根癌农杆菌甲氧基脱甲基酶Atu1420的酶学特性表征和定向进化  

Enzymatic Characterization and Directed Evolution of Agrobacterium tumefaciens O-demethylase Atu1420

作　　者：王浩 曹安妮 高欣怡 郭敏亮[1] WANG Hao;CAO An-ni;GAO Xin-yi;GUO Min-liang(College of Bioscience and Biotechnology,Yangzhou University,Yangzhou 225009)

机构地区：[1]扬州大学生物科学与技术学院,扬州225009

出　　处：《生物技术通报》2025年第3期319-329,共11页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32300151);江苏省高等学校自然科学研究项目(21KJB180019)。

摘　　要：【目的】原儿茶酸是一种应用广泛的酚类化合物,生物合成法生产原儿茶酸是替代高污染的化学合成法的潜在方案,寻找和改良合成原儿茶酸的酶是生物合成法的关键。根癌农杆菌甲氧基脱甲基酶Atu1420是一种能够将香草酸转化为原儿茶酸的酶,本研究旨在表征Atu1420的酶学特性,并对其进行改良。【方法】通过大肠杆菌表达了Atu1420,并利用高效液相色谱等方法测定了Atu1420的酶学特性。基于Atu1420的预测结构和催化机制,选择了19个位点对Atu1420进行了定点突变,利用4-氨基安替比林开发了一种可视化检测Atu1420催化活性的方法,用于筛选Atu1420定点突变的变异体。利用alpha Fold对Atu1420变异体的结构进行预测,分析了变异体催化效率提升的潜在机制。【结果】确定了Atu1420的V_(max)为33.5±1.6 nmol/(L·s),米氏常数K_(m)为82.7±3.5μmol/L,转换数k_(cat)为(6.7±0.3)×10^(-1) s^(-1),k_(cat)/K_(m)为8.1×10^(-3) L/(μmol·s),最适p H在7-8之间,最适温度为30℃。本研究在定点突变的变异体中筛选出了5个酶活力增强的变异体,其中活力最强的变异体为G35S,其催化速度比野生型提高66.0%。对这5个位点进行组合突变,在酶活力方面并没有产生明显的叠加效应。结构的比对分析暗示121位精氨酸残基角度的偏转可能是造成变异体催化效率提升的原因。【结论】Atu1420的酶学特性得到了有效的表征,获得了5个催化效率提高的Atu1420变异体,变异体催化效率的提高可能是121位精氨酸残基角度的偏转造成的。【Objective】Protocatechuic acid(PCA)is a widely used phenolic compound.Biosynthesis method of PCA is a potential alternative to the highly-polluting chemical synthesis.Finding and improving enzymes for synthesizing PCA is the key to biosynthesis.Agrobacterium tumefaciens O-demethylase Atu1420 is an enzyme that can convert vanillic acid(VA)to PCA.This work aims to characterize the enzymatic properties of Atu1420 and improve it.【Method】Atu1420 was expressed in Escherichia coli,and the enzymatic properties of Atu1420 were determined by methods such as high-performance liquid chromatography(HPLC).Based on the predicted structure and catalytic mechanism of Atu1420,19 sites were selected to conduct site-directed mutagenesis on Atu1420.A visual method for detecting the catalytic activity of Atu1420 was developed by using 4-aminoantipyrine to screen variants of Atu1420.The structure of Atu1420 variants was predicted by alphaFold,and the potential mechanism for the improvement of catalytic efficiency of variants was analyzed.【Result】The V_(max)of Atu1420 was determined to be 33.5±1.6 nmol/(L·s),the K_(m)is 82.7±3.5μmol/L,the k_(cat)is(6.7±0.3)×10^(-1) s^(-1),k_(cat)/K_(m)is 8.1×10^(-3)L/(μmol·s),the optimal pH is between 7 and 8,and the optimal temperature is 30℃.Five variants with enhanced enzyme activity were screened out from the variants obtained by site-directed mutagenesis.The variant with the strongest activity is G35S,and its catalytic activity is 66.0%higher than that of the wild type.Combined mutations at these five sites did not produce a significant additive effect on enzyme activity.Comparative analysis of the structures indicates that the deflection of the arginine residue at position 121 may be the reason for the improvement of the catalytic efficiency of the variants.【Conclusion】The enzymatic properties of Atu1420 have been characterized,and five Atu1420 variants with improved catalytic efficiency have been obtained.The increase in the catalytic efficiency of the variants may be c

关 键 词：甲氧基脱甲基酶 酶学特性 催化效率 原儿茶酸 香草酸 

分 类 号：R28[医药卫生—中药学]