Lactobacillus kefir

DeTemino, D.M., Hartmeier, W. and Ansorge-Schumacher, M.B. (2005) Entrapment of the alcohol dehydrogenase from Lactobacillus kefir in polyvinyl alcohol for the synthesis of chiral hydrophobic alcohols in organic solvents. Enzyme and Microbial Technology, 36 (1), 3-9. 

Similarly, whole-cell Lactobacillus kefir DSM 20587, which possesses two alcohol dehydrogenases for both asymmetric reduction steps, was applied in the reduction of tert-butyl 6-chloro-3,5-dioxohexanoate for asymmetric synthesis of ft rf-butyl-(31 ,5S)-6-chloro-dihydroxyhexanoate (Figure 7.5), a chiral building block for the HMG-CoA reductase inhibitor [ 17]. A final product concentration of 120 him and a specific product capacity of 2.4 mmol per gram dry cell were achieved in an optimized fed-batch process. Ado 99% was obtained for (3R,5S)- and (3.S, 55)-te/ f-butyl-6-chloro-dihydroxyhexanoate with the space-time yield being 4.7 mmolL-1 h-1. 

Figure 7.5 Reduction of tert-butyl 6-chloro-3,5-dioxohexanoate by whole-cell Lactobacillus kefir DSM 20587...

Lactobacillus kefir was also employed as the whole-cell biocatalyst for the asymmetric reduction of ethyl 4-chloroacetoacetate to ethyl (.S )-4-chloro-3-hydroxybutanoate, the chiral... 

Amidjojo, M., Franco-Lara, E., Nowak, A. et al. (2005) Asymmetric synthesis of tert-butyl (3R,5S) 6-chloro-dihydroxyhexanoate with Lactobacillus kefir. Applied Microbiology and Biotechnology, 69 (1), 9-15. 

Amidjojo, M. and Weuster-Botz, D. (2005) Asymmetric synthesis of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutanoate using Lactobacillus kefir. Tetrahedron Asymmetry, 16 (4), 899-901. 

Bradshaw, C.W., Hummel, W. and Wong, C.H. (1992) Lactobacillus kefir alcohol dehydrogenase a useful catalyst for synthesis. The Journal of Organic Chemistry, 57 (5), 1532—1536. 

Weckbecker, A. and Hummel, W. (2006) Cloning, expression, and characterization of an (/ (-specific alcohol dehydrogenase from Lactobacillus kefir. Biocatalysis and Biotransformation, 24 (5), 380-389. 

An example of a whole-cell process is the two-step synthesis of an enantiopure epoxide by asymmetric reduction of an a-chloro ketone (Scheme 6.4), catalyzed by recombinant whole cells of an Escherichia coli sp. overexpressing an (R)-KRED from Lactobacillus kefir and GDH from Thermoplasma acidophilum, to the corresponding chlorohydrin, followed by non-enzymatic base-catalyzed ring closure to the epoxide [17]. 

R)-Specific Alcohol Dehydrogenase from Lactobacillus kefir... 

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. 

Since cinnamyl aldehyde is the main component of cassia oil (approximately 90%) and Sri Lanka cinnamon bark oil (approximately 75%) [49], it is industrially more important to generate cinnamyl alcohol, which is less abundantly available from nature but is important as cinnamon flavour, by biotransformation of natural cinnamyl aldehyde than vice versa. Recently, a whole-cell reduction of cinnamyl aldehyde with a conversion yield of 98% at very high precursor concentrations of up to 166 g L was described [136]. Escherichia coli DSM 14459 expressing a NADPH-dependent R alcohol dehydrogenase from Lactobacillus kefir and a glucose dehydrogenase from Thermoplasma acidophilum for intracellular cofactor regeneration was applied as the biocatalyst (Scheme 23.8). 

Lactobacillus kefir alcohol dehydrogenase, NADPH Mortierella isabelllna... 

Organisms Lactobacillus kefir DSM 20587, Saccharomyces cerevisiae, Candida magnoliae, Bacillus megaterium, Thermoanaerobium brockii, Clostridium beijerinckii, Thermoanaerobacter ethanolicus, Rhodococcus ruber DSM 44541. Solvents ace = acetone iPr = i-PrOH. Substrates WM Wieland-Miescher ketone 4-Me-HP 4-methyl Hajos-Parrish ketone COBE ethyl 4-chloro-3-oxobutanoate. 

LKADH [86] (Lactobacillus kefir)a) TBADH [87, 88] (Thermoanaerobium brockii) Propandiol dehydrogenase [89] (E. coli)... 

Alcohol dehydrogenases (Coenzyme) Lactobacillus kefir (NADP) Mucor javanicus (NAD) yeast (NAD) not known... 

Diacetyl reductase (acetoin dehydrogenase) isolated from Lactobacillus kefir converts the prochiral diacetyl into optically pure (+)-acetoin (ee>94%) [145]. 

Table 7. Purification of alcohol dehydrogenase from Lactobacillus kefir. (Phenyl-and octylsepharoses are materials for hydrophobic interaction chromatography Mono Q is an anionic exchanger.)...

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 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)...

Fig. 2. Induction of (R)-alcohol dehydrogenase during growth of Lactobacillus kefir on increasing concentrations of glucose...

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]. 

Lactobacillus kefir alcohol dehydrogenase256 E2 Geotrichum candidum glycerol dehydrogenase256 6... 

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