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Improved yield of theaflavin-3,3'-digallate from Bacillus megaterium tyrosinase via directed evolution
Jinghui Zhou1, Changwei Liu1, Shimin Zhao2
1Key Laboratory of Tea Science of Ministry of Education, Hunan Agriculture University, Changsha 410128, People's Republic of China; National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients, Hunan Agriculture University, Changsha 410128, People's Republic of China; Co-Innovation Center of Education Ministry for Utilization of Botanical Functional Ingredients, Hunan Agriculture University, Changsha 410128, People's Republic of China.
Abstract:
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-1d-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.
