突变野油菜黄单胞菌α-淀粉酶基因在酿酒酵母中的整合表达  

Integration expression of a mutated alpha-amylase gene from Xanthomonas campestris in the ribosomal DNA of Saccharomyces cerevisiae

作　　者：马向东[1] 柯涛[2] 马立新[1] 何光源[3] 

机构地区：[1]湖北大学生命科学院,湖北武汉430062 [2]南阳师范学院生命科学与技术学院,河南南阳473061 [3]华中科技大学中英HUST-RRes基因工程和基因组学联合实验室,湖北武汉430074

出　　处：《华中科技大学学报（自然科学版）》2007年第5期116-119,共4页Journal of Huazhong University of Science and Technology(Natural Science Edition)

基　　金：国家重点基础研究发展规划资助项目(2002CB111302);国家自然科学基金资助项目(30370807);国家高技术研究发展计划资助项目(2002AA224031)

摘　　要：用定向进化的一种新方法,对野油菜黄单胞菌(Xanthomonas campestris pv.campestris)的α-淀粉酶基因(XAMY)进行PCR体外诱变后获得了一个编码的酶活性提高的突变基因,将其克隆到由rDNA序列介导的酵母整合型表达载体pHBM368上,得到重组质粒pHBM368XA.将pHBM368XA转化酿酒酵母S.cerevi-siaeINVScI,获得了整合型分泌表达α-淀粉酶的酵母重组菌株XA01.对选择的3个工程菌α-淀粉酶表达的稳定性进行了检测,结果表明在完全培养基中连续培养80代,淀粉酶的酶活性仍然保持稳定.在淀粉酶自身信号肽引导下,胞外分泌效率可达50%.A mutated Xanthornonas carnpestris pv. carnpestris alpha-amylase gene with encoded higher activity enzyme product was obtained through a new directed evolution method and cloned to the Saccharomyces cerevisiae expression vector pHBM368-a vector system for high-copy-number integration into the ribosomal DNA of the yeast. The recombinant plasmid was resigned pHBM368XA and transformed into an S. cerevisiae INVScI strain. Three transformants were selected, in which the integrated sequences were stable even after 80 generations of growth in nonselective medium cultivation. The transformants expressed and secreted alpha-amylase with a secretion efficiency of about 50 %.

关 键 词：Α-淀粉酶 rDNA介导 啤酒酵母 整合表达 

分 类 号：Q556.2[生物学—生物化学]