有机相微生物活细胞催化羰基还原反应的研究
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摘要
以有机相微生物活细胞催化不对称合成为背景，一方面考察了面包酵母对有机溶剂的耐受性，并建立了有机物存在时微生物的活力保留值模型，另一方面考察了面包酵母活细胞在有机介质中催化4-氯乙酰乙酸乙酯等含羰基化合物的不对称还原反应，对促进微生物活细胞生物转化法应用于手性有机合成领域具有一定的理论意义和实际价值。
首先，考察了固定化面包酵母在有机溶剂中浸泡24h后的糖代谢活力保留值，结果表明，固定化面包酵母对有机溶剂的耐受性比游离面包酵母有所增强；随着溶剂logP_(oct)值的增大固定化面包酵母对其耐受性增强；温度和酵母的预培养时间等也影响固定化面包酵母的溶剂耐受性，30℃时固定化面包酵母的耐受性最强，预培养10h的酵母耐受性有所降低。
其次，应用有机化合物定量结构—活性相关研究方法，根据“靶学说”理论，建立了有机物存在时微生物的活力保留值模型，分别得出了同一或同类有机物存在时细胞活力保留值与一些相关理化参数的关系式，并很好地关联了Rhizopus nigricans细胞的11α-羟化酶活力保留值和Monoraphidium braunii细胞的光合成活力保留值等数据。另外，考察了乙酸丁酯、仲丁醇、苯甲醛和4-氯乙酰乙酸乙酯等四种有机物存在时面包酵母的糖代谢活力保留值，结果表明，这四种有机物对面包酵母有毒害作用，随着其在水相中浓度的增大，对面包酵母的毒害作用增强。面包酵母的糖代谢活力保留值的实验测定值与模型计算值相符，进一步验证了微生物活力保留值模型的正确性。
第三，考察了有机介质中Sigma公司生产的面包酵母(Type Ⅱ)催化4-氯乙酰乙酸乙酯不对称羰基还原反应的影响因素，如溶剂选择、固定化条件、反应温度和底物浓度等。以邻苯二甲酸二丁酯作为反应介质，面包酵母可催化较高浓度的有毒底物4-氯乙酰乙酸乙酯发生还原反应，得到具有光学活性的手性药物中间体R-4-氯-3-羟基丁酸乙酯(ee=58％)。
第四，选用具有较高选择性的菌株——梅山酵母，固定化后在邻苯二甲酸二丁酯中催化4-氯乙酰乙酸乙酯的不对称还原反应，得到对映体过量值较高的R-4-氯-3-羟基丁酸乙酯(ee=76％)。不同缓冲体系、缓冲溶液pH值、温度等对梅山酵母和面包酵母(Type Ⅱ)在邻苯二甲酸二丁酯中催化4-氯乙酰乙酸乙酯不对称羰基还原反应的影响存在差异，不同来源的菌株表现出不同的反应特性。
第五，在面包酵母(TypeⅡ)催化4-氯乙酰乙酸乙酯还原反应体系中，分别加入
浙江大学博十学位论义 摘要
磷酸戊糖途径抑制剂（Na少O＊、糖酵解途径抑制剂（NaF）和三竣酸循环抑制剂（丙
二酸人 结果使个氯乙酚乙酸乙酯的还原反应都受到了抑制，反应产率大大下降。表
明糖酵解途径、磷酸戊糖途径和三竣酸循环的顺利进行对于面包酵母在有机介质中
催化4－氯乙酚乙酸乙酯还原反应都是十分重要的。
最后，考察了有机介质中面包酵母催化另两种含碳基化合物—一卜苯刁，2－丙二
酮和苯甲醛的还原反应，反应产率和选择性受到有机溶剂、反应温度和固定化所用
缓冲液pH值等因素的影响。
On the background of whole-cell biocatalysis for asymmetric synthesis in organic media, the solvent tolerance of baker's yeast was examined and a model for calculating activity retention of microbe in the presence of organic compounds was established. On the other hand, the asymmetric reductions of ethyl 4-chloroacetoacetate, l-phenyl-1,2-propanedione and benzaldehyde with baker's yeast in organic media were investigated in detail. The research work contributes to the application of whole-cell biocatalysis in the field of chiral synthesis in theory and practice.
