Improving process conditions of hydroxytyrosol synthesis by toluene-4-monooxygenase

Abstract

► Study focused on improving the process yield of hydroxytyrosol production from 2-phenylethanol. ► The biotransformation was scaled up to 1 L and optimized by evaluating substrate concentration, cell density, and different operational modes. ► A better yield was obtained by a fed batch mode and extended biotransformation time. ► Boric acid gel beads were used for the recovery and purification of hydroxytyrosol. Toluene-4-monooxygenase from Pseudomonas mendocina KR1 was recently engineered for the synthesis of hydroxytyrosol, a potent antioxidant. Following a 190-fold improvement in the enzyme activity by protein engineering means, improving the process conditions of this biocatalytic route was under taken for developing a liter-scale bioprocess. The growth stage was improved by selection of a rich media and harvesting the cells at the end of the logarithmic stage. The biotransformation stage was optimized by evaluating substrate concentration, cell density, and different operational modes. It was found that although reusing the cells in successive batch modes is feasible, their activity is dramatically decreased after the first use. In comparison, the activity of the cells following subsequent substrate addition in a fed batch mode was only slightly decreased. Furthermore, a better yield was obtained by extending the duration of the biotransformation stage, rather than adding more substrate. An overall concentration of 133 mg/L HTyr, corresponding to a volumetric productivity of 54 mg/L/h and a yield of 48% was achieved by a batch mode using 2 mM substrate. This is an order of magnitude improvement compared with the enzyme productivity before the process optimization. The use of beads conjugated with phenylboronic acid residues for adsorbing the product from the biotransformation bulk was evaluated. Though the recovery yield and purity were shown to be oppositely dependent, an average recovery procedure led to 2-fold purification of HTyr resulting in 84% purity with 70% recovery yield.