Stereoselective Reduction of Ethyl 3Oxobutanoate by Bakers Yeast 8 see Note

Reduction of ethyl 3-oxobutanoate to ethyl (¿>3-hydroxybutanoate was carried out with the doubly entrapped yeast cells corresponding to 4.7 g wet cells in 20 or 40 mL of isooctane containing 50 mM ethyl 3-oxobutanoate at 30°C with shaking (120 strokes/min). Gas chromatography was applied to calculate the conversion ratio and the stereoselectivity of the reaction was determined by measuring the optical purity of the product by HPLC.

Different kinds of yeasts were also applied for the asymmetric reduction of oxo-compounds (see Note 10).

4. Notes

1. Various types of photo-crosslinkable resin prepolymers (2,4) were synthesized by similar methods and applied to the entrapment of different biocatalysts. When

ENT and ENTP are mixed to control the hydrophilicity-hydrophobicity balance of gels, the mixtures are water miscible up to 30% (w/w) of ENTP.

2. A series of urethane resin prepolymers (PU) having different chain lengths and/or polyethylene glycol contents in polyether diol were prepared by the same method (2,4,14). All the prepolymers are water miscible and can be used in any mixing ratio.

3. Photo-crosslinkable resin prepolymers and urethane resin prepolymers have been used to entrap not only enzymes but also cells of various conditions (15,16).

4. Benzoin isobutyl ether can also be used as a photoinitiator at the same concentration as benzoin ethyl ether.

5. To induce steroid A*-dehydrogenase, 4-androstene-3, 17-dione was applied as an inducer.

6. Although only /-menthol was esterified by lipase OF 360 with 5-phenylvaleric acid, enantioselectivity of the reaction was dependent on the kind of acyl donors. Porcine pancreas lipase (19 U/mg) (Wako Pure Chemical Industries) was also applicable to this reaction. PU-3 and PU-6 gave almost the similar results on the enzymatic activity (9).

Lipase OF 360 entrapped with the prepolymers was used for the ester formation from oleic acid and n-heptanol or oleic acid and glycerol in organic solvents (17), esterification of citronellol in water-saturated cyclohexane (18), and hydrolysis of triglyceride in an oil-water emulsion system (19). In these cases, the enzyme entrapped with hydrophobic prepolymers (ENTP and PU-3) showed better activities than that entrapped with hydrophilic prepolymers (ENT and PU-6). As for the esterification of citronellol with 5-phenylvaleric acid, various kinds of lipases and esterases were found to be active (18).

7. In the interesterification of triglyceride by R. delemar lipase, entrapment of C-lipase with ENTP gave the highest and the most stable activity (10).

8. Nocardia rhodocrous showed the activities of steroid A'-dehydrogenation, 3p-hydroxysteroid dehydrogenation, and 17|3-hydroxysteroid dehydrogena-tion after appropriate induction of the enzyme systems and transformed a variety of steroids (6,20,21).

In general, the hydrophobic gel-entrapped cells showed higher activities of steroid transformation in organic solvents than the hydrophilic gel-entrapped cells, because of the high partition of substrates into the gels. However, the effect of the hydrophilicity-hydrophobicity balance of gels depends significantly on the hydrophobicity of substrates and the polarity of solvents (6,20-22). The water content of gels is another factor, as shown on esterification by lipase (17) and dehydrogenation of cholesterol by N. rhodocrous cells (23).

9. When the activity of the doubly entrapped cells becomes insufficient, reactivation is possible by cultivating the entrapped cells at 30°C for 8-12 h with shaking in the culture medium supplemented with 0.3% CaCl2. Thus, the entrapped cells survived and retained the activity for at least 50 d over repeated reactions in isooctane (8). Most of the cells were present in calcium alginate gel but not in polyurethane gel.

10. Prepolymer-entrapped yeasts, such as Saccharomyces delbrueckii, Saccharomy-ces fermentati, Candida albicans, and Kloeckera saturnus, were used for the asymmetric reduction of 2-methyl-3-oxo esters in water-saturated organic solvents (benzene, n-hexane, n-heptane, and isooctane) (24).

References

1. Fukui, S., Tanaka, A., Iida, T., and Hasegawa, H. (1976) Application of photo-crosslinkable resin to immobilization of an enzyme. FEBS Lett. 66, 179-182.

2. Fukui, S. and Tanaka, A. (1984) Application of biocatalysts immobilized by prepolymer methods. Adv. Biochem. Eng./Biotechnol. 29, 1-33.

3. Fukui, S. and Tanaka, A. (1985) Enzymatic reactions in organic solvents. Endeavour, New Series 9, 10-17.

4. Fukui, S., Sonomoto, K., and Tanaka, A. (1987) Entrapment of biocatalysts with photo-crosslinkable resin prepolymers and urethane resin prepolymers. Methods Enzymol. 135, 230-252.

