离子液体—水两相体系中固定化细胞选择性还原2’-氯苯乙酮的研究
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摘要
离子液体是指在室温下完全由离子组成的有机液体物质,因其具有无毒、无蒸气压、无可燃性、热稳定性高、可取代挥发性有机溶剂、可循环使用等独特性质而被用于分离工程、化学反应、生物催化等过程。离子液体符合绿色化学的原则,因而被称为绿色溶剂。
手性化合物由于其独特的光学活性,广泛应用于医药、食品、化工、农药等领域,随着对不同光学异构体药物药理作用的研究进展和对外消旋药物申报和使用的种种限制,手性药物成为近年来研究的热点和方向。光学纯醇类化合物是手性药物合成的重要中间体。利用微生物或其细胞内的氧化还原酶催化潜手性酮的不对称还原已成为制备高光学纯度手性醇的重要方法。本文以2′-氯-苯乙酮作为模型底物,研究了离子液体/水两相体系中酵母全细胞催化芳香酮类化合物不对称还原反应的特性,并通过条件优化提高了反应效率,强化了反应过程。
对各不同反应体系进行了比较,结果表明离子液体[BMIM]PF_6/水两相反应体系相比于有机溶剂/水两相体系和纯水相体系更有利于酵母细胞的还原反应,尤其是在较高的底物浓度下。当底物浓度高达64.72 mmol/L(10g·L~(-1))时,用游离酵母细胞作为生物催化剂,在最佳条件下,转化率可达到54.5%,是纯水相体系中反应转化率的300%,产物e.e.值为98.6%左右。
将酵母细胞用海藻酸钙固定化后催化能力有所提高。在此基础上对固定化酵母在离子液体/水两相体系中的反应条件进行了优化,确定较佳条件为:固定化细胞浓度360g/L,离子液体/水两相比率1:15,缓冲液pH7.2,摇床转速200rpm,反应时间45h,反应温度30℃,反应过程中分两次各添加10%的葡萄糖。在最优条件下,64.72 mmol/L的底物浓度时反应的最大转化率达到75.4%,明显提高了反应效率。产物的e.e.值保持在99.1%左右。
Ionic liquids are salts consisting of positively charged cations and negatively charged anions.They are liquids below 100℃.Depending upon their nontoxicity,low volatility,nonflammability,high thermal stability,recycle using and the substituent for other volatile organic solvents,they are widely used in processes of separation, chemical reaction and biocatalysis.For their specific properties.Ionic liquids are called "green solvent".
Chiral compounds have been widely applied in pharmaceuticals,food,pesticide and chemical industry due to their special optical activity.With the progress in pharmacology of chiral pharmaceuticals and the strict regulation on the application of new racemic drug in recent years,the research and development of chiral pharmaceuticals have become the new focus and direction.Enantiomerically pure alcohols are important intermediates for synthesis of pharmaceuticals.Asymmetric reduction of pro-chiral ketones catalyzed by microorganism or the oxidoreductase in the cell has been an important method of synthesis enantiomerically pure alcohols.In this work,using 2'-chloroacetophenone as the mode substrate,the reaction characteristics of the reduction of aromatic ketone by whole cell of yeast in Ionic liquid/aqueous biphasic system were studied.The conversion was improved by optimizing reaction conditions.
By the comparation of different system,it was proved that the ionic liquid [BMIM]PF_6/aqueous biphasic system is more suitable for whole cell of yeast's reduction than other organic solvent/aqueous biphasic system and pure aqueous phase system,especially when the substrate concentration was high.Under the substrate concentration of 64.72 mmol/L(10g·L~(-1))and catalyzed by free cells,the conversion was 54.5%after optimizing,nearly 300%as that in the pure water system.The production e.e.value was 98.6%.
After being immobilized by Sodium alginate-CaCl_2,the yeast cell's catalysis activity was increased.Based on this conclusion,the reaction conditions were optimized.The optimal conditions were:Immobilized cell's content of 360 g/L; V_(I.L)/V_(aq)of 1:15;buffer pH of 7.2;shaker frequency of 200rpm;reaction time of 45h; reaction temperature of 30℃;adding glucose of 10%twice.Under these conditions, the maximum conversion was 75.4%with the substrate concentration of 64.72 mmol/L.The production e.e.value was around 99.1%.
