Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis

The optimization of a continuous enzymatic reaction yielding (R)-phenylacet ylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presente d. We compare the suitability of three pyruvate decarboxylases (PDC), PDC f rom Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent muta nt of the latter, PDCW392M, with respect to their application in the biotra nsformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction co nditions of the carboligation reaction were investigated by determining ini tial rate velocities with varying substrate concentrations of both aldehyde s. From the resulting data a kinetic model was inferred which fits the expe rimental data with sufficient reliability to deduce the optimal concentrati ons of both substrates for the enzymatic process. The results demonstrate t hat the carboligation is most efficiently performed using a continuous reac tion system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L-1 d(-1) using a substrate concentration o f 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fer mentative process. (C) 2001 John Wiley & Sons, Inc.