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Acclimatization of Baker’s Yeast for Asymmetric Reduction at High Substrate Concentration Hongliang Ni and Shanjing Yao* Department of Chemical and Biochemical Engineering, Zhejiang UniVersity, Hangzhou 310027, People’s Republic of China
By gradually adding a low content of methyl acetoacetate into a solid medium, a new yeast strain, called MAA yeast, was separated from commercial baker’s yeast. The new yeast was used for catalyzing asymmetric reduction of methyl acetoacetate in the aqueous phase, and results showed that it was more efficient than regular baker’s yeast in the asymmetric reduction of methyl acetoacetate. The influence of external environment, pH, and temperature on MAA yeast was the same as on baker’s yeast. The yield of methyl β-hydroxybutanoate with the new strain was about 13% higher than with baker’s yeast at 0.05 M methyl acetoacetate. Similar results were also obtained in experiments, when MAA yeast was used to catalyze other systems, such as reducing ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydroxybutanoate and reducing ethyl acetoacetate to ethyl (S)-3-hydroxybutyrate. In addition, the main product was (S)-methyl β-hydroxybutanoate at low substrate concentration; however, (R)-methyl β-hydroxybutanoate would be obtained when the concentration of substrate exceeded 0.4 M. The morphology of the new strain is the same as that of baker’s yeast. Introduction Optically active compounds are greatly needed in pharmaceutical research and manufacturing. Chiral β-hydroxy esters are often used as chiral building blocks in the synthesis of pharmaceuticals, and many methods have been developed for the preparation of these optically active alcohols. However, asymmetric chemical synthesis1,2 and lipase-catalyzed kinetic resolution through alcoholysis or ammonolysis of racemoid 3-hydroxy esters3,4 cost a lot to get the expected target products. Chiral β-hydroxy esters with high enantiomeric excess (ee) values can be obtained when isolated reductase is applied,5,6 but high-value coenzymes, such as NAD(P)H, are needed in the reaction. This restricts application in industrial production. On the other hand, the biocatalytic asymmetric reduction of 3-oxo esters7,8 with whole cells is agreed to be a safe, economical, and mild method to prepare for chiral alcohols. The regeneration of NAD(P)H with whole cells can be easily carried out by adding carbohydrate during the reaction. According to the traditional view,9 organic solvents would harm living cells. When microorganisms are used as biocatalysts in asymmetric reduction in the aqueous phase, the yield of products declines rapidly as the substrate concentration increases. Therefore, only under low initial substrate concentration the microbial activity can be held at a high level; usually, the output of products is much less than expected.10 Several methods, such as immobilized cells,11,12 constructing genetic engineering microorganisms,13 isolating toluene-tolerant microorganisms,14-17 etc., were developed to improve the microorganism tolerance in organic solvents and increase the substrate and product concentrations. In our previous research,18 resin was used to adsorb the substrate and release it slowly during the reaction so that the toxicity of the substrates and products to cells was reduced to a low level. However, resin could not be applied to all kinds of substrates and products, and the yield of products would be reduced during the adsorption and absorption. In the research mentioned above, baker’s yeast was * To whom correspondence should be addressed. Tel.: +86-57187951982. Fax: +86-571-87951015. E-mail:
[email protected].