By determining the saccharide metabolic activity retention of baker's yeast after 24 h exposure to various organic solvents, the solvent tolerances of immobilized baker's yeast were studied. The results showed that the tolerance of immobilized baker's yeast was higher than that of free cells and increased with the increase of logP value of the organic solvent. The effects of temperature and pre-incubation time on the tolerance of immobilized baker's yeast were examined in detail. The tolerance of immobilized baker's yeast reached maximum at 30癈 and the activity retention of cells having been pre-incubated for 10 h decreased markedly.
Using the method of Quantitative Structure-Activity Relationship of organic compounds, a new model for calculating activity retention of microbe in the presence of organic compounds was established on the basis of "target theory" and the relationship between the activity retention and relevant parameters was gained for the same compound or the homologous compounds. The new model successfully correlated the data of 11 a -hydroxylase activity retention of Rhizopus nigricans cells incubated in the presence of eight primary alcohols and photosynthetic activity retention of Monoraphidium braunii cells incubated in the presence of several alcohols. In addition, the saccharide metabolic activity retentions of baker's yeast incubated in the presence of butyl acetate, sec-butanol, benzaldehyde and ethyl 4-chloroacetoacetate were determined. It showed that these compounds were toxic to baker's yeast and the toxicity increased with the increase of the compound concentration in aqueous phase. The calculated data of the
saccharide metabolic activity retention of baker's yeast using the new model accorded with the experimental data.
The effects of some factors were examined in detail on the asymmetric carbonyl reduction of ethyl 4-chloroacetoacetate with baker's yeast(Type II ,Sigma) in organic media, including the selection of organic solvents, the immobilization conditions, the reaction temperature and the substrate concentration. Ethyl 4-chloroacetoacetate was reduced with baker's yeast to obtain ethyl (R)-4-chloro-3-hydroxybutyrate (ee=58%) when dibutyl phthalate was used as reaction media.
Meishan yeast which showed higher stereoselectivity was used to catalyze the reduction of ethyl 4-chloroacetoacetate in dibutyl phthalate. As a result, (R)-4-chloro-3-hydroxybutyrate with higher ee value(ee=76%) was produced. The effects of the buffer used and its pH and the reaction temperature on the bioreductions with Meishan yeast or baker's yeast(Type II) were different.
When Na3PO4, NaF and malonic acid was added to the reaction system respectively, the reduction of ethyl 4-chloroacetoacetat with baker's yeast(Type II) was inhibited and the yield decreased notably. This reflected that BMP and HMP pathways and the tricarboxylic acid cycle(TCA cycle) were very important to the bioreduction.
Finally, the reductions of l-phenyl-l,2-propanedione and benzaldehyde with baker's yeast in organic media were investigated, too. The yield and chemical selectivity were influenced by organic solvents, temperature and pH of the buffer used for cell immobilization.
