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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (33): 194-198.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

Bacillus subtilis FS321的α-淀粉酶基因的克隆与表达

施碧红 温建新 吴伟斌 施巧琴 吴松刚   

  • 收稿日期:2011-09-19 修回日期:2011-10-21 出版日期:2011-12-25 发布日期:2011-12-25
  • 基金资助:

    福建省自然科学基金;福建省科技重点项目

Cloning and Expression of the α-amylase Gene from Bacillus subtilis FS321

  • Received:2011-09-19 Revised:2011-10-21 Online:2011-12-25 Published:2011-12-25

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摘要：

耐热α-淀粉酶被广泛用于食品等诸多行业，本研究从中国北方高温堆肥分离的枯草芽孢杆菌FS321中克隆了一中度耐热α-淀粉酶基因，并实现在大肠杆菌BL21（DE3）中的表达。通过PCR技术克隆Bacillus subtilis FS321的α-淀粉酶编码基因(BSA)，该基因全长1980 bp。并构建重组表达质粒pET-28a/BSA，转化大肠杆菌E. coli BL21(DE3)，经IPTG诱导表达，SDS-PAGE检测到大小约为73.0 kDa的重组融合蛋白，可溶性淀粉平板检测结果表明BSA在大肠杆菌中实现了有效表达。该重组α-淀粉酶的最适反应温度为50℃，最适反应pH为7.5。

关键词: 气相色谱分析, 气相色谱分析

Abstract:

The thermostable α-amylase was widely used in the fields of food industry etc., in the present study, a moderate thermostable α-amylase gene was cloned from Bacillus subtilis FS321, which was isolated from the dunghill soil in the northern of China, and expressed in Escherichia coli BL21 (DE3). The α-amylase gene from B.subtilis FS321 (BSA) was cloned using PCR method, the complete ORF of BSA was in a size of 1980 bp. The recombinant expression vector of pET-28a/BSA was constructed, and transformed to E. coli BL21 (DE3). A fusion protein with a size of about 73.0 kDa displayed in SDS-PAGE and the clear zones developed in the soluble starch plate demonstrated that the BSA was functional expressed in E. coli after induction by IPTG. The optimum reaction temperature of the recombinant α-amylase was of 50℃, and optimum reaction pH of 7.5.