often used in asymmetric reduction because of its cheapness, safeness for humans, and broad substrate types.19 In our previous asymmetric reduction experiments, after several hours in an aqueous phase, some yeast cells were not damaged by the organic solvent and still remained active when free baker’s yeast was used as biocatalyst. This phenomenon indicated that some yeast cells had more vitality than others. Therefore, it is possible to get excellent organic solvent tolerant yeast from common baker’s yeast. This paper describes how a new yeast strain was separated from baker’s yeast in solid media with the addition of a low content of methyl acetoacetate. This strain was then used to catalyze the asymmetric reduction of methyl acetoacetate and two other systems in order to improve the catalysis performance of the yeast in the aqueous phase. Materials and Methods Chemicals. Methyl acetoacetate and ethyl acetoacetate were purchased from China Medicine Shanghai Chemical Reagent Corp. Ethyl 4-chloro-3-oxobutanoate and decane were purchased from Sigma-Aldrich. NADPH (purity above 97%) was purchased from Roche Company. Microorganism. The active dried baker’s yeast was obtained from Meishan-Mauri Yeast Co. Ltd., China. Method of Activating Dried Yeast. The activation media was composed of 50 g/L glucose, 2.0 g/L(NH4)2SO4, 1.0 g/L K2HPO4, and 1.0 g/L citric acid. A 7.5 g sample of dried yeast was inoculated in 200 mL of 30 °C activation media and incubated in a shaker with a temperature controller at 30 °C, 170 rpm, for 2 h. Then yeast cells were collected by centrifugation at 8000 rpm for 10 min and washed twice with PBS (phosphate-buffered saline, 50 mM, pH 7.0). Culture Medium and Solid Medium. The culture medium was composed of 20 g/L glucose. 10 g/L yeast extract. and 20 g/L tryptone. It was sterilized at 121 °C for 15 min. Besides these compositions, 1.5% (m/v) agar was added in the solid medium. Screening the New Strain from Commercial Baker’s Yeast. The activated yeast was transferred to a 250 mL triangular flask containing 50 mL of the culture medium. Then,
10.1021/ie070140i CCC: $37.00 © 2007 American Chemical Society Published on Web 10/17/2007
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the medium was cultured at 170 rpm and 30 °C for 36 h under aerobic conditions. After that, the yeast was transferred with a inoculating loop to the solid medium with which 200 µL of methyl acetoacetate was mixed before use. Yeast on the solid medium propagated at 30 °C in thermostat. After 48 h, different colonies on the plate were collected, and they were cultured in the medium again under the conditions mentioned above for 36 h. Then, the cells were transferred from the triangular flask to solid medium again, the concentration of methyl acetoacetate in medium was increased gradually, and the culture process was repeated. When colonies succeeded in growing in solid medium with 1 mL of methyl acetoacetate being dissolved in, we finally separated MAA yeast, whose concentration in the same liquid culture medium was higher than that of other colonies during the same culture time. Growth Curves of Yeast. The yeast was inoculated in 100 mL of culture medium and incubated in a shaker at 170 rpm at 30 °C. Periodically, 1 mL of suspending liquid was taken from a flask and its optical absorbance was determined at 600 nm (OD600) with a spectrometer. Then the concentration of the microorganism in culture medium was calculated with standard curves. Preparation of Crude Reductase. A 5.0 g portion of wet yeast was suspended in 25 mL of PBS buffer, where 10 mM mercaptoethanol was added to protect the disulfide bond in reductase. The yeast cells in the suspension were disrupted by a Thermo French Press at 14 000 psi three times. Then the disrupted-cell suspension was centrifuged at 12 000 rpm for 30 min. The reductase for asymmetric reduction was in the supernate. All manipulations were carried out at 4 °C to protect the activity of the enzyme. Determination of Activity of Reductase. A 30 µL volume of 6 mmol/L NADPH solution and 10 µL of methyl acetoacetate were mixed with 1.0 mL of crude reductase solution in a 5.0 mL quartz colorimetric tube, with 3.0 mL of PBS added in. NADPH was consumed by reductase for the asymmetric reduction at 30 °C. The decrease of NADPH was detected by a spectrometer at 340 nm, the maximum absorbency, and the relative activity of reductase could be correlated to it. General Conditions of Biocatalysts. A 2.5 g portion of wet yeast was suspended in a 250 mL flask with 25 mL of PBS buffer (pH 7.0). Different amounts of substrate and glucose were added to the suspension. Then the flask was placed in a 160 rpm shaker at 30 °C for reaction. After 4 h, the reactive mixture was extracted with ethyl acetate twice (each time for 10 min). Before the organic phase was separated and dried with anhydrous MgSO4 for analysis, the mixture was centrifuged at 8000 rpm for 5 min to promote phase separation. Analytical Methods. The yield of different products was analyzed with an Agilent 6820 GC (gas chromatograph) equipped with different columns, HP-5MS (30 m × 0.32 mm × 0.25 µm) and CYCLODEXB (30 m × 0.25 mm × 0.25 µm). Flame ionization detection (FID) and programming temperature for the column were the conditions for GC. Decane was used as the internal standard substance. The enantiomeric excess value of the products was determined by an Agilent 1100 HPLC (highperformance liquid chromatograph), which was equipped with a chiral Chiralpak AD-H column (4.6 mm × 250 mm) (Daicel Chemicals, Japan). The optical rotation of products in the organic phase was determined with an AUTOPOL IV digital polarimeter (Rudolph Technologies, Inc., USA) at 589 nm. Results Growth of Two Strains. Because some baker’s yeasts could remain active after catalytic reaction when organic solvent
Figure 1. Growth curves of MAA yeast and baker’s yeast in culture medium with and without methyl acetoacetate. (2) Baker’s yeast in culture medium without methyl acetoacetate; (3) MAA yeast in culture medium without methyl acetoacetate; (b) MAA yeast in culture medium with methyl acetoacetate; (O) baker’s yeast in culture medium with methyl acetoacetate.
