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

• Published in: Molecular catalysis 2024-04, Vol.559, p.114059, Article 114059
• Main Authors: Luo, Shengkai, Hou, Yi, Hu, Song-Qing
• Format: Article
• Language: English
• Subjects: Enzymatic activity; Polyphenol oxidases; Semi-rational design; Theaflavin (TF1); Thermostability
• ISSN: 2468-8231; 2468-8231
• DOI: 10.1016/j.mcat.2024.114059

•Polyphenol oxidase (RcPPO) was semi-rational engineered at L173, A174 and N334.•Preference, activity and thermostability of mutants were improved by several times.•Improvement mechanism on enzyme was revealed by MD simulation and docking.•Yield of theaflavin (TF1) was improved to 70.65 % by catalysis of mutant. 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. [Display omitted]