Exploitation of a Laccase/Meldola's Blue System for NAD(+) Regeneration in Preparative Scale Hydroxysteroid Dehydrogenase-Catalyzed Oxidations

An enzymatic regeneration system consisting of laccase and the 2H+/2e- redox mediator Meldolas blue (MB) was developed for the efficient oxidation of reduced nicotinamide adenine dinucleotide (NADH) cofactor and employed in the gram-scale oxidation of cholic acid (1) to its 7-keto derivative (1a) by 7a-hydroxysteroid dehydrogenase (7a-HSDH) in an aqueous, buffered reaction system. The regenerating enzyme, laccase, reduces molecular oxygen as terminal 4H+/4e- acceptor to water and concomitantly reoxidizes NADH via MB at high turnover rates. The regeneration system was successfully applied to quantitatively convert 1 (50 mM, 20.4 g) into 1a with a space-time yield of 5.8 mmol?L-1?h-1. High total turnover numbers were achieved for 7a-HSDH (4.2x105) and laccase (1.1x106). Alternatively, the regeneration system was employed on the conversion of the methyl ester derivative of cholic acid (2, 200 mM, 5.9 g) dissolved in isopropyl acetate as organic solvent in a biphasic system. Due to the high concentration of 2 and the excellent performance of the enzymatic cascade reaction even under the harsh process conditions, space-time yields of up to 20 mmol?L-1?h-1 (8.5 g?L-1?h-1) of the produced 7-keto derivative 2a were obtained. The irreversibility and high driving force of the regenerating reaction allowed quantitative conversions and easy product recovery.