作　　者： ; ; ; ;

机构地区： 广西大学生命科学与技术学院广西亚热带生物资源保护利用重点实验室

出　　处： 《生物加工过程》 2010年第5期39-43,共5页

摘　　要： 利用RT-PCR从Rhizopus oryzaeGX-08总RNA中克隆到糖化酶的淀粉结合域(SBD)基因(sbd),将该基因片段插入α-淀粉酶(CN7A)基因cn7a的5′端构建融合表达质粒pSE-sbdcn7a。嵌合酶SBD-CN7A在Escherichia coliJM109表达,并经Ni-NTA、Sephacryl S300纯化。酶学性质研究表明:嵌合酶在最适作用条件方面与原始酶并无明显差别;在以生玉米粉为底物时,其比酶活提高了8.7倍,而以可溶性淀粉为底物时其比酶活是原始酶的1.8倍,Km也从3.784 g/L降低为2.234 g/L;嵌合酶在65℃下的半衰期从10 min缩短为4 min。结果表明,淀粉结合域SBD的融合赋予了α-淀粉酶CN7A水解生淀粉的能力。 The starch-binding domain gene sbd was amplified by RT-PCR from total RNA of Rhizopus oryzae GX-08.The fusion expression plasmid was constructed by inserting sbd to 5′ end of α-amylase gene cn7a.The chimeric enzyme（SBD-CN7A） was overexpressed in Escherichia coli JM109 and purified by Ni-NTA and Sephacryl S300.SBD-CN7A showed strong raw corn starch hydrolyzing activity,it was 8.7 times higher than parental enzyme CN7A.When taking soluble starch as a substrate,Km value decreased from 3.784 g/L to 2.234 g/L,and the specific activity increased from 17.8 U/nmol to 32.0 U/nmol.The half life at 65 ℃ declined from 10 min of CN7A to 4 min of SBD-CN7A.The fusion of SBD with α-amylase CN7A maked the enzyme more suitable for raw starch hydrolyzing.

关 键 词： 嵌合酶 淀粉结合域 热稳定性

领　　域： [轻工技术与工程] [轻工技术与工程] [生物学]