FDH from Candida boidinii

Since FDH from Candida boidinii now can be produced at pilot scale, this reaction can be generally used for NADH-regeneration. Recently, the same concept has been used for NADPH regeneration. This became possible because a NADPH-dependent FDH has been obtained by multipoint site-directed mutagenesis of the gene coding the enzyme from the bacterium sp. 101. (Seelbacheia/., 1996). 

In particular, FDH from Candida boidinii is often used for the regeneration of NADH. However, the low specific activity of this enzyme (4-6 U mg-1) is a considerable disadvantage. The KM values for NAD+ and formate are 0.09 and 13 mM, respectively. The enzyme has a broad pH optimum of 7.5-8.5 while 55°C is the optimal temperature [3]. 

The use of an LeuDH as an amino acid dehydrogenase showed a high L-enan-tiospecificity [24]. In this connection, an L-leucine dehydrogenase from Bacillus sphaericus has been applied very efficiently. The FDH from Candida boidinii is the preferred formate dehydrogenase for this process. The stability of this enzyme, which is available in technical quantities, has been remarkably improved by protein engineering and directed evolution [25], In particular the replacement of cys-... 

FDH from Candida boidinii is mostly used as regeneration enzyme. It found industrial application at Degussa-Hiils AG in a leucine dehydrogenase-catalyzed reductive animation of 2-keto acids yielding various amino acids (e.g. tert-leu-cine) l14-16l. Native FDH is very selective for NAD+. Recently a new FDH was developed by site-directed mutagenesis that shows all advantages of the NAD+-dependent enzymes and additionally accepts NADP+ as substrate1171. The activity of the mutant with NADP+ is about 60% of the wild-type FDH with NAD+[18 . 

The NADH-independent FDH enzymes from S. fumaroxidans are highly unstable and inactive in the presence of O2, limiting their practical application. Conversely, the NADH-dependent FDH from Candida boidinii (CbsEDH) was sufficiently stable for commercial use and has been used to enzymatically regenerate NADH. CbsFDH requires NADH, protons, and electrons to convert CO2 into formate, which can be supplied by the electrochemical system of the Cu electrode... 

Table 16. Chiral alcohols produced by continuous enzyme-catalyzed processes. The corresponding ketones are reduced with (S)-ADH from Rhodococcus erythropolis, NADH was regenerated by simultaneous coupling with formate dehydrogenase from Candida boidinii (FDH) and formate (data from [159])...

Wu, W.H., Zhu, D.M., and Hua, L. (2009) Site-saturation mutagenesis of formate dehydrogenase from Candida boidinii creating effective NADP -dependent FDH enzymes./. Mol. Catal. B., 61, 157-161. 

Candida boidinii (18) or Pichia pastoris (19) grown on methanol are useful sources of FDH. Expression of T. intermedins PDH in P. pastoris, inducible by methanol, yielded both enzymes from a single fermentation. Formate dehydrogenase activity per gram wet cells in P. pastoris was 2.7-fold greater than for C. boidinii and fermentor productivity was... 

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