A coupled enzymatic reaction of tyrosinase and glucose dehydrogenase for the production of hydroxytyrosol.

Hydroxytyrosol (HT) is a diphenolic compound prevalent mainly in olives with pronounced antioxidant activity and proven benefits for human health. Current production limitations have motivated studies concerning the hydroxylation of tyrosol to HT with tyrosinase; however, accumulation of the diphenol is restricted due to its rapid subsequent oxidation to 3,4-quinone-phenylethanol. In this study, a continuous two-enzyme reaction system of sol-gel-immobilized tyrosinase and glucose dehydrogenase (GDH) was developed for the synthesis of HT. Purified tyrosinase from Bacillus megaterium (TyrBm) and E. coli cell extract expressing GDH from B. megaterium were encapsulated in a sol-gel matrix based on triethoxysilane precursors. While tyrosinase oxidized tyrosol to 3,4-quinone-phenylethanol, GDH catalyzed the simultaneous reduction of the cofactor NAD(+) to NADH, which was the reducing agent enabling the accumulation of HT. Using 50 mM tyrosol, the immobilized system under optimized conditions, enabled a final HT yield of 7.68 g/L with productivity of 2.30 mg HT/mg TyrBm beads. Furthermore, the immobilized bi-enzyme system showed the feasibility for HT production from 1 mM tyrosol using a 0.5-L bioreactor as well as stable activity over 8 repeated cycles. The production of other diphenols with commercial importance such as L-dopa (3,4-dihydroxyphenylalanine) or piceatannol may be synthesized with this efficient approach.