existed in the aqueous phase, we isolated a new strain, MAA yeast, from baker’s yeast by adding organic solvent gradually into the solid medium. The process was described earlier. MAA yeast and baker’s yeast were inoculated in four bottles of 150 mL of liquid culture medium, respectively. A concentration of 0.1 M methyl acetoacetate was added to two bottles of culture medium. Figure 1 shows the growth curves of the two strains in the culture medium with or without methyl acetoacetate at 30 °C. MAA yeast grew well in the medium with 0.1 M methyl acetoacetate, which means it had a higher tolerance to methyl acetoacetate than baker’s yeast. Baker’s yeast grew very slowly in the same medium. Nevertheless, both strains had a typical growth curve, which presented an initial lag phase followed by a period of exponential growth. Also, the concentration of baker’s yeast was much lower than that of MAA yeast at the stationary phase. In the culture medium without organic solvent, the concentration of both stains was higher and their lag phase was not obvious. This is because there was no organic solvent in the culture medium that inhibited the growth of the two strains. To confirm whether the two strains had differences in cellular morphology, a microscope was used to observe the strains. Figure 2 shows that the morphology of MAA yeast growth with and without organic solvent was the same as that of baker’s yeast. Asymmetric Reduction with the Two Strains. Generally, the reaction of 3-oxo ester asymmetric reduction by baker’s yeast in the aqueous phase needs cofactor NAD(P)H as the electron donor, which transforms from NAD(P)H to oxidative-type NAD(P)+. A simplified scheme of the reduction reaction is shown in Figure 3. When the two strains were used as biocatalysts in the asymmetric reduction of methyl acetoacetate in PBS buffer, the GC detected different reaction processes at the same reaction condition. At 30 °C, substrate concentration of 0.05 M, and a 2.5 g portion of wet yeast suspended in 25 mL of PBS buffer, baker’s yeast took 4 h (Figure 4) while MAA yeast took 5 h (Figure 5) to completely consume the substrate in the suspension. In addition, the yield of product catalyzed by MAA yeast was 45% and by baker’s yeast was 32%. The productivity was 2.66 and 1.89 mg/g wet yeast, respectively. In these two reactions, the main products were the (S)-form, which was determined by a polarimeter. Effect of pH. We examined the influence of external environment on the yeast activity. This is particularly important in employing microorganisms in manufacturing processes. The
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Figure 3. Scheme of asymmetric reduction of 3-oxo ester to β-hydroxy esters by yeast with regeneration of the cofactor NAD(P)H in aqueous phase.
Figure 4. Reduction process curves of methyl acetoacetate with baker’s yeast at 30 °C, pH 7.0.
Figure 5. Reduction process curves of methyl acetoacetate with MAA yeast at 30 °C, pH 7.0.
