IN VITRO CONVERSION OF GLYCEROL TO LACTATE BY MICROBIALTHERMOPHILIC ENZYMES

Abstract

In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction. Particularly, employment of (hyper)thermophilic enzymes enables us a simple preparation of highly stable, selective biocatalytic modules and the construction of in vitro metabolic pathways. In this study, we designed and constructed an artificial in vitro metabolic pathway consisting of nine (hyper)thermophilic enzymes and applied it to the conversion of glycerol to lactate. We also assessed the compatibility of the in vitro bioconversion system with methanol, which is a major impurity in crude glycerol released from biodiesel production processes. The in vitro artificial pathway was designed to balance the intrapathway consumption and regeneration of energy and redox cofactors. All enzymes involved in the in vitro pathway exhibited an acceptable level of stability at high temperature (60°C) and their stability was not markedly affected by the co-existing of up to 100 mM methanol. The one-pot conversion of glycerol to lactate through the in vitro pathway could be achieved in an almost stoichiometric manner and 14.7 mM lactate could be produced in 7 h. Many thermophilic enzymes exhibit higher stability not only at high temperatures but also in the presence of denaturants than their mesophilic counterpart. In this study, compatibilities of thermophilic enzymes with methanol were demonstrated, indicating the potential applicability of in vitro bioconversion systems with thermophilic enzymes in the conversion of crude glycerol to value-added chemicals.