手性化合物由于其独特的光学活性,广泛应用于医药、食品、化工、农药等领域,随着对不同光学异构体药物药理作用的研究进展和对外消旋药物申报和使用的种种限制,手性药物成为近年来研究的热点和方向。光学纯醇类化合物是手性药物合成的重要中间体。利用微生物或其细胞内的氧化还原酶催化潜手性酮的不对称还原已成为制备高光学纯度手性醇的重要方法。本文以2′-氯-苯乙酮作为模型底物,研究了离子液体/水两相体系中酵母全细胞催化芳香酮类化合物不对称还原反应的特性,并通过条件优化提高了反应效率,强化了反应过程。
对各不同反应体系进行了比较,结果表明离子液体[BMIM]PF_6/水两相反应体系相比于有机溶剂/水两相体系和纯水相体系更有利于酵母细胞的还原反应,尤其是在较高的底物浓度下。当底物浓度高达64.72 mmol/L(10g·L~(-1))时,用游离酵母细胞作为生物催化剂,在最佳条件下,转化率可达到54.5%,是纯水相体系中反应转化率的300%,产物e.e.值为98.6%左右。
将酵母细胞用海藻酸钙固定化后催化能力有所提高。在此基础上对固定化酵母在离子液体/水两相体系中的反应条件进行了优化,确定较佳条件为:固定化细胞浓度360g/L,离子液体/水两相比率1:15,缓冲液pH7.2,摇床转速200rpm,反应时间45h,反应温度30℃,反应过程中分两次各添加10%的葡萄糖。在最优条件下,64.72 mmol/L的底物浓度时反应的最大转化率达到75.4%,明显提高了反应效率。产物的e.e.值保持在99.1%左右。
Ionic liquids are salts consisting of positively charged cations and negatively charged anions.They are liquids below 100℃.Depending upon their nontoxicity,low volatility,nonflammability,high thermal stability,recycle using and the substituent for other volatile organic solvents,they are widely used in processes of separation, chemical reaction and biocatalysis.For their specific properties.Ionic liquids are called "green solvent".
Chiral compounds have been widely applied in pharmaceuticals,food,pesticide and chemical industry due to their special optical activity.With the progress in pharmacology of chiral pharmaceuticals and the strict regulation on the application of new racemic drug in recent years,the research and development of chiral pharmaceuticals have become the new focus and direction.Enantiomerically pure alcohols are important intermediates for synthesis of pharmaceuticals.Asymmetric reduction of pro-chiral ketones catalyzed by microorganism or the oxidoreductase in the cell has been an important method of synthesis enantiomerically pure alcohols.In this work,using 2'-chloroacetophenone as the mode substrate,the reaction characteristics of the reduction of aromatic ketone by whole cell of yeast in Ionic liquid/aqueous biphasic system were studied.The conversion was improved by optimizing reaction conditions.
By the comparation of different system,it was proved that the ionic liquid [BMIM]PF_6/aqueous biphasic system is more suitable for whole cell of yeast's reduction than other organic solvent/aqueous biphasic system and pure aqueous phase system,especially when the substrate concentration was high.Under the substrate concentration of 64.72 mmol/L(10g·L~(-1))and catalyzed by free cells,the conversion was 54.5%after optimizing,nearly 300%as that in the pure water system.The production e.e.value was 98.6%.
After being immobilized by Sodium alginate-CaCl_2,the yeast cell's catalysis activity was increased.Based on this conclusion,the reaction conditions were optimized.The optimal conditions were:Immobilized cell's content of 360 g/L; V_(I.L)/V_(aq)of 1:15;buffer pH of 7.2;shaker frequency of 200rpm;reaction time of 45h; reaction temperature of 30℃;adding glucose of 10%twice.Under these conditions, the maximum conversion was 75.4%with the substrate concentration of 64.72 mmol/L.The production e.e.value was around 99.1%.
引文
[1]Seddon K R.Ionic liquids for clean technology[J].Chem.Tech.Biotech,1997,68:351-356.
[2]Welton T.Room-Temperature Ionic Liquids:solvents for synthesis and catalysis[J].Chem Rev.,1999,99(8):2071-2084.
[3]Koch V R,Miller L L,Osteryoung R A.Electroinitiated friedel-crafts transalkylations in a Room Temperature Molten-Salt medium[J].J.Ame.Chem.Soc.,1976,98(18):5 277-5 281.
