Journal
MOLECULES
Volume 28, Issue 9, Pages -Publisher
MDPI
DOI: 10.3390/molecules28093831
Keywords
polyphenol oxidase; protein engineering; transition state; theaflavin-3,3'-digallate
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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.
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.
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