首先，考察了固定化面包酵母在有机溶剂中浸泡24h后的糖代谢活力保留值，结果表明，固定化面包酵母对有机溶剂的耐受性比游离面包酵母有所增强；随着溶剂logP_(oct)值的增大固定化面包酵母对其耐受性增强；温度和酵母的预培养时间等也影响固定化面包酵母的溶剂耐受性，30℃时固定化面包酵母的耐受性最强，预培养10h的酵母耐受性有所降低。
其次，应用有机化合物定量结构—活性相关研究方法，根据“靶学说”理论，建立了有机物存在时微生物的活力保留值模型，分别得出了同一或同类有机物存在时细胞活力保留值与一些相关理化参数的关系式，并很好地关联了Rhizopus nigricans细胞的11α-羟化酶活力保留值和Monoraphidium braunii细胞的光合成活力保留值等数据。另外，考察了乙酸丁酯、仲丁醇、苯甲醛和4-氯乙酰乙酸乙酯等四种有机物存在时面包酵母的糖代谢活力保留值，结果表明，这四种有机物对面包酵母有毒害作用，随着其在水相中浓度的增大，对面包酵母的毒害作用增强。面包酵母的糖代谢活力保留值的实验测定值与模型计算值相符，进一步验证了微生物活力保留值模型的正确性。
第三，考察了有机介质中Sigma公司生产的面包酵母(Type Ⅱ)催化4-氯乙酰乙酸乙酯不对称羰基还原反应的影响因素，如溶剂选择、固定化条件、反应温度和底物浓度等。以邻苯二甲酸二丁酯作为反应介质，面包酵母可催化较高浓度的有毒底物4-氯乙酰乙酸乙酯发生还原反应，得到具有光学活性的手性药物中间体R-4-氯-3-羟基丁酸乙酯(ee=58％)。
第四，选用具有较高选择性的菌株——梅山酵母，固定化后在邻苯二甲酸二丁酯中催化4-氯乙酰乙酸乙酯的不对称还原反应，得到对映体过量值较高的R-4-氯-3-羟基丁酸乙酯(ee=76％)。不同缓冲体系、缓冲溶液pH值、温度等对梅山酵母和面包酵母(Type Ⅱ)在邻苯二甲酸二丁酯中催化4-氯乙酰乙酸乙酯不对称羰基还原反应的影响存在差异，不同来源的菌株表现出不同的反应特性。
第五，在面包酵母(TypeⅡ)催化4-氯乙酰乙酸乙酯还原反应体系中，分别加入
浙江大学博十学位论义 摘要
磷酸戊糖途径抑制剂（Na少O＊、糖酵解途径抑制剂（NaF）和三竣酸循环抑制剂（丙
二酸人 结果使个氯乙酚乙酸乙酯的还原反应都受到了抑制，反应产率大大下降。表
明糖酵解途径、磷酸戊糖途径和三竣酸循环的顺利进行对于面包酵母在有机介质中
催化4－氯乙酚乙酸乙酯还原反应都是十分重要的。
最后，考察了有机介质中面包酵母催化另两种含碳基化合物—一卜苯刁，2－丙二
酮和苯甲醛的还原反应，反应产率和选择性受到有机溶剂、反应温度和固定化所用
缓冲液pH值等因素的影响。
On the background of whole-cell biocatalysis for asymmetric synthesis in organic media, the solvent tolerance of baker's yeast was examined and a model for calculating activity retention of microbe in the presence of organic compounds was established. On the other hand, the asymmetric reductions of ethyl 4-chloroacetoacetate, l-phenyl-1,2-propanedione and benzaldehyde with baker's yeast in organic media were investigated in detail. The research work contributes to the application of whole-cell biocatalysis in the field of chiral synthesis in theory and practice.
By determining the saccharide metabolic activity retention of baker's yeast after 24 h exposure to various organic solvents, the solvent tolerances of immobilized baker's yeast were studied. The results showed that the tolerance of immobilized baker's yeast was higher than that of free cells and increased with the increase of logP value of the organic solvent. The effects of temperature and pre-incubation time on the tolerance of immobilized baker's yeast were examined in detail. The tolerance of immobilized baker's yeast reached maximum at 30癈 and the activity retention of cells having been pre-incubated for 10 h decreased markedly.
