热稳定性
蛋白质工程
突变体
化学
多酚氧化酶
突变
合理设计
生物化学
定点突变
生物催化
催化效率
催化作用
酶
立体化学
生物
基因
遗传学
反应机理
过氧化物酶
作者
Shengkai Luo,Yi Hou,Song‐Qing Hu
标识
DOI:10.1016/j.mcat.2024.114059
摘要
Improvement of activity and property in polyphenol oxidases (PPOs) by protein engineering may contribute to synthesis of theaflavin (TF1) in industrial processes. Here, the potential noncatalytic residues of PPO from Rosa chinensis (RcPPO) were selected by sequence alignment, structural analysis and surface residues prediction and the mutants were gained by semi-rational site-directed mutagenesis. Mutants L173F, A174K and N334I possessed 10.48, 9.74 and 7.13 times increases respectively in preference for diphenols over monophenols. Enzymatic activity, half-life at 40 and 50 °C of the most active mutant RcPPO-FKI increased to 2.67, 2.62 and 3.37 times respectively in comparison with wild type, implying that activity and thermostability of RcPPO through mutation were improved. The improvement of activity and thermostability in the mutant should be ascribed to more hydrogen bonds formed by the conserved catalytic histidine residues with substrate, reduced fluctuation of the loop regions and increased hydrogen network of the mutational amino acids, revealed by molecular dynamics simutation and molecular docking. Moreover, the yield of TF1 was improved by catalysis of mutant RcPPO-FKI with higher synthetic efficiency of TF1 in comparison with some reported catalysts and as a promising alternative to commercial mushroom tyrosinase. PPOs with enhanced performances provide the robust candidate for industrial application.
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