耐热糖化酶基因的克隆及其在枯草杆菌中的表达  

Cloning of a Thermostable Glucoamylase Gene and its Secretive Expression in Bacillus subtilis

作　　者：李颖[1] 宋存义[1] 邱并生[2] 汪兵 杨建国[2] 

机构地区：[1]北京科技大学,北京100083 [2]中国科学院微生物研究所,北京100080 [3]北京中天诺亚体育科技有限公司,北京100089

出　　处：《微生物学通报》2006年第5期45-49,共5页Microbiology China

基　　金：北京中天诺亚体育科技有限公司资助

摘　　要：应用PCR技术从嗜热古生菌硫磺矿硫化叶菌(Sulfolobussolfataricus)中扩增到大小约为1·9kb编码嗜热糖化酶的DNA片段,并将其插入枯草杆菌诱导型表达载体pSBPYF,获得含有该糖化酶基因的重组质粒pSGAYF,转化枯草芽孢杆菌DB1342。经蔗糖诱导后,该糖化酶在枯草杆菌DB1342(pSGAYF)中获得胞外分泌表达,酶活为3·6U/mL,最适温度为90℃,最适pH6·0。The gene encoding a putative thermostable glucoamylase in a hyperthermophilic archaeon, Sulfolobus solfataricus, was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive vector pSBPYF of Bacillus subtilis, which was constructed in this lab, and the resultant plasmid pSGAYF was then transformed into competent cells of the Bacillus subtilis strain DB1342. The positive transformant DB1342 （pSGAYF） was grown on Bacillus subtilis common fermentation medium, which contained 50μg/mL kanamycin. After 2h＇s cultivation, sucrose was added and increased to the final concentration of 2% for induction. The results showed that this glucoamylase was secreted into the medium, and the enzyme activity was 3.6U/mL, optimal temperature 90℃ and pH 6.0.

关 键 词：糖化酶 基因表达 枯草芽孢杆菌 

分 类 号：Q78[生物学—分子生物学]