Lactobacillus kefir ADH

Enzymatic reduction of 23a with recLBADH and CPCR resulted in unsatisfactory results (60% and 49% ee) as well. The results mentioned above indicate that a bulky substituent at the alkyne moiety results in a higher selectivity of the reduction. Furthermore, Bradshaw et al. reported that Lactobacillus kefir ADH, an enzyme highly homologous to LB ADH, affords (R)-4-trimethylsilyl-3-butyn-2-ol [(R)-25j with an ee of 94% in 25% yield [39bj. In our investigations ketone 23b was reduced by recLBADH with almost quantitative conversion. The enantiomeric excess and absolute configuration of the product were determined by desi-lylation with borax converting alcohol (R)-25 into enantiopure (R)-3-butyn-2-ol [(R)-24j (Scheme 2.2.7.14). 

Lactobacillus kefir (ADH E.C. 1.1.1.1) for use in organic solvents [11, 12]. Both biocatalysts are characterized by a very low stability in pure organic solvents or standard aqueous-organic two-phase systems [20], though their broad substrate ranges include many hydrophobic compounds [21, 22]. Figure 3.2.2 illustrates the denaturation of native BAL at the interface between a buffered aqueous solution and octanone. 

IkADH Lactobacillus kefir ADH SCR carbonyl reductase from Saccharomyces cerevisiae. 

Purification and Biochemical Characterization of [R)-ADH from Lactobacillus kefir... 

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...

Table 9. Taxonomic classification of Lactobacillus strains according to Bergey s Manual [163] and their reaction with the anti-L. kefir-ADH antibody. Boldface-typed strains were tested, underlined strains gave a positive reaction with the antibody (L. = Lactobacillus)...

Table 11. Growth of Lactobacillus kefir on various carbon sources (2%) and activity of (R)-ADH measured with acetophenone and NADPH. (Optical density was measured at 660 nm)...

The ADH from Lactobacillus brevis can be purified by exactly the same chromatographic procedures as applied for the purification of L. kefir ADH. 

Methods to produce chiral alcohols with ADHs are essentially described at a laboratory scale, namely those using HLADH, TBADH and the recently isolated enzymes from Rhodococcus erythropolis, and Lactobacillus kefir and L. brevis, respectively. 

R)-alcohols in high enantiomeric excess can be obtained with the aid of the NADP-dependent ADH from Lactobacillus kefir. Due to the broad substrate specificity of this enzyme, aromatic, cyclic, polycyclic as well as aliphatic ketones can be reduced. A simple method for the regeneration of NADPH is given by the simultaneously coupled oxidation of isopropanol by the same enzyme. Several chiral alcohols (Table 8) were synthesized at a 2.5 mmol scale within a reaction time of 12-36 h [160]. 

G-6-P-DH from Leuconostoc mesenteroides is used in organic synthesis for example to synthesize D-lactic acid or (S)-benzyl alcohol [43]. Commercially available G-6-P-DH was coupled with ADH from Lactobacillus kefir for the regeneration of NADPH to produce optically pure (/ )-phenylethanol [33]. 

Well characterized NADP+-dependent ADHs from microbial sources have been isolated from several sources (Table 1). ADHs from Lactobacillus kefir and L. brevis convert prochiral ketones into the corresponding (/ )-alcohols in a highly stereospecifically manner. They show a broad substrate spectrum, high specific activities (up to 100-500 U mg-1), and they both need the presence of Mg2+ ions to maintain their activity [30]. ADH from Thermoanaerobacter brockii (TBADH)... 

Fig. 46 One-pot two-step synthesis of allylic alcohols. The reductive step is catalyzed by (R)-ADH from Lactobacillus kefir or by (S)-ADH from Rhodococcus sp., respectively. The coenzyme is regenerated by an excess of isopropanol...

The first group is represented by the alcohol dehydrogenases (ADHs) that can be used for the synthesis of chiral alcohols. There are several commercially available ADHs isolated from yeast or horse liver (NADH dependent), or T. brockii (NADPH-dependent) that can be used for different types of substrates. Lactobacillus kefir produces an (/ )-ADH that accepts a broad variety of ketone substrates (ring halogenated, aliphatic, open-chain ketones, 2- and 3-ketoesters, and cyclic ketones), producing, for example, enantiomerically pure I -l-(2-pyridyl ethanol), / -(l-trimerhylsylyl)-l-butyn-3-ol or 5-phenylbutan-2-ol (Hummel 1990). 

Until now, several oxidoreductases or microorganisms have been used in the preparation of chiral alcohols including NAD -dependent alcohol dehydrogenases (ADHs) from yeast and horse liver [11] (EC 1.1.1.1), Candida parapsilosis [12] and Pseudomonas sp. [13], and NADP -dependent ADHs from yeast [14], Thermoanaer-obium brockii [15] and Lactobacillus kefir [16], aldehyde reductases from Sporobolo-myces salmonicolor (EC 1.1.1.2) [17] and Penicillium citrinum (EC 1.1.1.21) [18], and carbonyl reductase (EC 1.1.1.184) from Candida magnoliae [19]. Our research group has reported an efficient method for producing both enantiomers of chiral alcohols... 

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