Production of L-malic acid with fixation of HCO^-_3 by malic enzyme-catalyzed reaction based on regeneration of coenzyme on electrode modified by layer-by-layer self-assembly method(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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Malic enzyme prepared and purified from Brevundimonas diminuta IFO13182 catalyzed the decarboxylation reaction of malate to pyruvate and CO_2 using NAD^+ as the coenzyme, and the reverse reaction was used in the present study for L-malic acid production with fixation of HCO^-_3 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 HCO^-_3 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 11.9mmol and an HCO^-_3 fixation rate of nearly 47.4% in a buffer containing only KHCO_3 and pyruvic acid potassium salt, using a cation exchange membrane. The total turnover number of NADH within 48h 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 HCO^-_3 without addition of free coenzyme.
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