大肠杆菌嗜盐α-淀粉酶基因的分子改造及其酶学特性  

Escherichia coli Halophilic alpha Amylase Gene Molecular Modification and Enzymology Characteristics

作　　者：李剑龙[1,2] 杨媛[1,2] 吴昊[1,2] 汤宏赤 韦宇拓[1,2] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530005 [2]亚热带农业生物资源保护与利用国家重点实验室,广西南宁530005

出　　处：《广西科学》2016年第1期25-30,共6页Guangxi Sciences

基　　金：国家自然基金项目(31460437);广西研究生教育创新计划项目(YCSZ2014050)资助

摘　　要：【目的】对大肠杆菌Escherichia coli嗜盐α-淀粉酶基因进行改造,并探索嗜盐α-淀粉酶的嗜盐特性。【方法】从非嗜盐的大肠杆菌JM109中克隆到一个嗜盐α-淀粉酶基因k6并进行重组表达。通过同源建模,确定Na+结合位点上的氨基酸残基,并对相应位点进行定点突变。最后对突变酶的酶学性质进行研究。【结果】相对于野生酶,突变酶更加嗜盐,其最适NaCl浓度由2mol/L增加到3mol/L,最适pH值为7,最适温度为50℃,酶活力为4 831U/mg,提高近4倍。经HPLC检测,突变酶与2%(W/V)可溶性淀粉反应后的产物为葡萄糖、麦芽糖、麦芽三糖的混合物。【结论】嗜盐α-淀粉酶基因k6的嗜盐特性与Na+结合位点具有直接联系。【Objective】The modification of halophilic α-amylase gene-was conducted in Escherichia coli to explore halophilic properties of halophilic α-amylase.【Methods】Firstly,a halophilic amylase gene was cloned from the non-halophilic bacteria E.coli JM109,and the recombinant gene was successfully expressed.Secondly,the amino acid binding site of Na^+was determined by homology modeling,and site-directed mutagenesis was conducted at the corresponding sites.Finally,the enzymatic properties of the mutant enzymes were studied.【Results】The mutant enzyme was characterized by higher halophilic property than wild type,and the optimum concentration of NaCl increased from 2mol/L added to 3mol/L.The halophilic amylase had the optimum pH at 7and the optimum temperature at 50℃,under which its specific activity was 4 831 U/mg,revealing nearly fourfold increase than the original enzyme.HPLC testing showed that the products were mixed with glucose,maltose and maltotriose after the reaction with 2%(W/V)of soluble starch.【Conclusion】Halophilic characteristics of halophilic amylase gene k6 has direct relationship with Na^+ binding site.

关 键 词：非嗜盐菌 嗜盐淀粉酶 同源建模 定点突变 

分 类 号：Q93[生物学—微生物学]