[4]Wilkes J S,Zaworotko M J.Air and water stable l-ethyl-3-methylimidazolium based ionic liquids[J].J.Chem.Soc.Chem.Commun.,1992,(13):965-967.
[5]李汝雄,王建基.化工进展,2002,21(1),43.
[6]王明慧,吴坚平,杨立荣.有机化学,2005,25(4),364.
[7]Anthony J L,Maginn E J,Brennecke J F.Solution thermodynamics of imidazolium-based ionic liquids and water[J].J.Phys.Chem.B,2001,105(44):10 942-10 949.
[8]Ellis B,Keim W,Wassercheid P.Linear dimerization of But-l-ene in biphasic mode using buffered chloro-aluminate ionic liquid solvents[J].Chem.Commun.,1999,377-381.
[9]F.van Rantwijk,R.M.Lau,R.A.Sheldon,Trends in Biotechnology,2003,21,131-138.
[10]U.Kragl,M.Eckstein,N.Kaftzik,Current Opinion in Biotechnology,2002,13,565-571.
[11]Magnusson D.K.,Bodey J.and Adams D.F.,J.Sol.Chem.,1984,13(2),583-587.
[12]Cull S.G.,et al.,J.Biotechnol.Bioeng.,2000,69(2),227-229.
[13]Erbeldinger M.,Mesiano A.J.,Biotechnol.Progr.,2000,16,1 129-1 131.
[14]Lozano P.,et al.,Biotechnol.Bioeng.,2001,75,563-569.
[15]Eckstein M.,Sesing M.,Kragl U.,Adlercreutz P.,Biotechnol.Lett.,2002,24,867-872.
[16]Laszlo J.A.,Compton D.L.,Biotechnol.Bioeng.,2001,75,181-186.
[17]Zhao H.,Malhotra S.V.,Biotechnol.Lett.,2002,24,1 257-1 260.
[18]Park S.,Kazlanskas R.J.,Org.Chem.,2001,66(25),8 395-8 401.
[19]Schofer S.H.,Kaftzik N.,Wasserscheid P.,Kragl U.,Chem.Commun.,2001,425-426.
[20]Itoh T.,Akasak E.,Kudo K.,Shirakami S.,Chem.Lett..,2001,262-263.
[21]Nara S.J.,Mohile S.S.,Harjani J.R.,Naik P.U.,Mol.Catal.B,2004,28,39-43.
[22]Lau R.M.,et al.,Org.Lett.,2000,26(2),4 189-4 191.
[23]Park S.,Viklund F.,Hult K.,Kazlauskas R.J.,Green Chem.,2003,5,715-719.
[24]Hinckley G.,Mozhaev V.,Budde C.,Biotechnol.Lett.,2002,24,2 083.
[25]Okrasa K.,et al.,Tetrahedron Asymmetry,2003,14,2 487-2 490.
[76]Howarth J,James P,Dai J.Immobilized baker's yeast reduction of ketones in an ionic liquid,[bmim]PF6 and water mix.Tetrahedron Lett.2001,42:7 517-7 519.
[27]Wen-Yong Lou,Min-Hua Zong,Thomas J.Smith.Use of ionic liquids to improve whole-cell biocatalytic asymmetric reduction of acetyltrimethylsilane for efficient synthesis of enantiopure (S)- 1-trimethylsilylethanol.Green Chem.,2006,8,147-155.
[28]Holger Pfruender,Ross Jones,Dirk Weuster-Botz.Water immiscible ionic liquids as solvents for whole cell biocatalysis.Journal of Biotechnology,2006(124):182-190.
[29]Beroza P,Suto M.I.Designing chiral libraries of drug discovery.DDV.2000,5(8):364.
[30]Oti-Amoako K,Vozeh S,Ha H R.The relative potency of major metabolites and enantiomers of propafenone in an experimental reperfusion arrhythmia mode.J Cardiovasc Pharm.1990,15(1):75-81.
[31]Agranat I,Caner H,Caldwell J.Putting chirality to work;the strategy of chiral switches.Nat Rev Drug Discov.2002,1(10):753-768.
[32]芮建中,吴锦芳,庞晓东.手性药物对映体选择性的药代动力学和药效学与临床合理用药.中国药理学通报,1998,14(1):14-18.
