Production of Fumaric Acid from l-Malic Acid by Solvent Engineering Using a Recombinant Thermostable Fumarase from Thermus thermophilus HB8

Currently, fumaric acid is produced by catalytic isomerization of maleic acid in aqueous solutions at low pH. Being petroleum based, requiring catalyst, and producing vast amounts of by-products and wastewater, the production of fumaric acid from renewable resources by a “green” process is increasingly attractive. In an aqueous solution, the reaction equilibrium constant of the fumarase-mediated conversion of l-malic acid to fumaric acid is 1:4.2 (fumaric acid to l-malic acid). To shift the reaction equilibrium to fumaric acid, solvent engineering was carried out by varying hydrophilic solvents and their concentrations. Generally, organic solvents may denature fumarase. Therefore, fumarase from Thermus thermophilus was employed to overcome this problem. Ethylene glycol was found more suitable than other solvents. This fumarase was shown to be more stable in 50 % than in 70 % ethylene glycol. Therefore, a preparation was carried out in 50 % ethylene glycol. Under this condition, 54.7 % conversion was observed using fumarase for transforming 1 mmol l-malic acid. After precipitation by adapting the pH, and washing to remove residual solvent and substrate, 27 % total yield was obtained with 99 % purity. The results demonstrated that the alternative green route to produce bio-based fumaric acid via l-malic acid is feasible and viable.