Formate dehydrogenase preparation

Groger, H., Hummel, W., Rollmann, C. et al. (2004) Preparative asymmetric reduction of ketones in a biphasic medium with an (5)-alcohol dehydrogenase under in f/w-cofactor-recycling with a formate dehydrogenase. Tetrahedron, 60 (3), 633-640. 

Biocatalysts based on hydrolases (E.C. class 3, Table 5.2) ate mostly used as (purified) enzymes since they are cofactor independent, since these preparations are commercially available and because a number of hydrolases can be applied in organic solvents. Oxidoreductases (E.C. class 1) however, are relatively complex enzymes, which require cofactors and frequently consist of more than one protein component. Thus, despite the fact that efficient cofactor regeneration systems for NADH based on formate dehydrogenase (FDH) have been developed (Bradshaw et al, 1992 Chenault Whitesides, 1987 Wandrey Bossow, 1986, chapter 10) and that also an NADPH dependent FDH has been isolated (Klyushnichenko, Tishkov Kula, 1997), these enzymes are still mostly used as whole-cell biocatalysts. 

Table 8. Preparation of chiral alcohols by enzyme-catalyzed reduction of the corresponding ketones with ADH from Lactobacillus kefir. The production of phenylethanol with formate and formate dehydrogenase (FDH) for coenzyme regeneration was carried out continuously in an enzyme-membrane-reactor...

A simple procedure was established for the synthesis of various D-amino adds by means of four types of thermostable enzymes alanine racemase, D-amino acid aminotransferase 49, 501, L-alanine dehydrogenase 51, and formate dehydrogenase (Fig. 17-4) 171. The commercial preparation of formate dehydrogenase from Candida boidinii used by Wichmanri et al. 38 is not sufficiently stable. However, Galkin et al.1521 doned and expressed the gene of thermostable formate dehydrogenase in E. coli. 

Thermoactinomyces intermedius and endogenous formate dehydrogenase. The reductive amination process was further scaled up using a preparation of the two enzymes expressed in single recombinant E. coli. The amino acid 7 can be directly protected as its boc derivative without isolation to afford intermediate 8. Yields before isolation were close to 98% with 100% EE. 

Enzymatic Synthesis of At lysine Ethylene Acetal. (S)-2-Amino-5-(l,3-dioxolan-2-yl)-pentanoic acid (5 )-allysine ethylene acetal (7) (Fig. 3A) is one of three building blocks used in an alternative synthesis of Omapatrilat (1). It previously had been prepared via an eight-step chemical synthesis from 3,4-dihydro [2H] pyran (16). An alternate synthesis of (7) was demonstrated by reductive amination of ketoacid acetal (8) using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedins (17). The reaction required ammonia and NADH NAD produced during the reaction was recycled to NADH by the oxidation of formate to CO using formate dehydrogenase (FDH). 

Proteoliposomes containing polysulfide reductase and either hydrogenase or formate dehydrogenase isolated from W. succinogenes do not catalyze polysulfide sulfur respiration unless 8-methyl-menaquinone is present (Table 3). Menaquinone with a side chain consisting of six or four isoprene units, or vitamin Ki served in reconstituting fumarate respiration, but did not replace 8-methyl-menaquinone in polysulfide sulfur respiration. The low activities of polysulfide sulfur respiration observed without added 8-methyl-menaquinone were probably due to the small amounts of this quinone associated with the enzyme preparations used. Maximum activity of polysulfide sulfur respiration required 10 p.mol 8-methyl-menaquinone per gram phospholipid [O. Klimmek and W. Dietrich, unpublished results]. 

Under the optimized reaction conditions, co-transami-nase was used as the enzyme (ATA-113 for preparing the (5)-enantiomers and ATA-117 for synthesizing the (R)-enantiomers of the product amines), sodium phosphate buffer for maintaining the pH at 7, and ammonium formate and formate dehydrogenase to recycle the alanine. The variety of synthesized of chiral amines are shown in the following scheme (Scheme 39.42). 

Applications of lactate dehydrogenase L-Lactate dehydrogenases (LDH) prepared from mammalian and bacterial sources, are available commercially. They are known to produce (2S)-a-hydoxy acids with different hydrocarbon side-chains by reduction of a-keto acids in high enantiomeric purities and good chemical yields (Figure 12.10). As NADH-dependent enzymes, LDHs are used in combination with other enzymes, such as formate dehydrogenase (FDH), to regenerate the co-factor in situ. 

The factor in glucose dehydrogenase preparations which can stimulate tyrosine formation can be replaced by crystalline catalase when tetrahydropteridines are used while catalase cannot replace glucose dehydrogenase with the cofactor. It is possible that the glucose dehydrogenase fractions contain another factor, besides catalase, which is required for the activity of the cofactor. This additional factor may also be involved in the conversion of the cofactor to an active form. 

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