NAD-dependent formate dehydrogenase from Candida boidinii is well known as an effective enzyme for cofactor regeneration during the synthesis of chiral compounds using dehydrogenases. The enzyme is used in several industrial applications like the reductive amination of a-keto acids leading to enantiomerically pure L-amino acids as well as D- and L-hydroxy acids [1,2]. The application of Cb-FDH in a continuously operated enzyme-membrane reactor allows regeneration of the expensive cofactor NADH and recycling of the enzymes thereby reducing the production costs [3,4]. Although FDH is stable under storage conditions (50% v/v glycerol, -20°C) for several years, the native enzyme from the yeast shows a low operational stability resulting in higher enzyme consumption [5].

Cloning and recombinant expression of the fdh gene from C. boidinii in E.coli resulted in an expression of the enzyme up to 20 % of the soluble cell protein [6].The goal of the work described here was to stabilize the recombinant FDH (rec-FDH) by mutagenesis for its use as an NADH-regenerating enzyme for the reductive amination of a-keto acids. In a first approach oxidation-sensible amino acid residues in rec-FDH were replaced by site-directed mutagenesis resulting in two mutants with a significantly higher stability under biotransformation conditions. Additionally, the presentation intends to elucidate the inactivation mechanism of FDH and discusses future aspects for a further stabilization of rec-FDH for its industrial application.

[1] Krix, G.; Bommarius, A., Drauz, K., Kottenhahn, M, Schwarm, M and Kula, M.-R., J. Biotechnol., 53, 29-39, 1997

[2] Bommarius, A.S., Schwarm M. and Drauz, K., J.Mol. Catal. B: Enz., 5, 1-11, 1998

[3] Wichmann, R., Wandrey, C., Bückmann, A.F. and Kula, M.-R., Biotechnol. Bioeng., 23, 2789-2802, 1981

[4] Kragl, U., Vasic-Racki, D and Wandrey, C., Bioprocess Eng., 14, 291-297, 1996

[5] Kragl, U., Kruse, W., Hummel, W. and Wandrey, C, Biotechnol. Bioeng, 52, 309-319, 1996

[6] Slusarczyk, H., Pohl, M. and Kula, M.-R., German patent application DE 19753350.7