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  

Improvement of Thermal Stability and Substrate Affinity of Aryl‐alcohol Oxidase by Directed Introduction of N‐glycosylation Site

作　　者：曹查 朱作华[1] 龚文兵[1] 周映君 谢纯良[1] 彭源德[1] CAO Cha;ZHU Zuohua;GONG Wenbing;ZHOU Yingjun;XIE Chunliang;PENG Yuande(Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha 410205,Hunan,China)

机构地区：[1]中国农业科学院麻类研究所,湖南长沙410205

出　　处：《食品研究与开发》2024年第7期165-173,共9页Food Research and Development

基　　金：湖南省自然科学基金项目(2020JJ5641)。

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。Aryl⁃alcohol oxidase plays a crucial role in lignin degradation,and its enzymatic properties are in⁃fluenced by N⁃glycosylation modification.This study aimed to investigate the N⁃glycosylation of Pleurotus eryn⁃gii aryl⁃alcohol oxidase in order to enhance its thermal stability and substrate affinity.In this study,the expres⁃sion system of Pichia GS115 and site⁃directed mutagenesis were used to construct the expressions of six aryl⁃al⁃cohol 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 aryl⁃alcohol oxidase caused reduced optimum temperature and enzymatic thermal stability at 70℃.However,intro⁃ducing 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 p⁃coumaric alcohol as a substrate,the mutant[AAO(F⁃X⁃N⁃X⁃T)]exhibited the highest substrate affinity.N⁃gly⁃cosylation 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.

关 键 词：芳基醇氧化酶 N-糖基化 定点突变 重组表达 酶学性质 

分 类 号：Q814[生物学—生物工程]