Proteolytic activation and characterization of recombinant polyphenol oxidase from Rosa chinensis for efficient synthesis of theaflavins

Although the oxidation of tea polyphenols catalyzed by polyphenol oxidases (PPOs) is an attractive method for the synthesis of theaflavins (TFs), PPOs for synthesizing TFs were impure, and either not activated or activated by classical chemical methods. Herein, high pure PPO from Rosa chinensis (RcPPO) was cleanly and efficiently activated, and the MBP-RcPPO zymogen was successfully expressed in E. coli BL21 (DE3) with a high pure yield of 37.20 mg/L. Enzymatic activity of the RcPPO after proteolytic activation with trypsin in vitro increased to 5.52 times compared with the MBP-RcPPO zymogen, obtaining a 1625.00 U/L yield of activated enzyme with a specific activity of 480.77 U/mg for 25 mmol/L catechol, and could be enhanced by CuSO 4 , MgCl 2 , DMSO and ethanol. The MBP-RcPPO zymogen was specifically cleaved at the peptide bond of R (−5)-I (−4) for N-terminal, but not specifically cleaved at multiple cleavage sites for C-terminal during the activation with trypsin. RcPPO has been classified as tyrosinase with the obvious preference for diphenol compound substrates. Moreover, the four major TFs were synthesized from tea polyphenols extract by RcPPO with a higher efficiency than native PPO from Camellia sinensis , and the yield of TFs monomers could be furtherly improved using the corresponding purified tea polyphenols as substrate. The results indicated that activated RcPPO may serve as a promising biocatalyst for commercial-scale production of TFs.