活性位点附近氨基酸对解淀粉芽孢杆菌核糖核酸酶Barnase酶学性质的影响  

Influence of amino acids near the active site on the enzymatic properties of Barnase from Bacillus amyloliquefaciens

作　　者：李鹏龙 王雷[2] 李兴江 吴学凤 洪泂[3] 穆冬冬 LI Penglong;WANG Lei;LI Xingjiang;WU Xuefeng;HONG Jiong;MU Dongdong(School of Foodand Biological Engineering,Hefei University of Technology,Hefei 230601,China;Medical Insurance Division,The First Affiliated Hospital of USTC(Anhui Provincial Hospital),Hefei 230001,China;School of Life Sciences,University of Scienceand Technology of China,Hefei 230026,China)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230601 [2]中国科学技术大学附属第一医院(安徽省立医院)医保处,安徽合肥230001 [3]中国科学技术大学生命科学学院,安徽合肥230026

出　　处：《安徽大学学报（自然科学版）》2024年第5期71-81,共11页Journal of Anhui University(Natural Science Edition)

基　　金：合肥工业大学岳西兴岳产业投资有限公司校地合作产业创新引导资金资助项目(W2023JSKF0788);安徽省自然科学基金资助项目(2108085MC123);安徽省重大科技专项资助项目(202103b06020019)。

摘　　要：Barnase活性位点附近氨基酸与底物之间的相互作用会影响酶与底物的识别从而影响酶的催化,为了研究活性位点附近氨基酸如何影响催化从而获得更高活性和稳定性的Barnase,采用定点突变技术对解淀粉芽孢杆菌BH072核糖核酸酶Barnase活性位点附近氨基酸进行突变,将突变体酶分别诱导表达、纯化并表征,检测其比酶活和热稳定性.得到最高比酶活的突变酶为N131A,比酶活为41.65 kU·mg^(-1),相较野生型提高了43.82%.该突变体在60℃放置12 h后仍能保持80%的酶活力.为了探究活性位点附近残基突变对催化反应造成影响的原因,笔者使用同源建模及分子对接分析了催化口袋与底物的相互作用,发现N131A突变提高了酶分子结合底物的能力.该研究所获得的突变体N131A进一步丰富了核糖核酸酶突变体酶库,具有良好应用前景.The interaction between substrates and amino acids near the active site of Barnase can affect the recognition of Baranse and substrates,thereby affecting enzyme catalysis.In order to study how amino acids near the active site affect catalysis and obtain higher activity and stability of Barnase,site-specific mutation has been used to mutate amino acids near the active site of Barnase from Bacillus amyloliquefaciens BH072.The mutant enzymes were induced for expression,purified,and characterized,and their specific enzyme activity and thermal stability were measured.The mutant enzyme with the highest specific enzyme activity was N131A,with a specific enzyme activity of 41.65 kU·mg^(-1),which was 43.82%higher than the wild-type.The mutant could still maintain 80%enzyme activity after being placed at 60℃for 12 hours.In order to investigate the reasons for the impact of residue mutations near the active site on catalytic reactions,homologous modeling and molecular docking have been used to analyze the interaction between catalytic pockets and substrates.It was found that the N131A mutation improved the enzyme molecule's ability to bind to substrates.The mutant N131A obtained in this study further enriches the ribonuclease mutant enzyme library and has good application prospects.

关 键 词：核糖核酸酶 定点突变 稳定性 催化活性 

分 类 号：Q556.1[生物学—生物化学]