SUMO融合表达对淀粉酶N-Amy活性的影响  

Effect of SUMO fusion expression on amylase N-Amy activity

作　　者：王美星 向腊 胡慧贞 巫攀 张桂敏[1] WANG Meixing;XIANG La;HU Huizhen;WU Pan;ZHANG Guimin(College of Life Sciences,Hubei University,Wuhan 430062,China)

机构地区：[1]湖北大学生命科学学院,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2019年第3期217-222,共6页Journal of Hubei University：Natural Science

基　　金：国家自然科学基金(31670069);湖北省自然科学基金杰出青年人才基金(2018CFA042)资助

摘　　要：前期解析了一个来自嗜碱菌Bacillus pseudofirmus 703的新型的碱性淀粉酶Amy703,该酶含有一个未知功能的N端结构域,将该结构域缺失后得到的突变体N-Amy相对野生型Amy703比酶活提高了35倍;底物结合实验显示Amy703可以结合不可溶性底物,单独的N端结构域和N端结构域缺失突变体N-Amy都不能结合,推测N端结构域会造成催化结构域的结构变化从而引起比酶活的大幅提高.为了进一步探究Amy703特异的N端结构域造成酶活显著下降的原因,将Amy703的N端结构域替换为SUMO蛋白,实验结果显示裂解液上清的SUMO/N-Amy条带比Amy703粗亮,验证了SUMO蛋白的促可溶性作用;但纯化后的融合蛋白SUMO/N-Amy的比酶活显著低于N-Amy,仅和野生型Amy703的比酶活相当,表明SUMO与N-Amy的融合显著降低了酶活,同时也预示着N端多一段结构域会影响到催化结构域的结构变化.此外,对SUMO/N-Amy融合蛋白是否与底物结合进行了探讨,为进一步解析Amy703的N端结构域功能提供了实验依据.A novel alkaline amylase Amy703 from Bacillus pseudofirmus 703 was analyzed in the previous work. This enzyme contained an N-terminal domain of unknown function,and an N-terminal domain deletion mutant N-Amy was obtained,which showed that the enzymatic activity was increased by 35-fold compared to the wild-type. Substrate binding experiments showed that Amy703 could bind insoluble amylose substrate,while N-Amy and N-terminal domain alone could not bind it. To further explore the function of the N-terminal domain of Amy703,the N-terminal region of Amy703 was replaced with the SUMO protein. The results showed that the lysate supernatant band of SUMO/N-Amy was thicker than that of Amy703,confirming that SUMO protein has a pro-soluble effect. The enzymatic activity of the fusion protein SUMO/N-Amy was much lower than that of N-Amy,which was similar with the wild-type Amy703. It indicated that the fusion of SUMO and N-Amy reduced enzyme activity. In addition,we discussed whether SUMO/N-Amy fusion protein were combined with substrates. This paper provides an experimental basis for the further analysis of the N-terminal structure domain function of Amy703.

关 键 词：碱性淀粉酶 N端结构域 SUMO蛋白 底物结合能力 比酶活 

分 类 号：Q556[生物学—生物化学]