Phosphate-induced enhancement of fumarate production from a CO2 and pyruvate with the system of malate dehydrogenase and fumarase

Abstract

Fumarate is a useful unsaturated dicarboxylate and utilized as a raw material for unsaturated polyester resin. As a fumarate is produced using a petroleum-derived material, thus, it is required to establish a synthesis from renewable raw materials such as CO₂ and biomass derived substances. In this work, the synthesis of fumarate from CO₂ and pyruvate in an aqueous medium using a multi-biocatalytic system of malate dehydrogenase (oxaloacetate-decarboxylating; ME; EC 1.1.1.38) from Sulfobus tokodaii and fumarase from porcine heart (FUM; EC 4.2.1.2) in the presence of NADH is established. In this system, it is important to improve the efficiency of fumarate production based on FUM-catalyzed dehydration of L-malate in aqueous media. It was found that saturation of the additional substrate binding site present in FUM with phosphate promotes fumarate production based on the dehydration of L-malate. Under the reaction condition of L-malate (1.0 mM) and FUM (1.3 nM) in HEPES buffer (pH 7.0) for fumarate production, the addition of 70 mM phosphate improved the fumarate production rate up to 2.4 times compared to no addition of phosphate. On the other hand, no effect of phosphate addition on the ME-catalyzed L-malate production from pyruvate and CO₂ in the presence of NADH was observed. In particular, the conversion yield for pyruvate to fumarate with the system of ME and FUM was improved up to 1.6 times due to the improvement in the catalytic activity of FUM caused by the addition of phosphate in this system.