C端非催化免疫球蛋白样结构域对假交替单胞菌κ-卡拉胶酶的酶学性质与结构的影响  

Effect of C-terminal Non-catalytic Immunoglobulin-Like Domain on Enzymatic Properties and Structure ofκ-Carrageenanase from Pseudoalteromonas tetraodonis

作　　者：吴婷 朱艳冰[1,2] 李鹤宾 陈艳红 洪涛[1,2] 姜泽东 倪辉 WU Ting;ZHU Yanbing;LI Hebin;CHEN Yanhong;HONG Tao;JIANG Zedong;NI Hui(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China;Fujian Key Laboratory of Food Microbiology and Enzyme Engineering,Xiamen 361021,China;Department of Pharmacy,Xiamen Medical College,Xiamen 361023,China)

机构地区：[1]集美大学海洋食品与生物工程学院,福建厦门361021 [2]福建省食品微生物与酶工程重点实验室,福建厦门361021 [3]厦门医学院药学系,福建厦门361023

出　　处：《食品科学》2025年第9期156-164,共9页Food Science

基　　金：福建省科技计划高校产学合作项目(2024Y4008);国家自然科学基金面上项目(22178142);福建省促进海洋与渔业产业高质量发展专项(FJHYF-L-2023-25)。

摘　　要：目的:分析假交替单胞菌(Pseudoalteromonas tetraodonis)来源κ-卡拉胶酶的结构域,构建结构域截短突变体,探究非催化结构域对假交替单胞菌κ-卡拉胶酶的酶学性质与结构的影响。方法:利用大肠杆菌异源表达假交替单胞菌κ-卡拉胶酶WT和仅包含糖苷水解酶催化结构域的κ-卡拉胶酶截短体GH(缺失免疫球蛋白(immunoglobulin,Ig)样结构域),利用3,5-二硝基水杨酸法研究κ-卡拉胶酶WT和GH的酶学性质,利用分子对接和分子动力学模拟研究酶微观结构的变化。结果:成功对假交替单胞菌κ-卡拉胶酶WT及其截短体GH进行体外表达,WT和GH的分子质量分别为44.0 kDa和35.0 kDa,均能专一性地酶解κ-卡拉胶底物。酶学性质研究发现,假交替单胞菌κ-卡拉胶酶的Ig样结构域不影响酶的底物特异性、最适反应温度、最适反应pH值和κ-卡拉胶酶解产物的组成,但降低了酶的热稳定性、强酸(pH 4.0)和强碱(pH 11.0)稳定性、酶对底物的亲和力。酶的分子对接及分子动力学模拟分析结果表明,Ig样结构域截短后,κ-卡拉胶酶与κ-卡拉胶四糖底物的相互作用增强,酶结构的刚性增强,κ-卡拉胶酶F1、F3和F5指状区环状结构柔性降低,F2和F6指状区β片层结构柔性升高,这些结构的改变可能是截短体GH催化活性和热稳定性提高的原因。结论:本研究解析了假交替单胞菌κ-卡拉胶酶的非催化Ig样结构域与酶性质的构效关系,可为该酶结构与功能研究及酶的应用提供理论基础。Objective:To analyze the domains ofκ-carrageenase from Pseudoalteromonas tetraodonis,construct domain truncation mutants,and investigate the effect of non-catalytic domains on the enzymatic properties and structure of P.tetraodonisκ-carrageenase.Methods:Escherichia coli was used to heterologously express the wild-type(WT)κ-carrageenase and the truncated mutant GH,containing only the catalytic domain of glycoside hydrolase(immunoglobulin(Ig)-like domain-deleted).We determined the enzymatic properties of WT and GH by the 3,5-dinitrosalicylic acid(DNS)method,and studied the changes in the microscopic structure of the enzyme using molecular docking and molecular dynamics simulation.Results:Both WT and its truncated mutant were successfully expressed in vitro.The molecular mass of WT and GH were 44.0 and 35.0 kDa,respectively,and both of them specifically cleavedκ-carrageenan.Enzymatic characterization revealed that deletion of the Ig-like domain did not affect the substrate specificity,optimal reaction temperature,optimal reaction pH,or hydrolysate composition,but decreased the enzyme’s thermal,strong acid(pH 4.0)and strong base(pH 11.0)stability,as well as its substrate affinity.Molecular docking and molecular dynamics simulation analyses showed that after the truncation of the Ig-like domain,the interaction betweenκ-carrageenanase and its substateκ-carrageenan tetrasaccharide was enhanced,the structural rigidity of the enzyme was increased,and the cyclic structure of the F1,F3,and F5 finger regions became less flexible,and theβ-sheet structure of the F2 and F6 finger regions became more flexible.These structural changes might be the cause of the improved catalytic activity and thermal stability of GH.Conclusion:Our findings on the relationship between the non-catalytic Ig-like domain and enzyme properties of P.tetraodonisκ-carrageenase provide a theoretical basis for research on the structure and function of the enzyme,as well as its application.

关 键 词：假交替单胞菌 κ-卡拉胶酶 免疫球蛋白样结构域 酶学性质 结构稳定性 

分 类 号：TS201.2[轻工技术与工程—食品科学]