Figure 2. Microscopic examinations: MAA yeast grown with methyl acetotateace (A) or without (B) in the culture medium, and baker’s yeast (C).
pH of the reaction solution may affect the activity of most enzymes in the yeast. Changes in pH may also alter the result of asymmetric reduction. Most bacteria grow in the range pH 6.5-7.5, while yeast can tolerate lower pH. Therefore, the pH of the reaction solution was controlled from 3.0 to 8.0 and was adjusted by PBS buffer. The substrate was 0.05 M methyl acetoacetate. Figure 6 shows that the optimum pH of the reaction was between 6.0 and 7.0. At pH 6.0 and 7.0, the yield of products catalyzed by MAA yeast was higher than the yield of
products catalyzed by baker’s yeast. However, there was little discrepancy in the yields of products catalyzed by baker’s yeast and MAA yeast at lower and higher pH, which may be caused by denaturation of enzymes in the yeast and hydrolyzation of substrate and product. Effect of Temperature. In general, eukaryotes show a narrow range of growth temperature. At lower temperature, the growth rate of the microorganism was very slow and the same for the activity of enzymes in the microorganism. At higher temperature, enzyme activity will decrease with temperature rise because of enzyme denaturation. At pH 7.0, the reaction temperature was changed from 20 to 40 °C, and the effect on the yield of the products is shown in Figure 7. The concentration of methyl acetoacetate was 0.05 M. The yield at 20 °C was much lower than that at higher temperature. Both baker’s yeast and MAA yeast had higher catalytic activity between 30 and 35 °C. In addition, MAA yeast had more catalytic efficiency than baker’s yeast at the optimum temperature. At 40 °C, the yield decreased obviously for the denaturation of enzymes in the microorganisms.
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Figure 6. Effect of pH on reduction of methyl acetoacetate catalyzed by baker’s yeast and MAA yeast (glucose concentration 12 g/L; cell concentration 100 g/L; reaction temperature 30 °C).
Figure 7. Effect of temperature on reduction of methyl acetoacetate catalyzed by baker’s yeast and MAA yeast (glucose concentration 12 g/L; cell concentration 100 g/L; pH 7.0).
Activity of Reductase. In the asymmetric reduction, the reductase in the yeast was required to reduce the carbonyl group to hydroxyl group, which was the chiral center of the product. During the reaction, cofactor NAD(P)H helped reductase to remain the reducing force. The more reductase that existed in the cells, the more NAD(P)H was consumed. When the whole cell was used as catalyst, cosubstrate, such as glucose, sucrose, and glycerol, was added to the reactant mixture to regenerate NAD(P)H spontaneously through the metabolic pathway in the cells. The activity of reductase in the two strains of yeast was investigated. The solution of NADPH, crude reductase, and substrate were mixed and reacted at 30 °C for 5 min in the thermostatic spectrophotometer. During this period, the absorbance of NADPH at 340 nm decreased gradually and the absorbance at 5 min was recorded to compare the activity of reductase in the two strains (Figure 8). At 5 min, the absorbance of NADPH in crude reductase solution of baker’s yeast was 0.35 while that of MAA yeast was 0.27. This indicated that the consumption of NADPH by reductase of MAA yeast was greater than that of baker’s yeast. Reaction with Other Substrates. MAA yeast was prepared from culture medium with addition of methyl acetoacetate, one of the 3-oxo ester compounds. The reductase of the MAA strain was more active than that of baker’s yeast during catalysis of methyl acetaoacetate in Figure 8. Other 3-oxo ester compounds had a similar configuration, except that the compounds had different group R1 or R2 (Figure 3) and different toxicity to the cells. Using ethyl 4-chloro-3-oxobutanoate and ethyl acetoacetate as substrate, respectively, the reduction results showed
Figure 8. Absorbance of NADPH at 340 nm after reaction with reductase of baker’s yeast and MAA yeast (30 °C, 5 min).
