解淀粉芽孢杆菌中温α-淀粉酶基因的克隆、表达与酶学性质分析

以中温α-淀粉酶生产菌株Bacillus amyloliquefaciens M23基因组DNA为模板。PCR扩增得到了2．0kb α-淀粉酶基因全长序列。该基因由上游启动子220bp，结构基因1544bp和终止序列320bp构成。将无信号肽的α-淀粉酶结构基因amyQ，克隆入表达载体pET28a，转化E．coli BL21（DE3），经诱导，测定α-淀粉酶活性。结果表明：α-淀粉酶基因amyQ获得了活性表达，酶活力为2．297U／mL，SDS-PAGE电泳结果显示出分子量约为58kDa特异性蛋白质条带。酶学性质分析表明，重组α-淀粉酶的最适反应温度为60℃，最适反应pH为6．5，在60℃保温15min保持85％以上活性，超过15min，酶迅速失活，在pH5．5～10．0环境下稳定。水解产物分析表明：淀粉水解终产物主要为麦芽寡糖和糊精和少量葡萄糖。

Cloning,expression and characterization of α-amylase gene from Bacillus amyloliquefaciens M23

An α-amylase gene with 2.0kb was cloned from an amylase industrial producing strain Bacillus amyloliquefaciens M23, which was composed of 1544 bp structural gene fragment, a 220 bp promoter and 320 bp terminal sequence. A 1.6 kb structural region devoid of signal peptide was amplified and inserted into expression vector pET-28a. The recombinant plasrnid was transformed into E. coli BL21 （DE3）. It showed 2. 297 U/mL of amylase activity in supematant when induced by IPTG. The molecular weight of the recombinant enzyme was 58 kDa showed in SDS-PAGE. The recombinant enzyme showed the optimum temperature was at 60℃ and the optimum pH was at 6.5. It kept stable within 15 min and lost activity rapidly over 15 min at 60℃. The enzyme was stable at pH range of 5.5 - 10.0. The main hydrolyze products to soluble starch were some oligasaccharides, dextrin and glucose. Key words