Improving Theaflavin-3,3′-digallate Production Efficiency Optimization by Transition State Conformation of Polyphenol Oxidase

Theaflavins (TFs) are good for health because of their bioactivities. Enzymatic synthesis of TFs has garnered much attention; however, the source and activity of the enzymes needed limit their wide application. In this study, a microbial polyphenol oxidase from Bacillus megaterium was screened for the synthesis of theaflavin-3,3 '-digallate (TFDG). Based on structural and mechanistic analyses of the enzyme, the O-O bond dissociation was identified as the rate-determining step. To address this issue, a transition state (TS) conformation optimization strategy was adopted to stabilize the spatial conformation of the O-O bond dissociation, which improved the catalytic efficiency of tyrosinase. Under the optimum transformation conditions of pH 4.0, temperature 25 degrees C, (-)-epigallocatechin gallate/epicatechin gallate molar ratio of 2:1, and time of 30 min, Mu(4) (BmTyr(V218A/R209S)) produced 960.36 mg/L TFDG with a 44.22% conversion rate, which was 6.35-fold higher than that of the wild type. Thus, the method established has great potential in the synthesis of TFDG and other TFs.

Automated summary

In this study, a microbial polyphenol oxidase from Bacillus megaterium was used to synthesize theaflavin-3,3 '-digallate (TFDG). By optimizing the conformation of the O-O bond dissociation, the catalytic efficiency of the enzyme was improved. The optimized conditions resulted in a significantly higher production of TFDG, indicating the great potential of this method in the synthesis of TFDG and other TFs.