南京工业大学学报（自然科学版）2012，Vol.34Issue(3)：20-26,7.DOI:10.3969/j.issn.1671-7627.2012.03.004
耐酸耐温α-淀粉酶基因在大肠杆菌中的功能表达
Codon optimization, expression and characterization of aciduric and thermostable α-amylase in E.coli
摘要
Abstract
Considering the codon bias in Escherichia coli and Bacillus suhlilis, one gene amyCN1 encoding an aciduric and thermostable α-amylase was optimized and functionally expressed in E. coli. Using the one-step purification procedure of Ni-NTA, the C-terminal 6xHis tag fused AMY1 was purified to purily of 95% assessed by SDS-PACE. The purified recombinant AMY1 showed appealing enzymatic properties under optimum pH of 5. 5, optimum temperature of 75 ℃, Km of 20. 93 g/L and catalytic efficiency (kcat/Km) of 98. 20 L/(g·s) ; low concentration of Co2+ increased the amylase activity by approx. 30% , but Mn + inhibited more than a half of the activity, and other metal ions including Ca2+ showed hardly anything significant to the activities of AMY1, EDTA deteriorated the AMY1 activity, indicating that the metal ions were required for the structural stability and function. The AMY1 adopted the typical arrangement of three domains in a-amylase family; domain A consisting of one (α/β)s barrel locating the catalytic amino acid residual D198 and E222, domain B protruding between the third β strand and third a helix, and domain C composing of all beta sheets. Further theoretical structural model analysis disclosed that there were one Ca2+ binding site and five Zn2+ binding sites located on the AMY1. The aciduric and thermostable features of AMY1 provided the potentiality for the simultaneous liquefaction and saccharifica-tion process for the ethanol production from the cassava starch.关键词
淀粉酶/密码子偏好/耐酸/耐温/表达Key words
amylase/ codon bias/ aciduric/ thermostable/ expression分类
生物科学引用本文复制引用
王成华,王青艳,熊伍平,廖思明,何冰芳,黄日波..耐酸耐温α-淀粉酶基因在大肠杆菌中的功能表达[J].南京工业大学学报（自然科学版）,2012,34(3):20-26,7.基金项目
广西自然科学基金重点资助项目(2010GXNSFD013030) （2010GXNSFD013030）
广西科学研究与技术开发计划资助项目(桂科攻1099070) （桂科攻1099070）
广西科学院基金资助项目(09YJ17SW01) （09YJ17SW01）
