Improved yield of theaflavin-3,3′-digallate from Bacillus megaterium tyrosinase via directed evolution

Theaflavin-3,3'-digallate (TFDG) in black tea possesses several health benefits. However, low TFDG yields limit its application. Herein, tyrosinases from Bacillus megaterium (Bmtyrc) were used to synthesize TFDG. To improve the catalytic efficiency of tyrosinase, a directed evolution strategy and a high-throughput screening method was employed. Compared with the wild type, mutant Bmtyrc-3 (N205D/D166E/D167G/F197W) showed 6.46 and 4.91-folds higher specific activity and 51.97- and 1.95-folds higher Vmax values towards epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), respectively. Moreover, Bmtyrc-3 displayed significantly enhanced catalytic efficiencies, and the space-time yield of TFDG was 35.35 g L-1 d(-1). Bmtyrc-3 presents a broader substrate binding area, caused by a mutation (N205D) encompassing the active site. Changes in the potential of the substrate binding site and hydrogen bonds, and the electrostatic effect on the protein surface resulted in an increased activity of the substrates EGCG and ECG.

Automated summary

Theaflavin-3,3'-digallate (TFDG) from black tea has various health benefits, but its application is limited due to low yields. In this study, tyrosinases from Bacillus megaterium were used to synthesize TFDG through directed evolution and high-throughput screening. Mutant Bmtyrc-3 showed significantly improved catalytic efficiency and higher production of TFDG. The broader substrate binding area of Bmtyrc-3 resulted in increased activity of substrates EGCG and ECG.