枯草芽孢杆菌漆酶与阴离子型聚丙烯酰胺微观作用机理  

Microscopic action mechanism of laccase from Bacillus subtilis with anionic polyacrylamide

作　　者：王方略 张力雯 张东晨[3] 吴学凤[4] 邓胜松 WANG Fanglue;ZHANG Liwen;ZHANG Dongchen;WU Xuefeng;DENG Shengsong(School of Bioengineering Huainan Normal University,Huainan,232038,China;School of Mechanical and Electrical Engineering Huainan Normal University,Huainan,232038,China;School of Material Science and Engineering Anhui University of Science and Technology,Huainan,232001,China;School of Food and Bioengineering Hefei University of Technology,Hefei,230009,China)

机构地区：[1]淮南师范学院生物工程学院,淮南232038 [2]淮南师范学院机械与电气工程学院,淮南232038 [3]安徽理工大学材料科学与工程学院,淮南232001 [4]合肥工业大学食品与生物工程学院,合肥230009

出　　处：《环境化学》2024年第11期3873-3885,共13页Environmental Chemistry

基　　金：国家自然科学基金(51274012)资助.

摘　　要：阴离子型聚丙烯酰胺(HPAM)常用于煤泥水的澄清处理,产生大量的含聚污水将对选煤厂周边环境造成不利影响.为探究漆酶降解HPAM的微观作用机理,采用对接模拟了HPAM及其脱氨产物聚丙烯酸(PAA)结构模型与枯草芽孢杆菌漆酶(Lac)的结合,根据-CDOCKER_Energyscore打分最高原则筛选出Lac的最适底物,然后对该最适复合物分别进行基于亲和力虚拟突变和不同温度下的分子动力学(MD)模拟.结合模式分析表明,Lac对HPAM-3享有最高亲和力,且其结合最好,因此HPAM-3是该酶的最适底物;HPAM比PAA易被Lac降解;Lac可容纳一定碳链长度的HPAM和PAA.相互作用分析表明,Lac-HPAM-3亲和力最大主要原因是盐桥;TYR133通过形成氢键以稳定Lac-HPAM-2、Lac-HPAM-3和Lac-HPAM-4,而ARG487通过形成疏水以稳定所有的酶-底物复合物.基于亲和力虚拟突变分析表明,TYR118、TYR133、ARG487和LYS135是Lac降解HPAM-3的关键残基;LYS135和ARG487分别通过形成盐桥和疏水来最大限度地影响酶同底物的亲和力.MD分析表明,Lac-HPAM-3在298 K时总相互作用能、酶骨架RMSD及所有残基RMSF皆最低,因此该复合物在298 K时结合稳定性最佳;308 K时由于酶骨架RMSD最大,导致底物偏离最初对接位置,因此Lac-HPAM-3在308 K时结合稳定性最差.这些数据为揭示HPAM酶降解过程奠定基础,为将来突变试验来改造酶提供位点支持.Anionic polyacrylamide(HPAM)is often used to clarifying the coal slime water.A large amount of polymer-containing wastewater produced will have adverse effects on the surrounding environment of coal preparation plant.In order to explore the microscopic action mechanism of HPAM by laccase,docking was used to simulate the binding of HPAM and its deamination product polyacrylic acid(PAA)with Bacillus subtilis laccase(Lac).The optimal substrate of Lac was screened according to the highest principle of-CDOCKER_Energy score.Then,molecular dynamics(MD)simulation at different temperatures and virtual mutation based on affinity were carried out by this optimal complex,respectively.The binding model analysis showed that Lacacquired the highest affinity for HPAM-3,and their binding was the optimal.Hence,HPAM-3 was the most suitable substrate for this enzyme;HPAM was more easily degraded by Lac than PAA;Lac could accommodate HPAM and PAA with a certain carbon chain length.The interaction analysis showed that Lac-HPAM-3 had the highest affinity mainly due to the salt bridge;TYR133 stabilized Lac-HPAM-2,Lac-HPAM-3,and Lac-HPAM-4 by forming hydrogen bonds,while ARG487 stabilized all enzyme-substrate complexes by forming hydrophobicity.The virtual mutation based on affinity analysis showed that TYR118,TYR133,ARG487 and LYS135 in Lac were key residues for degrading HPAM-3;LYS135 and ARG487 affected the affinity of enzyme with substrate to the utmost extent by forming salt bridge and hydrophobicity,respectively.MD analysis showed that the total interaction energy,RMSD of enzyme skeleton and RMSF of all residues were all the lowest for Lac-HPAM-3 at 298 K,so this complex had the optimal binding stability at 298 K;RMSD of the enzyme skeleton was the maximum at 308 K causing the substrate deviated from the initial docking position.Hence,Lac-HPAM-3 had the worst binding stability at 308 K.These data laid the foundation for revealing the enzymatic degradation process of HPAM,and provided site support for mutagenesis tests to modify the

关 键 词：枯草芽孢杆菌漆酶(Lac) 阴离子型聚丙烯酰胺(HPAM) 对接 基于亲和力虚拟突变 分子动力学(MD) 

分 类 号：X-1[环境科学与工程] O6[理学—化学]