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食品研究与开发:2024,45(7):165-173
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定向引入N-糖基化位点促进芳基醇氧化酶热稳定性及底物亲和力
(中国农业科学院 麻类研究所,湖南 长沙 410205)
Improvement of Thermal Stability and Substrate Affinity of Aryl-alcohol Oxidase by Directed Introduction of N-glycosylation Site
(Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha 410205,Hunan,China)
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投稿时间:2022-09-23    
中文摘要: 芳基醇氧化酶在木质素降解过程中发挥重要作用,N-糖基化修饰影响其酶学性质。该文旨在通过研究刺芹侧耳(Pleurotus eryngii)来源的芳基醇氧化酶N-糖基化,来提高其热稳定性和底物亲和力。利用毕赤酵母GS115 表达系统和定点突变技术,构建表达6 种芳基醇氧化酶突变体蛋白,并对纯化后的野生型和突变体酶进行酶学性质和热稳定性分析。结果表明,芳基醇氧化酶N89 和N249 糖基化位点突变导致最适温度和70 ℃时酶热稳定性降低;在这个过程中,将其引入新的糖基化位点后的突变体,其最适酸碱度没有变化,最适温度以及70 ℃下的热稳定程度都明显优于野生型;以藜芦醇为底物时,突变体[AAO(F-X-N-X-T)]与底物亲和力最高。N-糖基化主要影响芳醇氧化酶的热稳定性,其中N89 和N249 位点的N-糖基化对酶的热稳定性起重要作用;引入N-糖基化位点[AAO(F-X-N-X-T)]能获得具有高活力和高稳定性的芳醇氧化酶。
Abstract:Aryl-alcohol oxidase plays a crucial role in lignin degradation,and its enzymatic properties are influenced by N-glycosylation modification.This study aimed to investigate the N-glycosylation of Pleurotus eryngii aryl-alcohol oxidase in order to enhance its thermal stability and substrate affinity.In this study,the expression system of Pichia GS115 and site-directed mutagenesis were used to construct the expressions of six aryl-alcohol oxidase mutant proteins.The purified wild-type and mutant enzymes were also analyzed for enzymatic properties and stability.The results indicated that the N-glycosylation mutations at sites N89 and N249 of arylalcohol oxidase caused reduced optimum temperature and enzymatic thermal stability at 70 ℃.However,introducing a new glycosylation site through mutagenesis did not affect the optimum pH but significantly improved both the optimum temperature and thermal stability at 70 ℃compared to the wild-type enzyme.When using pcoumaric alcohol as a substrate,the mutant[AAO(F-X-N-X-T)]exhibited the highest substrate affinity.N-glycosylation primarily affects the thermal stability of aryl-alcohol oxidase,with glycosylation at sites N89 and N249 playing a critical role in enzyme stability.The introduction of N-glycosylation site [AAO(F-X-N-X-T)]results in aryl-alcohol oxidase with enhanced activity and stability.
文章编号:202407023     中图分类号:    文献标志码:
基金项目:湖南省自然科学基金项目(2020JJ5641)
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