Whole-cell biocatalysts alcohol

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

Matsuyama, A. Yamamoto, H. and Kobayashi, Y. Practical Apphcation of Recombinant Whole-cell Biocatalysts for the Manufacturing of Pharmaceutical Intermediates such as Chiral Alcohols. Org. Process Res. Dev. 2002, 6, 558-561. 

Based on the (/ )-specific ADH from L. kefir, a recombinant E. coli strain was constructed as a whole-cell biocatalyst, and co-expressed GDH was used for regeneration of NADPH [157]. These designer cells were applied for the reduction of 4-fluoroacetophenone to the corresponding optically active (/ )-4-fluorophe-nylethan-l-ol at 0.5 M educt concentration [158]. After a reaction time of 23 h, a conversion of >95% has been achieved, and the purified isolated chiral alcohol showed an ee value of >99% (87% yield). (S)-p-Halohydrins were obtained with this whole-cell catalyst by means of an enantioselective reduction of the corresponding ketones with both high conversions of >95% and enantioselectivities of >99% (Fig. 40). Base-induced cyclization of the [S-halohydrin led to enantiomeri-cally pure (S)-epoxides in high yield and enantiomeric purity (>99% ee) [159]. 

In industrial biotransformations, hydrolytic reactions occupy a prominent position for the production of optically active amines, alcohols, and carboxylic acids. Compared with other reactions, hydrolytic reactions are feasible to scale up because they are cofactor-free, relatively simple, and chemically tunable systems. In addition to home-made whole-cell biocatalysts, which are considered to be more cost-effective for specific syntheses, some commercially available hydrolases, including lipases/esterases, epoxide hydrolases, nitrilases, and glycosidases, are also employed for the enantioselective production of chiral chemicals. 

In summary, ketoreductases have emerged as valuable catalysts for asymmetric ketone reductions and are preparing to enter the mainstream of synthetic chemistry of chiral alcohols. These biocatalysts are used in three forms wild-type whole-cell microorganism, recombinant... 

Enzymes are natural biocatalysts that are becoming increasingly popular tools in synthetic organic chemistry [1]. The major areas of exploration have involved the use of hydrolases, particularly esterases and lipases [2]. These enzymes are readily available, robust and inexpensive. The second most popular area of investigation has been the reduction of carbonyl compounds to chiral secondary alcohols using either dehydrogenases (with co-factors) or a whole-cell system such as bakers yeast [3]. 

Lactobacillus brevis whole-cell biotransformation When the reduction of diketo ester la was performed with whole cells of Lactobacillus brevis or L. kefir, formation of the 3,5-dihydroxy ester (3R,5S)-5a was observed [10, 22]. This was surprising since it is known that the prevailing alcohol dehydrogenase in I. brevis is the one described as LBADH [23] and since, moreover, this enzyme does not reduce P-keto 5-hydroxy ester 2a to the corresponding dihydroxy ester (Scheme 2.2.7.6). Under the conditions tested, further alcohol dehydrogenase activity is clearly present in I. brevis and I. kefir. Pfruender et al. optimized the production of L. kefir cells and used this biocatalyst for the one-pot synthesis of dihydroxy ester syn-(3R,5S)-5a using diketo ester la as starting material [24]. 

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). 

The presence of a biocatalyst, either whole cells [ 126] or enzymes [ 157], or any other biological surface-active materials either produced or present as substrates in the bioconversion system, such as fatty acids or long chain alcohols [ 127,184], were expected to lower interfacial tension and hence breakthrough pressure [126, 157,184]. A threefold decrease in the interfacial tension was observed in an aque-ous-tetradecane system when either Pseudomonas putida or bakers yeast cells were used, as compared to the cell-free system [126]. A decrease in the breakthrough pressure due to the presence of a surface-active agent, lauric acid, was also cited [184]. 

Prior to the widespread awdlabdity of recombiant carbonyl reductases enzymes, the use of microbial reductions using either actively growing or dormant cells was commonplace Bakers yeast in particular, was a readily available source of stereoselective carbonyl reductases enzymes. Even with the widespread knowledge of the power of recombinant CRED biocatalysts, the literature is still rife with wild-type whole-cell microbial reductions. The reductions presented have advanced well beyond the early Bakers yeast reduction and have an apphcation even today. When the whole-cell fermentation is developed and finely tuned, high titers of product alcohol are possible and Scheme 6.4 shows m example of a keto-amide 12 bioreduction performing at 100 g/L with more than 98% ee with multi-kg isolation [12]. The bioprocess was performed over 8 days at pH 7 using the yeast Candida sorbophila. 

Besides wild-type strains, more recently the use of recombinant whole cells has gained increasing popularity for application in asymmetric ketone reduction. When overexpressing the ADFi only, in situ cofactor recycling based on a "substrate-coupled approach" represents a favorite approach as demonstrated in an early contribution by the Itoh group [86] utilizing a recombinant ADH from a Corynebacterium overexpressed in E. coli. This concept has been also applied by Daicel researchers in the presence of an E. coli catalyst with recombinant ADH from Candida parapsilosis. This biocatalyst catalyzes the reduction of p-ketoester 28 at a 36.6 g/1 substrate loading and fimiished the alcohol (R)-29 in 95.2% yield and with 99%ee (Scheme 23.12) [87]. 

---