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[1]王成华,王青艳,熊伍平,等.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(03):20-26.[doi:10.3969/j.issn.1671-7627.2012.03.004]
 WANG Chenghua,WANG Qingyan,XIONG Wuping,et al.Codon optimization, expression and characterization of aciduric and thermostable α-amylase in E.coli[J].Journal of NANJING TECH UNIVERSITY(NATURAL SCIENCE EDITION),2012,34(03):20-26.[doi:10.3969/j.issn.1671-7627.2012.03.004]
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耐酸耐温α-淀粉酶基因在大肠杆菌中的功能表达()
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《南京工业大学学报(自然科学版)》[ISSN:1671-7627/CN:32-1670/N]

卷:
34
期数:
2012年03期
页码:
20-26
栏目:
出版日期:
2012-05-20

文章信息/Info

Title:
Codon optimization, expression and characterization of aciduric and thermostable α-amylase in E.coli
文章编号:
1671-7627(2012)03-0020-07
作者:
王成华12王青艳23熊伍平23廖思明23何冰芳1黄日波123
1. 南京工业大学 生物与制药工程学院,江苏 南京 211816; 2. 广西科学院 国家非粮生物质能源工程技术研究中心,非粮生物质酶解国家重点实验室,广西 南宁 530007; 3. 广西大学 生命科学与技术学院,广西 南宁 530004
Author(s):
WANG Chenghua12 WANG Qingyan23 XIONG Wuping23 LIAO Siming23 HE Bingfang1 HUANG Ribo123
1. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China; 2. State Key Laboratory of Non-Food Biomass and Enzyme Technology, National Engineering Research Center for Non-food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China; 3. College of Life Science and Technology, Guangxi University, Nanning 530004, China
关键词:
淀粉酶 密码子偏好 耐酸 耐温 表达
Keywords:
amylase codon bias aciduric thermostable expression
分类号:
Q556.2
DOI:
10.3969/j.issn.1671-7627.2012.03.004
文献标志码:
A
摘要:
兼顾大肠杆菌与枯草芽孢杆菌密码子偏好性,优化获得了1个耐酸耐温淀粉酶的基因amyCN1,并将其在大肠杆菌中实现了功能表达。纯化后的重组α-淀粉酶(AMY1)表征结果表明:在最适pH 5.5和最适温度75 ℃条件下表观米氏常数(Km)值和催化效率(kcat/Km)值分别为20.93 g/L和98.20 L/(g·s); 低浓度的Co2+(1 mmol/L)可以提高30%的淀粉酶活力,Mn2+抑制了大部分活力,包括Ca2+在内的其他金属离子影响不显著; 高浓度的EDTA(≥50 mmol/L)抑制其活力; 生物信息学分析表明,AMY1具有α-淀粉酶家族典型的3-结构域分布,具有1个Ca2+结合位点和5个Zn2+结合位点,活性中心催化氨基酸残基D198和E222位于(β/α)8桶中β折叠片C端一侧。AMY1优良的低pH耐受性和耐温性,有助于木薯淀粉生产燃料乙醇工业液化与糖化同步发酵工艺的实现。
Abstract:
Considering the codon bias in Escherichia coli and Bacillus subtilis, 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 purity of 95% assessed by SDS-PAGE. 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 Mn2+ 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 α-amylase family: domain A consisting of one(β/α)8 barrel locating the catalytic amino acid residual D198 and E222, domain B protruding between the third β strand and third α 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 saccharification process for the ethanol production from the cassava starch.

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备注/Memo

备注/Memo:
收稿日期:2011-05-30
基金项目:广西自然科学基金重点资助项目(2010GXNSFD013030); 广西科学研究与技术开发计划资助项目(桂科攻1099070); 广西科学院基金资助项目(09YJ17SW01)
作者简介:王成华(1983—),男,河南南阳人,博士生,主要研究方向为工业生物催化; 黄日波(联系人),教授,E-mail:rbhuang@gxas.ac.cn..
更新日期/Last Update: 2012-05-20