Figure 9. Effect of substrate concentration on bioreduction of methyl acetoacetate by cells of baker’s yeast (glucose concentration 12 g/L; cell concentration 100 g/L; reaction temperature 30 °C). Table 1. Asymmetric Reduction of Different Substrates with Baker’s Yeast and MAA Yeast at 0.1 M baker’s yeast
MAA yeast
substrate
yield, %
ee, %
yield, %
ee, %
methyl acetoacetate ethyl acetoacetate 4-chloro-3-oxobutanoate
18 13 21
70 84 45
25 20 36
60 75 39
that MAA yeast could improve the yield of (S)-4-chloro-3hydroxybutanoate and ethyl (S)-3-hydroxybutyrate by 7% and 15%, respectively (Table 1). Otherwise, the enantiomeric excess value of three products decreased by 10%, 9% and 6%, respectively. It is suggested that MAA yeast can improve the yield of 3-oxo substrate while the enantiomeric excess value would decrease at the same time. Effect of Substrate Concentration. Methyl acetoacetate can be easily dissolved in water. This is an advantage for yeast catalysis in the aqueous phase. However, Figures 9 and 10 shows that the yield decreased while the substrate concentration increased. Moreover, the change of substrate concentration also had an effect on the enantiomeric excess value of the product. These results might be interpreted as improving the substrate concentration also increased the toxicity to both yeast strains to some extent. In Figures 9 and 10, at each substrate concentration, the yield of products catalyzed by MAA yeast was higher than that by baker’s yeast. When the substrate concentration was increased to 0.4 M, the yield of products catalyzed by MAA yeast was 7% while the products yield catalyzed by baker’s yeast was 3%. This proved that the cells of MAA yeast could tolerate high substrate concentration and retain considerable biotransformation activity.
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Figure 10. Effect of substrate concentration on bioreduction of methyl acetoacetate by cells of MAA yeast (glucose concentration 12 g/L; cell concentration 100 g/L; reaction temperature 30 °C).
On the other hand, the enantiomeric excess value of the products catalyzed by MAA yeast was lower than by baker’s yeast at each substrate concentration. These results were matched well with the ones in Table 1. However, something interesting happened: when the substrate concentration was increased to 0.4 M, the main product catalyzed by MAA yeast was the (R)form with 25% enantiomeric excess value while the product catalyzed by baker’s yeast was the (S)-form with 35% enantiomeric excess value. Discussion MAA yeast we acclimated from baker’s yeast had more catalytic activity than baker’s yeast. It could grow better than baker’s yeast when methyl acetoacetate existed in the culture medium. The resistance of MAA yeast to organic solvent may be due to some difference in the cytoplasmic membrane which makes it more active in the presence of organic solvent.15 In the research of Kawamoto17 and Keweloh,20 they analyzed the fatty acid composition of phospholipids extracted from the membrane of an organic solvent tolerant microorganism. The saturated fatty acid occupancy of the tolerant microorganism was higher than that of a nontolerant microorganism. That said, the fatty acid composition of a cell membrane might play an important role in its tolerance to organic solvent;20 at the same time the composition of the cell membrane may influence the growth of yeast. In the same culture medium without organic solvent, MAA yeast’s slow growth might be ascribed to more carbon source needed to synthesize fatty acid in the cell membrane. Furthermore, we found that during the culture of MAA yeast, if more peptone was added to the culture medium, the growth rate would be as quick as that of baker’s yeast with less peptone in the medium. This indicated that the compositions of the two strains, baker’s yeast and MAA yeast, not only in membrane but also in cytoplasmic inclusions, such as enzymes, are different though they have the same shape in morphology. The oxidoreductase needed NAD(P)H as cofactor during reaction in cells was necessary in asymmetric reduction. The consumption of NAD(P)H can reflect the amount and activity of oxidoreductase. In Figures 4 and 5, both conversions of the substrate were 100% while different yields were obtained. In Chin-Joe et al.,21 enzyme hydrolysis was one of the main courses responsible for the low yield. In Table 1, MAA yeast improved the yield of products of 3-oxo esters in a limited concentration range, which suggested that there would be more oxidoreductase or less hydrolase in MAA yeast than in common baker’s yeast. Two types of enzymes, D-oxidoreductase and L-oxidoreductase, were isolated from baker’s yeast by Nakamura et al.,22
