Coenzymes are always required in the oxidoreductases-catalyzed reactions and especially nicotinamide coenzymes, NAD+ or NADH, are the most widely used in biotransformation reactions. The efficient regeneration of nicotinamide coenzymes is considered an important process for industrial applications due to their high cost and stoichiometric requirements. There are several cofactor regeneration methods including enzymatic, chemical, electrochemical and photochemical methods. In particular, enzymatic method has advantages over the other methods. Since it requires mild reaction conditions and is environmentally friendly. NADH oxidase (NOX, EC 1.6.3.1) and NAD-dependent formate dehydrogenase (FDH, EC 1.2.1.2) have been most widely used for NAD+ and NADH regenerations, respectively. While NOX requires oxygen as electron acceptor to regenerate NAD+, NAD-dependent FDH requires formate as electron donor to regenerate NADH. Renewable electrical current can be used as electron source in electro-enzymatic method for more efficient NADH regeneration. However, Lipoamide dehydrogenase (diaphorase, EC 1.8.1.4) is the only known enzyme used for electro-enzymatic NADH regeneration. In this study, the beta subunit of FDH1 from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. Interestingly, it exhibited NAD+ reduction activity as well as NADH oxidation activity in the absence of its alpha subunit. It showed higher NADH oxidation activities with the artificial electron mediators. The beta subunit of FDH1 without mediator showed relatively low activity (0.28 Umg-1 enzyme). With 250 �M benzyl viologen, however, it exhibited 35.6-fold (9.98 Umg-1 enzyme) increased activity by mediating electron transfer from the enzyme to oxygen. Cyclic voltammetry was applied to confirm the electro-enzymatic NADH regeneration through beta subunit. We confirmed that NADH was regenerated by beta subunit through coupling reaction with D-Lactate dehydrogenase to produce D-lactate from pyruvate. Furthermore, we expect that direct electron transfer (DET) without mediator can be possible because beta subunit has iron-sulfur cluster in its structure. Beta subunit will be used as a key biocatalyst to regenerate NADH coenzyme by using electrons supplied from cathode. In addition, it will replace conventionally used Rh-complex for the regeneration of NADH coenzyme since this biocatalyst showed much higher turnover rate as well as economically competitiveness.

Application of the beta subunilt of FDH1 from <i>Methylobacterium extorquens</i> AM1 as a cofactor regeneration biocatalyst

247th National Meeting (2014)

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Application of the beta subunilt of FDH1 from Methylobacterium extorquens AM1 as a cofactor regeneration biocatalyst