米根霉α-淀粉酶保守区域组氨酸突变及功能性质  

Mutation and functional properties of the histidine residues existed in the conserved regions of Rhizopus oryzae α-amylase

作　　者：杨倩 汤斌 李松 陈阿娜 汤文晶 YANG Qian;TANG Bin;LI Song;CHEN A-na;TANG Wen-jing(School of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu 241000, China;Anhui Engineering Technology Research Center of Microbial Fermentation, Wuhu 241000, China)

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000 [2]微生物发酵安徽省工程研究中心,安徽芜湖241000

出　　处：《食品与发酵工业》2017年第11期22-29,共8页Food and Fermentation Industries

基　　金：国家自然科学基金青年项目(31401630);安徽省自然科学基金(1708085QC63)

摘　　要：有关真菌α-淀粉酶的研究主要集中在酶的底物催化形式等生化性质方面,该类酶中的大量氨基酸残基组成与酶功能性质之间的关系尚不明确。利用分子对接和同源比对等生物信息学分析方法发现米根霉α-淀粉酶中有3个特殊的组氨酸残基(H120,H200和H286),并利用定点突变的方法构建了系列突变体。性质研究表明,286位氨基酸残基结构与该酶的最适反应温度、最适反应pH、酸耐受性以及麦芽糖生成能力均有关联;突变体H286L的酸耐受性和水解淀粉的麦芽糖生成能力得到显著提高;120位和200位氨基酸残基的变化不改变该酶的最适反应温度和最适反应pH,而两者的组合突变可显著提升该酶的酸耐受性。此外,数据表明该酶的高麦芽糖生成能力与酶对麦芽三糖的亲和力不直接相关。研究结果可为真菌α-淀粉酶高麦芽糖生成能力的原因及其定向进化提供一定理论参考。The research on fungal α-amylase mainly focused on the biochemical properties such as the substrate catalytic mechanism,and the relationship between the composition of amino acids residues and the functional properties of this enzyme is unclear.In this paper,three special conserved histidine residues（ H120,H200 and H286） that existed in the catalytic domain of Rhizopus oryzae α-amylase were identified by bioinformatics analysis methods of molecular docking and comparative analysis.A series of mutants were constructed on the basis of above three identified sites using site-directed mutagenesis.The results showed that the structure of the amino acid residue at position 286 had been proved to be related with the optimum temperature,optimum pH,acid resistance and maltose-forming ability of the enzyme.The acid resistance and maltose-forming ability on soluble starch of the mutant H286 L were significantly improved.Changes of amino acid residues at position 120 and position 200 exhibited no effects on the optimal temperature,optimum pH and maltose-forming ability,but the combined mutations at both sites of 120 and 200 could improve its acid resistance.The results indicated that the high maltose-forming ability of the enzyme was not directly related to its affinity on maltotriose,which might provide a theoretical reference for study on the mechanism of high maltose-forming ability of fungal α-amylase and its directed evolution.

关 键 词：真菌Α-淀粉酶 分子对接 定点突变 结构与功能 麦芽糖 

分 类 号：TS201.25[轻工技术与工程—食品科学]