5. Fukushima, S., Nagai, T., Fujita, K., Tanaka, A., and Fukui, S. (1981) Hydrophilic urethane prepolymers: Convenient materials for enzyme entrapment. Biotechnol. Bioeng. 20, 1465-1469.

6. Omata, T., Iida, T., Tanaka, A., and Fukui, S. (1979) Transformation of steroids by gel-entrapped Nocardia rhodocrous cells in organic solvent. Eur. J. Appl. Microbiol. Biotechnol. 8, 143-155.

7. Omata, T., lwamoto, N., Kimura, T., Tanaka, A., and Fukui, S. (1981) Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent. Eur. J. Appl. Microbiol. Biotechnol. 11, 199-204.

8. Kanda, T., Miyata, N., Fukui, T., Kawamoto, T., and Tanaka, A. (1998) Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent. Appl. Microbiol. Biotechnol. 49, 377-381.

9. Koshiro, S., Sonomoto, K., Tanaka, A., and Fukui, S. (1985) Stereoselective esterification of dl-menthol by polyurethane-entrapped lipase in organic solvent. J. Biotechnol. 2, 47-57.

10. Yokozeki, K., Yamanaka, S., Takinami, K., Hirose, Y, Tanaka, A., Sonomoto, K., and Fukui, S. (1982) Application of immobilized lipase to regio-selective interesterification of triglyceride in organic solvent. Eur. J. Appl. Microbiol. Biotechnol. 14, 1-5.

11. Fukunaga, K., Minamijima, N., Sugimura, Y, Zhang, Z., and Nakao, K. (1996) Immobilization of organic solvent-soluble lipase in nonaqueous conditions and properties of the immobilized enzymes. J. Biotechnol. 52, 81-88.

12. Goto, M., Kameyama, H., Goto, M., Miyata, M., and Nakashio, F. (1993) Design of surfactants suitable for surfactant-coated enzymes as catalysts in organic media. J. Chem. Eng. Jpn. 26, 109-111.

13. Fukui, S. and Tanaka, A. (1987) Optical resolution of dl-menthol by entrapped biocatalysts. Methods Enzymol. 136, 293-302.

14. Sonomoto, K., Jin, I.-N., Tanaka, A., and Fukui, S. (1980) Application of urethane prepolymers to immobilization of biocatalysts: A'-dehydrogenation of hydrocor-

tisone by Arthrobacter simplex cells entrapped with urethane prepolymers. Agric. Biol. Chem. 44, 1119-1126.

15. Fukui, S. and Tanaka, A. (1989) Application of living microbial cells entrapped with synthetic resin prepolymers. Experientia 45, 1055-1061.

16. Tanaka, A. and Nakajima, H. (1990) Application of immobilized growing cells. Adv. Biochem. Eng./Biotechnol. 42, 97-131.

17. Fukui, S., Tanaka, A., and Iida, T. (1986) Immobilization of biocatalysts for bioprocesses in organic solvent media, in Biocatalysis in Organic Media (Laane, C., Tramper, J., and Lilly, M. D., eds.), Elsevier, Amsterdam. pp. 21-41.

18. Kawamoto, T., Sonomoto, K., and Tanaka, A. (1987) Esterification in organic solvents: selection of hydrolases and effects of reaction conditions. Biocatalysis 1, 137-145.

19. Kimura, Y., Tanaka, A., Sonomoto, K., Nihira, T., and Fukui, S. (1983) Application of immobilized lipase to hydrolysis of triacylglyceride. Eur. J. Appl. Microbiol. Biotechnol. 17, 107-112.

20. Yamane, T., Nakatani, H., Sada, E., Omata, T., Tanaka, A., and Fukui, S. (1979) Steroid bioconversion in water-insoluble organic solvents: A1 -dehydrogenation by free microbial cells and by cells entrapped in hydrophilic or lipophilic gels. Biotechnol. Bioeng. 21, 2133-2145.

21. Fukui, S., Ahmed, S. A., Omata, T., and Tanaka, A. (1980) Bioconversion of lipophilic compounds in non-aqueous solvent. Effect of gel hydrophobicity on diverse conversion of testosterone by gel-entrapped Nocardia rhodocrous cells. Eur. J. Appl. Microbiol. Biotechnol. 10, 289-301.

22. Omata, T., Tanaka, A., and Fukui, S. (1980) Bioconversion under hydrophobic conditions: effect of solvent polarity on steroid transformations by gel-entrapped Nocardia rhodocrous cells. J. Ferment. Technol. 58, 339-343.

23. Sonomoto, K., Hosokawa, Y., and Tanaka, A. (1987) Effect of water content in cell-entrapping gels on enzyme activity in organic solvents. Ann. NY Acad. Sci. 501,343-346.

24. Akita, H., Matsukura, H., Sonomoto, K., Tanaka, A., and Oishi, T. (1987) Asymmetric reduction of a-methyl p-keto esters by microbial cells immobilized with prepolymer. Chem. Pharm. Bull. 35, 4985-4987.

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