Using the method of Quantitative Structure-Activity Relationship of organic compounds, a new model for calculating activity retention of microbe in the presence of organic compounds was established on the basis of "target theory" and the relationship between the activity retention and relevant parameters was gained for the same compound or the homologous compounds. The new model successfully correlated the data of 11 a -hydroxylase activity retention of Rhizopus nigricans cells incubated in the presence of eight primary alcohols and photosynthetic activity retention of Monoraphidium braunii cells incubated in the presence of several alcohols. In addition, the saccharide metabolic activity retentions of baker's yeast incubated in the presence of butyl acetate, sec-butanol, benzaldehyde and ethyl 4-chloroacetoacetate were determined. It showed that these compounds were toxic to baker's yeast and the toxicity increased with the increase of the compound concentration in aqueous phase. The calculated data of the
saccharide metabolic activity retention of baker's yeast using the new model accorded with the experimental data.
The effects of some factors were examined in detail on the asymmetric carbonyl reduction of ethyl 4-chloroacetoacetate with baker's yeast(Type II ,Sigma) in organic media, including the selection of organic solvents, the immobilization conditions, the reaction temperature and the substrate concentration. Ethyl 4-chloroacetoacetate was reduced with baker's yeast to obtain ethyl (R)-4-chloro-3-hydroxybutyrate (ee=58%) when dibutyl phthalate was used as reaction media.
Meishan yeast which showed higher stereoselectivity was used to catalyze the reduction of ethyl 4-chloroacetoacetate in dibutyl phthalate. As a result, (R)-4-chloro-3-hydroxybutyrate with higher ee value(ee=76%) was produced. The effects of the buffer used and its pH and the reaction temperature on the bioreductions with Meishan yeast or baker's yeast(Type II) were different.
When Na3PO4, NaF and malonic acid was added to the reaction system respectively, the reduction of ethyl 4-chloroacetoacetat with baker's yeast(Type II) was inhibited and the yield decreased notably. This reflected that BMP and HMP pathways and the tricarboxylic acid cycle(TCA cycle) were very important to the bioreduction.
Finally, the reductions of l-phenyl-l,2-propanedione and benzaldehyde with baker's yeast in organic media were investigated, too. The yield and chemical selectivity were influenced by organic solvents, temperature and pH of the buffer used for cell immobilization.
引文
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55. Jayasinghe L Y, Smallridge A J, Trewhella M A. The yeast mediated reduction of ethyl acetoacetate in petroleum ether. Tetrahedron Lett., 1993, 34(24): 3949～3950
56. Griffin D R., Yang F X, Carta G, Gainer J L. Asymmetric reduction of acetophenone with calcium-alginate-entrapped baker's yeast in organic solvent. Biotechnol. Prog., 1998, 14:588～593
57．胡忠策，郑晓冬．固定化细胞在有机相中的催化反应．生物技术，1999，9(3)：39～41
58. Medson C, Smallridge A J, Trewhella M A. Baker's yeast activity in an organic solvent system. J. Mol. Cat. B: Enzymatic, 2001, 11:897～903
59. Naoshima Y, Nakamura A, Nishiyama T, Haramaki T, Mende M, Munakata Y. Asymmetric bioreduction of ethyl 3-oxobutanoate by immobilized Baker's yeast entrapped in calcium alginate beads. Application of the immobilized biocatalyst to the synthesis of (5Z,13S)-5-tetradecen-13-olide,aggregation pheromone of cryptolestes grain beetle. Chem. Lett., 1989:1023～1026
