深海放线菌生淀粉酶基因的克隆表达及酶学特性研究

摘要

The glycoside hydrolase family 13 gene amy032, deduced amino acid sequence of which exhibited highest identity of 67% with known protein in GenBank,was amplified from a deep sea strain Streptomyces sp. SCSIO 03032. The mature gene was inserted into pET32a 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 Vmax was 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.%从深海放线菌Streptomyces sp�SCSIO 03032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�02 g／L， Vmax为70 mg／（ 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。