FDH from Candida

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... 

First applied by Whitesides and Shaked in the 1980s [29], the FDH from Candida hoidinii has ever since been receiving great interest as an NADH regeneration system (Table 8.4). Variants with improved stability [30] or an enlarged cofactor spectrum (accepting also NADP ) [31] have been reported. Due to its popularity, FDH is commercially available from various suppliers [lb]. Also, sources for novel FDHs are constantly being explored [32]. 

Two groups in particular have pioneered the practical application of PheDH from various bacterial species, namely those of Asano etal in Japan and Hummel etal. in Germany. Asano and his colleagues initially explored the application of PheDH to the chiral synthesis of the physiological substrate L-phenylalanine. The PheDH of Bacillus sphaericus was overexpressed in E. coll The issue of cofactor recycling was tackled by using the FDH of Candida hoidinil Importantly, these authors showed that both catalytic activities could be successfully... 

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])...

Detection of oligosaccharides (e.g., stachyose, raffinose, sucrose, and fructose) in a soybean extract using invertase hydrolysis of p-o-fructo-fructoside to fructose, and further oxidation of this sugar by hexacyanoferrate (III) ion in the presence of fructose dehydrogenase (FDH). This analysis is based on a coimmobilization of invertase from Candida utilis and FDH from Gluconobacter on poly(vinyl alcohol) (PVA) beads and coulometric quantification of the hexacyanoferrate(II) ions formed. 

As a new option, for the bioconversion of poorly soluble substrates the classical EMR-concept can be extended to an Emulsion Membrane Reactor , comprising a separate chamber for emulsification (with a hydrophilic ultrafiltration membrane), an EMR-Ioop with a normal ultrafiltration module, and a circulation pump. This approach has been successfully demonstrated for the enzymatic reduction of poorly soluble ketones [107]. Using this device, e.g., for the enantioselective reduction of 2-octanone to (S)-2-octanol (e.e. >99.5%) with a carbonyl reductase from Candida parapsilosis under NADH-regeneration with FDH/for-mate, the total turnover number was increased by a factor 9 as compared with the classical EMR. 

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. 

In the enzymatic part of the process, a one-pot conversion was achieved by using Candida lipase (Lip) to hydrolyze the ester and then LeuDH to catalyze the reductive amination (with or without N labeling). In this case, the coenzyme recycling was accomplished by adding FDH and formate. The same group used a similar enzymatic strategy to prepare labeled L-threonine and L-allothreonine starting from the a-keto methyl ester. ... 

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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