60. Naoshima Y, Hasegawa H, Nishiyama T, Nakamura A. Synthesis of both the enantiomers of phoracantholideⅠ,a defensive secretion of the eucarypt longicorn(phoracantha synónyma),
employing microbial asymmetric reduction with immobilized Baker's yeast. Bull. Chem. Soc. Jpn., 1989, 62:608～610
61. Naoshima Y, Maeda J, Munakata Y. Bioreduction with immobilized baker's yeast in hexane using alcohols as an energy source. J. Chem. Soc., Chem. Commun., 1990, 14:964～965
62. Haag T, Arslan T, Seebach D. Preparative β-ketoester reductions and ester hydrolyses by yeast, using free cells in organic media. Chimia, 1989, 43(11): 351～353
63. Nakamura K, Kondo S, Nakajima N, Ohno A. Mechanistic study for stereochemical control of microbial reduction of α-keto esters in an organic solvent. Tetrahedron, 1995, 51(3): 687～694
64. Naoshima Y, Maeda J, Munakata Y. Control of the enantioselectivity of the bioreduction with immobilized baker's yeast in a hexane solvent system. J. Chem. Soc., Perkin Trans. I,1992, 6: 659～660
65. Dao D H, Okamura M, Akasaka T, Kawai Y, Hida K, Ohno A. Stereochemical control in microbial reduction. Part 31:Reduction of alkyl 2-oxo-4-arylbutyrates by baker's yeast under selected reaction conditions. Tetrahedron:Asymmetry, 1998, 9(15): 2725～2737
66. Zaks A, Klibanov A M. Enzymatic catalysis in organic media at 100℃. Science, 1984, 224: 1249～1251
67. Kirchner G, Scollar M P, Klibanov A M. Resolution of racemic mixtures via lipase catalysis in organic solvents,J. Am. Chem. Soc., 1985, 107:7072～7076
68. Kazandlian R Z, Dordick J S, Klibanov A M. Enzymatic analyses in organic solvents. Biotechnol. Bioeng., 1986, 28:417～421
69．邱树毅，姚汝华．有机溶剂中的酶催化——水和有机溶剂的影响，工业微生物，1996，26(3)：40～45
70．黄中祥．有机溶剂中的酶催化反应，生命的化学，1987，7：18～20
71. Zaks A, Klibanov A M. Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. U.S.A, 1985, 82(10): 3192～3196
72. Vermüe M, Sikkema J, Verheul A, Bakker R, Tramper J. Toxicity of homologous series of organic solvents for the Gram-positive bacteria Arthrobacter and Nocardia sp. and the Gram-negative bacteria Acinetobacter and Pseudomonas sp. Biotechnol. Bioeng., 1993, 42:747～758
73. Brink L E S, Tramper J. Optimization of organic solvent in multiphase biocatalysis. Biotechnol Bioeng., 1985, 27:1258～1269
74. Laane C, Boeren S, Vos K, Veeger C. Rules for the optimization of biocatalysis in organic solvents. Biotechnol. Bioeng., 1987, 30:81～87
75. Bruce L J, Daugulis A J. Solvent selection strategies for extractive biocatalysis. Biotechnol. Prog., 1991,7:116～124
76. Inoue A, Horikoshi K. A pseudomonas putida thrives in high concentration of toluene. Nature, 1989, 338:264～266
77. Bassetti L, Tramper J. Organic solvent toxicity in Morinda citrifolia cell suspensions. Enzyme Microb. Technol., 1994, 16:642～648
78. 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(organics-aqueous) medium. Appl. Environ. Microb., 1992, 58:2723～2729
79. Le6n R, Garbayo I, Hernádez R, Vigara J, Vilchez C. Organic solvent toxicity in photoautotrophic unicellular microorganisms. Enzyme Microb. Technol., 2001, 29:173～180
80.Rajagopal A N. Growth of Gram-negative bacteria in the presence of organic solvents. Enzyme Microb. Technol., 1996, 19 (8): 606～613
81.Ogino H, Miyamoto K, Yasuda M, Ishimi K, Ishikawa H. Growth of organic solvent-tolerant Pseudomonas aeruginosa LST-03 in the presence of various organic solvents and production of lipolytic enzyme in the presence of cyclohexane. Biochemical Engineering Journal, 1999, 4:1～6
