深海放线菌生淀粉酶基因的克隆表达及酶学特性研究  

Cloning,expression and characterization of raw amylase gene fromdeep-sea actinomycete

作　　者：李丽珍[1,2] 杨键[1] 田新朋[1] 麦志茂[1,2] 苏宏飞[1,2] 龙丽娟[1,2] 张偲[1,2] 

机构地区：[1]中国科学院南海海洋研究所热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室 [2]中国科学院大学

出　　处：《生物加工过程》2015年第2期35-40,共6页Chinese Journal of Bioprocess Engineering

基　　金：国家高技术研究发展计划(863计划)(2012AA092104);国家自然科学基金重点项目(41230962);国家海洋公益专项(201305018);广东海洋经济创新发展区域示范项目(GD2012-D01-002);中国科学院重点部署项目(KSCX2-EW-B-13)

摘　　要：从深海放线菌Streptomyces sp.SCSIO03032基因组中扩增到1条含淀粉结合域的水解糖苷13家族基因amy032,该基因编码氨基酸与已知蛋白一致性最高为67%。将amy032插入表达载体pET32a启动子下游,构建重组载体pET-amy。重组质粒导入大肠杆菌Rosseta(DE3)菌株中,SDS-PAGE分析结果显示目的基因成功实现异源表达。Ni-NTA对重组酶进行纯化,并对其酶学性质进行表征。结果表明:重组淀粉酶AMY032的最适作用温度为50℃,最适pH为8.0,以可溶性淀粉为底物时的比酶活为(276±57)U/mg,Km为0.02g/L,Vmax为70mg/(L·min)。Ca2+能提高该酶的催化活性,Ni2+、Cu2+、Zn2+和Mn2+对该酶有抑制作用。AMY032对生玉米淀粉和生大米淀粉具有水解活性,其比酶活分别为(49±12)U/mg和(39±11)U/mg;扫描电镜结果显示AMY032使生玉米淀粉的表面产生明显凹陷。The glycoside hydrolase family 13 gene amy 032,deduced amino acid sequence of which exhibited highest identity of 67% with known protein in Gen Bank,was amplified from a deep sea strain Streptomyces sp.SCSIO 03032.The mature gene was inserted into pET32 a under T7 promoter.Recombinant vector was transformed in Escherichia coli Rosetta（DE3） cells,and SDS-PAGE analysis results showed that the amylase gene was successfully expressed.Enzyme AMY032 was then purified by nickel-affinity chromatography and characterized.The optimum temperature of the purified enzyme was 50℃,and optimum pH 8.0.With soluble starch as substrate,specific activity was（276 ± 57） U/mg,Km was 0.02 g/L and Vmaxwas 70 mg /（L·min）.The catalytic activity of the enzyme was slightly improved by Ca2 ＋and restrained by Ni2 ＋,Cu2 ＋,Zn2 ＋and Mn2 ＋.AMY032 could hydrolyze raw corn starch and raw rice starch,with specific activity of（49 ± 12） U/mg and（39 ± 11） U/mg,respectively.Scanning electron microscope showed AMY032 generate obviously dents on raw corn starch surface.

关 键 词：深海放线菌 基因组挖掘 有机物降解 淀粉结合域 克隆表达 

分 类 号：Q93[生物学—微生物学] TQ925.1[轻工技术与工程—发酵工程]