Candida magnoliae

Two interesting yeast carbonyl reductases, one from Candida magnoliae (CMCR) [33,54] and the other from Sporobolomyces salmonicolor (SSCR) [55], were found to catalyze the reduction of ethyl 4-chloro-3-oxobutanoate to give ethyl (5)-4-chloro-3-hydroxybutanoate, a useful chiral building block. In an effort to search for carbonyl reductases with anti-Prelog enantioselectivity, the activity and enantioselectivity of CMCR and SSCR have been evaluated toward the reduction of various ketones, including a- and /3-ketoesters, and their application potential in the synthesis of pharmaceutically important chiral alcohol intermediates have been explored [56-58]. 

The carbonyl reductase from Candida magnoliae catalyzed the enantioselective reduction of a diversity of ketones, including aliphatic and aromatic ketones and a- and /3-ketoesters (Figure 7.17), to anti-Prelog configurated alcohols in excellent optical purity (99% ee or higher) [56]. 

The usefulness of the carbonyl reductase from Candida magnoliae as an enzyme catalyst in the synthesis of chiral alcohol intermediates has been demonstrated by carrying out the reduction of several ketones on a preparative scale [56]. The isolated yields and enantiomeric excess of the product alcohols are summarized in Table 7.1, from which it can be seen that these chiral alcohols were obtained in essentially optically pure forms in excellent yields. These chiral alcohols are important intermediates in the synthesis of pharmaceuticals and agrichemicals. For example, optically active 2-hydroxy-3-methylbutyrate is an important chiral synthon... 

Yasohara, Y., Kizaki, N., Hasegawa, J. et al. (2001) Stereoselective reduction of alkyl 3-oxobutanoates by carbonyl reductase from Candida magnoliae. Tetrahedron Asymmetry, 12 (12), 1713-1718. 

Wada, M., Kataoka, M., Kawabata, H. et al. (1998) Purification and characterization of NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae. Bioscience Biotechnology and Biochemistry, 62 (2), 280-285. 

Zhu, D., Yang, Y. and Hua, L. (2006) Stereoselective enzymatic synthesis of chiral alcohols with the use of a carbonyl reductase from Candida magnoliae with anti-Prelog enantioselectivity. The Journal of Organic Chemistry, 71 (11), 4202-4205. 

L. brevis Lactobacillus brevis L. lactis Lactococcus lactis Candida magnoliae ... 

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. 

Wada M, Kawabata H et al (1999) Occurrence of multiple ethyl 4-chloro-3-oxobutanoate-reducing enzymes in Candida magnoliae. J Biosci Bioeng 87 144-148... 

Fig. 42 Stereospecific reduction of 4-chloro-3-oxobutanoate ester (COBE) to (K) and (S)-4-chloro-3-hydroxybutanoate ester (CHBE) by aldehyde reductase from Sporobolomyces salmonicolor and carbonyl reductase from Candida magnoliae, respectively...

Fig. 3.49 Biocatalytic production of ethyl (S)-4-ch loro-3-hydroxybutanoate using alcohol dehydrogenase from Candida magnoliae.

Another example is the use of heat treatment as a supplement to the screening process. The enantioselectivity of the reduction of ethyl 4-chloro-3-oxobutanoate by Candida magnoliae was improved from 96.6% ee (S) using untreated cells to 99% ee (S) with heat treated cells [67L... 

Besides these examples, many other important enzymes for biocatalytic reductions, such as the NADPH-dependent carbonyl reductase from Candida magnoliae U2 the ketoreductase from Zygosaccharomyces rowxii11431, and the aldehyde reductase from Sporobolomyces salmonicolor AKU442911441, etc. have also been expressed in E. coli etc. and shown to be active. 

Ethyl (5J-4-chloro- Carbonyl reductase Candida magnoliae... 

Other remarkable stepwise transformations make use of the combination of several biocatalysts to obtain valuable compounds. In particular, a sequential process employing a recombinant enoate reductase (ER) and a carbonyl reductase has been used for the stereoselective redudion of both enantiomers of carvone to dihydrocarveol (Scheme 4.21) [72]. In the first step, hydrogenation of the endocydic C=C double bond of (R)- or (S)-carvone was achieved by ER from Lactobacillus casd (LacER). In the second step, wild-type or mutant reductases from Candida magnolia (CMCR) and Sporobolomyces sahnoni-color (SSCR) were employed, respectively. First, experiments were performed in a sequential manner and later the one-pot synthesis was tested with a substrate concentration of 0.1 M, obtaining the final compound with excellent conversions and de. 

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... 

Isolated carbonyl reductases from either Candida magnoliae (CMCR reductase) or S. cerevisiae (Ymr266c reductase) were first employed for the biocatalytic... 

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