82.Inoue A, Horikoshi K. Estimation of solvent tolerance of bacteria by solvent parameter logP. J. Ferm. Bioeng., 1991, 3:194～196
83.Sikkema J, Weber F J, Heipieper H J, de Bont J A M. Cellular toxicity of lipophilic compounds: Mechanisms, implications, and adaptations. Biocatalysis, 1994, 10:113～122
84.Bar R. Effect of interphase mixing on a water organic solvent two-phase microbial system: Ethanol fermentation. J. Chem. Tech. Biotechnol., 1988, 43: 49～62
85.Bassetti L, Hagendoorn M, Tramper J. Surfactant-induced nonlethal release of anthraquinones from suspension cultures of Morinda citrifolia, J. Biotechnol., 1995, 39(2): 149～155
86.Osborne S J, Leaver J, Turner M K. Solvent selection for whole cell biotransformation in organic media. In: Biocatalysis in Non-Conventional Media(Tramper J, Vermtüe M H, Beeftink H H, Von Stockar U Eds.), Elsevier Science, Amsterdam, 1992:31～36
87.Osborne S J, Leaver J, Turner M K, Dunnill P. Correlation of biocatalytic activity in an organic-aqueous two-liquid phase system with solvent concentration in cell membrane. Enzyme Microb. Technol., 1990, 12:281～291
88.Heipieper H J, de Bont J A M. Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl. Environ. Microb.,1994, 60:4440～4444
89.Weber F J, Ooijkaas L P, Schemen R M W, Hartmans S, de Bont J A M. Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents. Appl. Environ. Microb., 1993, 59:3502～3504
90.Heipieper H J, Diefenbach R, Keweloh H. Conversion of cis unsaturated fatty acids to trans, a possible mechamism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl. Environ. Microb., 1992, 58:1847～1852
91.Weber F J, Isken S, de Bont J A M. Cis/trans isomerization of fatty acids as a defense mechanism of Pseudomonas putida strains to toxic concentration of toluene. Microbiology, 1994, 140: 2013～2017
92.Keweloh H, Weyracuch G, Rehm H J. Phenol-induces membrane changes in free and immobilized Escherichia coli. Appl. Microbiol. Biotechnol., 1990, 33:66～71
93.Heipieper H J, Weber F J, Sikkema J, Keweloh H, de Bont J A M. Mechanisms of resistance of whole cells to toxic organic solvents. Trends Biotechnol., 1994, 12:409～415
94.Ramos J L, Duque E, Rodriguez-Herva J-J, Godoy P, Haidour A, Reyes F, Fernández-Barrero A. Mechanisms for solvent tolerance in Bacteria. J. Biol. Chem., 1997, 272:3887～3890
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47. Nikolova P, Ward O P. Biotransformation of benzaldehyde to benzyl alcohol by whole cells and cell extracts of baker's yeast in two-phase systems. Prog. Biotechnol., 1992, 8(Biocatalysis in Non-Conventional Media): 667～673
48. Naoshima Y, Nishiyama T, Munakata Y. Bioreduction of Ethyl 3-oxobutanoate with immobilized baker's yeast in organic-water solvent systems. Chem. Lett., 1989:1517～1518
49. Naoshima Y, Munakata Y, Nishiyama T, Maeda J, Kamezawa M, Haramaki T, Tachibana H. Asymmetric bioreduction of keto esters by immobilized baker's yeast entrapped in calcium alginate beads in both water and organic/water solvent systems. World J. Microbiol. Biotechnol., 1991, 7(2): 219～224
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52. Nakamura K, Inoue K, Ushio K, Oka S, Ohno A. Stereochemical control on yeast reduction of α -keto esters. Reduction by immobilized baker's yeast in hexane, J. Org. Chem., 1988, 53(11): 2589～2593
