Production of L-malic acid with fixation of HCO3- by malic enzyme-catalyzed reaction based on regeneration of coenzyme on electrode modified by layer-by-layer self-assembly method

Malic enzyme prepared and purified from Brevundimonas diminuta IFO13182 catalyzed the decarboxylation reaction of malate to pyruvate and CO2 using NAD(+) as the coenzyme, and the reverse reaction was used in the present study for L-malic acid production with fixation of HCO3- as a model compound for carbon source. The L-malic acid production was based on electrochemical regeneration of NADH on a carbon plate electrode modified by layer-by-layer adsorption of polymer-bound mediator (Alginic acid bound viologen derivative, Alg-V), polymer-bound coenzyme (Alginic acid bound NAD(+), Alg-NAD(+)), and lipoamide dehydrogenase (LipDH). Electrochemical reduction of immobilized NAD(+) catalyzed by LipDH in a multilayer film was achieved, and the L-malic acid production with HCO3- fixation system with layer-by-layer immobilization of Alg-V/LipDH/Alg-NAD(+)/malic enzyme multilayer film on the electrode gave an L-malic acid production of nearly 11.9 mmol and an HCO3- fixation rate of nearly 47.4% in a buffer containing only KHCO3 and pyruvic acid potassium salt, using a cation exchange membrane. The total turnover number of NADH within 48 h was about 19,000, which suggests that efficient NADH regeneration and fast electron transfer were achieved within the multilayer film, and that the modified electrode is a potential method for the fixation of HCO3- without addition of free coenzyme. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.