis harboring the hybrid plasmid can digest amylase into maltose and maltotriose at first, then turn them to maltose and glucose, as incubation time extended. It also can digest maltotriose to maltose and glucose. As a control, the extracts from the broth of recipient strain have no detectable amylase activities- Therefore the enzyme coded by this gene is defined as saccharifying type α-amylase. Its molecular weight is about 58 000 daltons.