53. Rupley J A ,Careri G. Protein hydration and function. Adv. Protein Chem., 1991, 41:37～172
54. Affieck R, Haynes C A, Clark D S. Solvent dielectric effects on protein dynamics. Proc. Natl. Acad. Sci. U.S.A, 1992, 89(11): 5167～5170
55. Jayasinghe L Y, Smallridge A J, Trewhella M A. The yeast mediated reduction of ethyl acetoacetate in petroleum ether. Tetrahedron Lett., 1993, 34(24): 3949～3950
56. Griffin D R., Yang F X, Carta G, Gainer J L. Asymmetric reduction of acetophenone with calcium-alginate-entrapped baker's yeast in organic solvent. Biotechnol. Prog., 1998, 14:588～593
57．胡忠策，郑晓冬．固定化细胞在有机相中的催化反应．生物技术，1999，9(3)：39～41
58. Medson C, Smallridge A J, Trewhella M A. Baker's yeast activity in an organic solvent system. J. Mol. Cat. B: Enzymatic, 2001, 11:897～903
59. Naoshima Y, Nakamura A, Nishiyama T, Haramaki T, Mende M, Munakata Y. Asymmetric bioreduction of ethyl 3-oxobutanoate by immobilized Baker's yeast entrapped in calcium alginate beads. Application of the immobilized biocatalyst to the synthesis of (5Z,13S)-5-tetradecen-13-olide,aggregation pheromone of cryptolestes grain beetle. Chem. Lett., 1989:1023～1026
60. Naoshima Y, Hasegawa H, Nishiyama T, Nakamura A. Synthesis of both the enantiomers of phoracantholideⅠ,a defensive secretion of the eucarypt longicorn(phoracantha synónyma),
employing microbial asymmetric reduction with immobilized Baker's yeast. Bull. Chem. Soc. Jpn., 1989, 62:608～610
61. Naoshima Y, Maeda J, Munakata Y. Bioreduction with immobilized baker's yeast in hexane using alcohols as an energy source. J. Chem. Soc., Chem. Commun., 1990, 14:964～965
62. Haag T, Arslan T, Seebach D. Preparative β-ketoester reductions and ester hydrolyses by yeast, using free cells in organic media. Chimia, 1989, 43(11): 351～353
63. Nakamura K, Kondo S, Nakajima N, Ohno A. Mechanistic study for stereochemical control of microbial reduction of α-keto esters in an organic solvent. Tetrahedron, 1995, 51(3): 687～694
64. Naoshima Y, Maeda J, Munakata Y. Control of the enantioselectivity of the bioreduction with immobilized baker's yeast in a hexane solvent system. J. Chem. Soc., Perkin Trans. I,1992, 6: 659～660
65. Dao D H, Okamura M, Akasaka T, Kawai Y, Hida K, Ohno A. Stereochemical control in microbial reduction. Part 31:Reduction of alkyl 2-oxo-4-arylbutyrates by baker's yeast under selected reaction conditions. Tetrahedron:Asymmetry, 1998, 9(15): 2725～2737
66. Zaks A, Klibanov A M. Enzymatic catalysis in organic media at 100℃. Science, 1984, 224: 1249～1251
67. Kirchner G, Scollar M P, Klibanov A M. Resolution of racemic mixtures via lipase catalysis in organic solvents,J. Am. Chem. Soc., 1985, 107:7072～7076
68. Kazandlian R Z, Dordick J S, Klibanov A M. Enzymatic analyses in organic solvents. Biotechnol. Bioeng., 1986, 28:417～421
69．邱树毅，姚汝华．有机溶剂中的酶催化——水和有机溶剂的影响，工业微生物，1996，26(3)：40～45
70．黄中祥．有机溶剂中的酶催化反应，生命的化学，1987，7：18～20
71. Zaks A, Klibanov A M. Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. U.S.A, 1985, 82(10): 3192～3196
72. Vermüe M, Sikkema J, Verheul A, Bakker R, Tramper J. Toxicity of homologous series of organic solvents for the Gram-positive bacteria Arthrobacter and Nocardia sp. and the Gram-negative bacteria Acinetobacter and Pseudomonas sp. Biotechnol. Bioeng., 1993, 42:747～758
73. Brink L E S, Tramper J. Optimization of organic solvent in multiphase biocatalysis. Biotechnol Bioeng., 1985, 27:1258～1269
74. Laane C, Boeren S, Vos K, Veeger C. Rules for the optimization of biocatalysis in organic solvents. Biotechnol. Bioeng., 1987, 30:81～87
