食品科学 ›› 2004, Vol. 25 ›› Issue (9): 78-82.

• 基础研究 • 上一篇    下一篇

淀粉酶基因的克隆及其在工业啤酒酵母中的表达

 刘增然, 何秀萍, 龙章富, 张博润, 刘世贵   

  1. 四川大学生命科学院,中科院微生物研究所
  • 出版日期:2004-09-15 发布日期:2011-10-24

Study on Cloning of α-Amylase Gene and Its Yeast Expression

 LIU  Zeng-Ran, HE  Xiu-Ping, LONG  Zhang-Fu, ZHANG  Bo-Run, LIU  Shi-Gui   

  1. 1.College of Life Science, Sichuan University;2.Institute of Microbiology, Chinese Academy of Science
  • Online:2004-09-15 Published:2011-10-24

摘要: 以扣囊复膜孢酵母菌总DNA为模板, 通过PCR扩增克隆到约1.5kb的α-淀粉酶基因(AMY)。用酵母菌穿梭载体YEp352为出发质粒,磷酸甘油酸激酶基因1(PGK1)启动子和乙醇脱氢酶基因1(ADH1)终止子为调控元件构建了含α-淀粉酶基因编码序列的酵母菌重组表达质粒pLA8。pLA8导入工业啤酒酵母菌,在以淀粉为唯一碳源的YNBS培养基上筛选阳性转化子,转化子培养液中α-淀粉酶活性为1.1U/ml,细胞破碎液的酶活性为0.3U/ml,而受体菌培养液及细胞破碎液中未检测到α-淀粉酶活性。

关键词: 工业啤酒酵母菌, 淀粉酶, 基因的克隆和表达

Abstract: A 1.5kb α-amylase gene was prepared by PCR with Saccharomycopsis fibuligera genomic DNA as the template.The gene, together with phosphoglycerate kinase1 promoter and ethanol dehydrogenase1 terminator from S. cerevisiae was clonedinto a yeast expression vector (YEp352), generating a recombinant plasmid pLA8. The recombinant plasmid was introduced intoS. cerevisiae and the transformants were screened on the medium in which starch was the sole carbon source. The average α-amylase activities of the transformants in cellular extract and culture supernatants were 0.3U/ml and 1.1U/ml respectively. Noα-amylase activity was detected in those of the host strain.

Key words: industrial strain of S. cerevisiae, amylase, gene cloning and expression