Enhancement of preference, catalytic activity and thermostability of polyphenol oxidase from Rosa Chinensis by semi-rational engineering

被引:1
作者
Luo, Shengkai [1 ]
Hou, Yi [2 ]
Hu, Song-Qing [1 ]
机构
[1] South China Univ Technol, Sch Food Sci & Engn, Guangzhou 510641, Guangdong, Peoples R China
[2] South China Univ Technol, State Key Lab Pulp & Paper Engn, Guangzhou 510640, Peoples R China
来源
MOLECULAR CATALYSIS | 2024年 / 559卷
基金
中国国家自然科学基金;
关键词
Polyphenol oxidases; Enzymatic activity; Thermostability; Theaflavin (TF1); Semi-rational design; ENZYMATIC BIOTRANSFORMATION METHOD; SINENSIS CELL-CULTURE; AMINO-ACIDS; THEAFLAVINS; BLACK; MECHANISM; TYROSINASE; GREEN; MICE;
D O I
10.1016/j.mcat.2024.114059
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
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 degrees 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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页数:11
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