摘要
【目的】提升α-L-鼠李糖苷酶(EC 3.2.1.40)的催化活性、热稳定性和pH稳定性。【方法】基于已改良的单点突变体,构建了3个联合突变体:K423R-R307Y-D508N、K423R-R307Y-N561D和K389R-R307Y-N561D。【结果】相比于原始酶(wild type,WT),K423R-R307Y-D508N在温度为65、70℃时的半衰期均提高了0.24 min,在pH为7.0、8.0时的半衰期分别提高了145.64、4.91 min,但其相对酶活力仅为WT的41.01%;K423RR307Y-N561D在pH为7.0、8.0时的半衰期分别提高了51.42、20.65 min,相对酶活力为WT的63.26%;K389R-R307Y-N561D的相对酶活力是WT的167.20%,在温度为65、70℃时的半衰期分别提高了1.60、1.34 min,但在碱性环境下的pH稳定性未发生明显变化。通过分子对接、分子动力学模拟和MM-PBSA分析发现,K389R-R307Y-N561D的催化结构域Loop区和底部部分α螺旋结构的柔性增大,使酶与底物结合更充分,提升了亲和性;但R307突变引发局部扰动,破坏氢键网络及π-π疏水相互作用,导致结构稳定性下降。【结论】联合突变触发位点补偿机制是α-L-鼠李糖苷酶催化活性与结构稳定性难以协同提升的主要原因。
[Objective]This study aims to enhance the catalytic activity,thermal stability,and pH stability ofα-Lrhamnosidase(EC 3.2.1.40).[Method]Three combined mutants(K423R-R307Y-D508N,K423R-R307Y-N561D,and K389R-R307Y-N561D)were constructed based on improved single-point mutants.[Result]Compared with the wild type(WT),the combined mutants K423R-R307Y-D508N exhibited an increased half-life of 0.24 min at both 65℃and 70℃.Additionally,its half-life improved by 145.64 min and 4.91 min at pH 7.0 and pH 8.0,respectively.However,its relative enzyme activity was only 41.01%of that of the WT.The combined mutant K423R-R307Y-N561D exhibited an increased half-life of 51.42 min and 20.65 min at pH 7.0 and pH 8.0,respectively,and its relative enzyme activity were 63.26%of that of the WT.The combined mutant K389RR307Y-N561D achieved an relative enzyme activity 167.20%of that of the WT,with an increased half-life of 1.60 min and 1.34 min at 65℃and 70℃,respectively.However,its stability in alkaline environments did not show significant improvement.Molecular docking,molecular dynamics simulations,and MM-PBSA analyses revealed that the increased flexibility of the catalytic domain Loop region and partialαhelix structure at the bottom of K389R-R307Y-N561D enhanced substrate binding and affinity.However,the R307 mutation induced local perturbations,disrupting the hydrogen bond network andπ-πhydrophobic interactions,leading to decreased structural stability.[Conclusion]The site compensation mechanism triggered by combined mutations is the primary reason for the difficulty in simultaneously enhancing the catalytic activity and structural stability ofα-Lrhamnosidase.
作者
唐艺玲
刘小琴
李乐
李利君
倪辉
TANG Yiling;LIU Xiaoqin;LI Le;LI Lijun;NI Hui(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China;Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering,Jimei University,Xiamen 361021,China;Research Center of Food Biotechnology of Xiamen City,Jimei University,Xiamen 361021,China;Faculty of Marine Biology,Xiamen Ocean Vocational College,Xiamen 361100,China)
出处
《食品与生物技术学报》
北大核心
2025年第2期23-32,共10页
Journal of Food Science and Biotechnology
基金
国家自然科学基金面上项目(32170056)。
