生物技术通报2025，Vol.41Issue(3)：319-329,11.DOI:10.13560/j.cnki.biotech.bull.1985.2024-0993
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
摘要
Abstract
[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 Vmax of Atu1420 was determined to be 33.5±1.6 nmol/(L·s),the Km is 82.7±3.5 μmol/L,the kcat is(6.7±0.3)×10-1 s-1,kcat/Km 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 caused by the deflection of the arginine residue at position 121.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Key words
O-demethylase/enzymatic properties/catalytic efficiency/protocatechuic acid/vanillic acid引用本文复制引用
王浩,曹安妮,高欣怡,郭敏亮..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[J].生物技术通报,2025,41(3):319-329,11.基金项目
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) （32300151）