which could reduce the 3-oxo ester. These two types of enzymes could catalyze the substrate to different stereo configuration products separately,23 (S)-products and (R)-products. Because two types of enzymes had different properties when the temperature was changed or some inhibitors were added, Yang et al.24 had improved the stereoselectivity of asymmetric reduction of 3-oxo ester to chiral 3-hydroxy ester by pretreating yeast cells with heating or allyl compounds. When MAA yeast and baker’s yeast were used to obtain methyl β-hydroxybutanoate by asymmetric reduction in aqueous phase at low substrate concentration, the main product was (S)-methyl β-hydroxybutanoate. However, when substrate was increased to 0.4 M, the main product transformed to (R)-methyl β-hydroxybutanoate catalyzed by MAA yeast. This indicated that L-oxidoreductase enzyme was greater in MAA yeast than in baker’s yeast. What was more, in other research by Yang,25 baker’s yeast pretreated with allyl bromide could turn over the stereoselectivity of the asymmetric reduction reaction from the (S)-form to the (R)-form. This method would do great harm to baker’s yeast, and the added organic solvent would cause difficulty in separation. If using MAA yeast in the asymmetric reduction, the (R)-form product could be obtained by increasing the substrate concentration to some extent. Conclusions We succeeded in preparing an organic solvent tolerant strain from commercial baker’s yeast by adding organic solvent in solid culture medium gradually. The strain MAA yeast could improve the yield of asymmetric reduction, and a reverse form of the product could be obtained at a higher substrate concentration. External environment, pH, and temperature had the same effect on both strains. The method of acclimatization is expected to be useful for preparation of other solvent-tolerant microorganisms. However, the mechanisms and reasons for such preparation need more research. Acknowledgment We thank the Chinese National Natural Science Foundation (No. 20576118) for financial support. Literature Cited (1) Madec, J.; Pfister, X.; Phansavath, P.; Ratovelomanana-Vidal, V.; GeneA ˜ t, J.-P. Asymmetric hydrogenation reactions using a practical in situ generation of chiral ruthenium diphosphine catalysts from anhydrous RuCl3. Tetrahedron 2001, 57, 2563. (2) Berthod, M.; Mignanib, G.; Lemaire, M. New perfluoroalkylated BINAP usable as a ligand in homogeneous and supercritical carbon dioxide asymmetric hydrogenation. Tetrahedron: Asymmetry 2004, 15, 1121. (3) Fishman, A.; Eroshov, M.; Dee-Noor, S. S.; van Mil, J.; Cogan, U.; Effenberger, R. A Two-Step Enzymatic Resolution Process for Large-Scale Production of (S)- and (R)-Ethyl-3-Hydroxybutyrate. Biotechnol. Bioeng. 2001, 74, 256. (4) Garcia-Urdiales, E.; Rebolledo, F.; Gotor, V. Enzymatic Ammonolysis of Ethyl (()-4-Chloro-3-Hydroxybutanoate. Chemoenzymatic Syntheses of Both Enantiomers of Pyrrolidin-3-ol and 5-(chloromethyl)-1,3-oxazolidin2-one. Tetrahedron: Asymmetry 1999, 10, 721. (5) Nakamura, K.; Matsuda, T. Asymmetric Reduction of Ketones by the Acetone Powder of Geotrichum candidum. J. Org. Chem. 1998, 63, 8957. (6) Wada, M.; Kawabata, H.; Kataoka, M.; Yasohara, Y.; Kizaki, N.; Hasegawa, J.; Shimizu, S., Purification and characterization of an aldehyde reductase from Candida magnoliae. J. Mol. Catal. B: Enzym. 1999, 6, 333. (7) Salvi, N. A.; Chattopadhyay, S. Rhizopus arrhizus mediated asymmetric reduction of alkyl 3-oxobutanoates. Tetrahedron: Asymmetry 2004, 15, 3397.