75. Bruce L J, Daugulis A J. Solvent selection strategies for extractive biocatalysis. Biotechnol. Prog., 1991,7:116～124
76. Inoue A, Horikoshi K. A pseudomonas putida thrives in high concentration of toluene. Nature, 1989, 338:264～266
77. Bassetti L, Tramper J. Organic solvent toxicity in Morinda citrifolia cell suspensions. Enzyme Microb. Technol., 1994, 16:642～648
78. 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(organics-aqueous) medium. Appl. Environ. Microb., 1992, 58:2723～2729
79. Le6n R, Garbayo I, Hernádez R, Vigara J, Vilchez C. Organic solvent toxicity in photoautotrophic unicellular microorganisms. Enzyme Microb. Technol., 2001, 29:173～180
80.Rajagopal A N. Growth of Gram-negative bacteria in the presence of organic solvents. Enzyme Microb. Technol., 1996, 19 (8): 606～613
81.Ogino H, Miyamoto K, Yasuda M, Ishimi K, Ishikawa H. Growth of organic solvent-tolerant Pseudomonas aeruginosa LST-03 in the presence of various organic solvents and production of lipolytic enzyme in the presence of cyclohexane. Biochemical Engineering Journal, 1999, 4:1～6
82.Inoue A, Horikoshi K. Estimation of solvent tolerance of bacteria by solvent parameter logP. J. Ferm. Bioeng., 1991, 3:194～196
83.Sikkema J, Weber F J, Heipieper H J, de Bont J A M. Cellular toxicity of lipophilic compounds: Mechanisms, implications, and adaptations. Biocatalysis, 1994, 10:113～122
84.Bar R. Effect of interphase mixing on a water organic solvent two-phase microbial system: Ethanol fermentation. J. Chem. Tech. Biotechnol., 1988, 43: 49～62
85.Bassetti L, Hagendoorn M, Tramper J. Surfactant-induced nonlethal release of anthraquinones from suspension cultures of Morinda citrifolia, J. Biotechnol., 1995, 39(2): 149～155
86.Osborne S J, Leaver J, Turner M K. Solvent selection for whole cell biotransformation in organic media. In: Biocatalysis in Non-Conventional Media(Tramper J, Vermtüe M H, Beeftink H H, Von Stockar U Eds.), Elsevier Science, Amsterdam, 1992:31～36
87.Osborne S J, Leaver J, Turner M K, Dunnill P. Correlation of biocatalytic activity in an organic-aqueous two-liquid phase system with solvent concentration in cell membrane. Enzyme Microb. Technol., 1990, 12:281～291
88.Heipieper H J, de Bont J A M. Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl. Environ. Microb.,1994, 60:4440～4444
89.Weber F J, Ooijkaas L P, Schemen R M W, Hartmans S, de Bont J A M. Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents. Appl. Environ. Microb., 1993, 59:3502～3504
90.Heipieper H J, Diefenbach R, Keweloh H. Conversion of cis unsaturated fatty acids to trans, a possible mechamism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl. Environ. Microb., 1992, 58:1847～1852
91.Weber F J, Isken S, de Bont J A M. Cis/trans isomerization of fatty acids as a defense mechanism of Pseudomonas putida strains to toxic concentration of toluene. Microbiology, 1994, 140: 2013～2017
92.Keweloh H, Weyracuch G, Rehm H J. Phenol-induces membrane changes in free and immobilized Escherichia coli. Appl. Microbiol. Biotechnol., 1990, 33:66～71
93.Heipieper H J, Weber F J, Sikkema J, Keweloh H, de Bont J A M. Mechanisms of resistance of whole cells to toxic organic solvents. Trends Biotechnol., 1994, 12:409～415
94.Ramos J L, Duque E, Rodriguez-Herva J-J, Godoy P, Haidour A, Reyes F, Fernández-Barrero A. Mechanisms for solvent tolerance in Bacteria. J. Biol. Chem., 1997, 272:3887～3890