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(8) Yasohara, Y.; Kizaki, N.; Hasegawa, J.; Takahashi, S.; Wada, M.; Kataoka, M.; Shimizu, S. Synthesis of Optically Active Ethyl 4-Chloro-3Hydroxybutanoate by Microbial Reduction. Appl. EnViron. Microbiol. 1999, 51, 847. (9) Sikkema, J.; Weber, F. J.; Heipieper, H. J.; Bont, J. D. Cellular toxicity of lipophilic compounds: mechanisms, implications, and adaptations. Biocatalysis 1994, 10, 113. (10) Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T. Recent Developments in Asymmetric Reduction of Ketones with Biocatalysts. Tetrahedron: Asymmetry 2003, 14, 2659-2681. (11) Jiang, Q.; Yao, S. J.; Mei, L. H. Tolerance of immobilized baker’s yeast in organic solvents. Enzyme Microb. Technol. 2002, 30, 721. (12) Buque, E. M.; Chin-Joe, I.; Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Immobilization affects the rate and enantioselectivity of 3-oxo ester reduction by baker’s yeast. Enzyme Microb. Technol. 2002, 31, 656. (13) Yamamoto, H.; Matsuyama, A.; Kobayashi, Y. Synthesis of Ethyl (R)-4-Chloro-3-hydroxybutanoate with Recombinant Escherichia coli Cells Expressing (S)-Specific Secondary Alcohol Dehydrogenase. Biosci. Biotechnol. Biochem. 2002, 66, 481. (14) Aono, R.; Ito, M.; Inoue, A.; Horikoshi, K. Isolation of novel toluene-tolerant strain of Pseudomonas aeruginosa. Biosci. Biotechnol. Biochem. 1992, 56, 145. (15) Cruden, D. L.; Wolfram, J. H.; Rogers, R. D.; Gibson, D. T. Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium. Appl. EnViron. Microbiol. 1992, 58, 2723. (16) Nakajima, H.; Kobayashi, H.; Aono, R.; Horikoshi, K. Effective isolation and identification of toluene-tolerant Pseudomonas strains. Biosci. Biotechnol. Biochem. 1992, 56, 1872. (17) Kawamoto, T.; Kanda, T.; Tanaka, A. Preparation of an organic solvent-tolerant strain from Baker’s yeast. Appl. Microbiol. Biotechnol. 2001, 55, 476.
(18) Yang, Z. H.; Yao, S. J.; Guan, Y. X. A Complex Process of Asymmetric Synthesis of β-Hydroxy Ester by Baker’s Yeast Accompanied by Resin Adsorption. Ind. Eng. Chem. Res. 2005, 44, 5411. (19) Servi, S. Baker’s Yeast as a Reagent in Organic Synthesis. Synthesis 1990, 1990, 1. (20) Keweloh, H.; Diefenbach, R.; Rehm, H. J. Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids. Arch. Microbiol. 1991, 157, 49. (21) Chin-Joe, I.; Nelisse, P. M.; Straathof, A. J. J.; Jongejan, J. A.; Pronk, J. T.; Heijnen, J. J. Hydrolytic Activity in Baker’s Yeast Limits the Yield of Asymmetric 3-Oxo Ester Reduction. Biotechnol. Bioeng. 2000, 69, 370. (22) Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. Stereochemical Control of Microbial Reduction. 17. A Method for Controlling the Enantioselectivity of Reductions with Bakers’ Yeast. J. Org. Chem. 1991, 56, 4778. (23) Nakamura, K.; Kondo, S.; Nakajima, N.; Ohno, A. Study for Stereochemical Control of Microbial Reduction of R-Keto Esters in an Organic Solvent. Tetrahedron 1995, 51, 687. (24) Yang, Z. H.; Yao, S. J.; Lin, D. Q. Improving the Stereoselectivity of Asymmetric Reduction of 3-Oxo Ester to 3-Hydroxy Ester with Pretreatments on Bakers’ Yeast. Ind. Eng. Chem. Res. 2004, 43, 4871. (25) Yang, Z. H.; Yao, S. J.; An effective method for controlling the stereoselectivity in asymmetric reduction of beta-oxo ester with yeast cells. Chin. J. Catal. 2004, 25, 805.
ReceiVed for reView January 23, 2007 ReVised manuscript receiVed June 18, 2007 Accepted August 29, 2007 IE070140I