[33]Sanchez C,Bogeso K P,Ebert B.Escitalopram versus citalopram;the surprising role of the R-enantiomer.Psychopharmacology.2004,174(2):163-176.
[34]Strong M.FDA policy and regulation of stereojsomers;paradigm shift and the future of safer,more effective drugs.Food Drug Law J.1999,54(3):463-487.
[35]Rentsch KM.The importance of stereoseclecetive determination of drugs in the clinical laboratory.Biochem Biophys Methods.2002,54:1.
[36]舒之罡.制药领域是手性化合物的最大市场.医药中间体及其化工原料,2004,2:23-26.
[37]制药原料及中间体信息.2006,11:20-23.
[38]Cushman D W,Cheung M S,Sabo E F,Ondetti M A.Biochemistry.1977,16:5 484-5 491.
[39]Patel R N,Banerjee A,McNamee C G,Syarka L J.Appl Micrebiol Biotechnol.1993,40:241-245.
[40]Wada M,Kataoka M,Kawabata H.Purification and Characterization of NADPH-Dependent Carbonyl Reductase,Involved in Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate,from Candida magnoliae.Biosci Biotechnol Biochem.1998,62(2):280-285.
[41]Wei ZL,Li ZY,Lin GQ.Anti-Prelog microbial reduction of aryl α-halomethyl or α-hydroxymethyl ketones with Geotrichum sp.38.Tetrahedron.1998,54:13 059- 13 072.
[42]Srebrik M,Ramachandran P V,Brown H C.A Highly Enantioselective Synthesis of Both Optical Isomers of Tomoxetine,Fluoxetine,and Nisoxetine.J Org Chem.1988,53:2 916-2 920.
[43]Fronza G,Fuganti C,Grasselli P,Mele A.On the mode of bakers' yeast transformation of 3-chloropropiophenone and related ketones.Synthesis of(2S)-[2-2H]propiophenone,(R)-Fluoxetine,and(R)- and(S)-Fenfluramine.J Org Chem.1991,56:6 019-6 023.
[44]Corey E J,Reichard G A.Enantioselective and practical syntheses of R- and S-fluoxetines. Tetrahedron Lett.1989,30(39):5 207-5 210.
[45]Shimizu S,Hattori S,Hata H,Yamada H.Stereoselective enzymatic oxidation and reduction system for the production of D(-)-pantoyl lactone from a racemic mixture of pantoyl lactone.Enzyme Microb Technol.1987,9(7):411-416.
[46]Shimizu S,Hattori S,Hata H,Yamada H.One-step microbial conversion of a racemic mixture of pantoyl lactone to optically active D-(-)-pantoyi lactone.Appl Environ Microb.1987,3:519-522.
[47]Glanzer B I,Faber K,Griengi H.Microbial resolution of O-acetylpantoyl lactone.Enzyme Microb Technol.1988,10(11):689-690.
[48]王明慧,吴坚平,杨立荣,陈新志.相转移催化法合成2-氯-3-氯甲基-噻吩及噻康唑.应用化学,2006,23(1):106-108.
[49]黄蓓,杨立荣,吴坚平.手性拆分技术的工业应用,化工进展,2002,21(6):375-380.
[50]Nozaki H,Monut S,Noyori R.Asymmetric induction in carbeniod reaction by means of a dissymmetric copper chelate.Tetrahedron Lett.1966,7:5 239-5 244.
[51]Ohkuma T,Ooka H,Hashiguchi S.Practical enantioselective hydrogenation of aromatic ketones.J Am Chem Soc.1995,117(9):2 675-2 676.
[52]Noyori R,Tomino I,Tanimoto Y.Highly enantioselective reduction of aromatic ketones by binaphthol-modified lithium aluminum hydride reagents.J Am Chem Soc.1984,106(22):6 709-6716.
[53]廖永卫,陈卫平.手性唑硼烷催化酮的不对称还原反应.中国药物化学杂志,1994,4(3),202-208.
[54]Wei Z L,Li Z Y,Lin G Q.anti-Prelog Microbial Reduction of Aryl α -Halomethyl or α-Hydroxymethyl Ketones with Geotrichum sp.38.Tetrahedron.1998,54:13 059-13 072.
[55]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):2 725-2 737.
[56]Forni A,Moretti I,Pratt F,Torre G.Microbial synthesis of(+)and(-)methyl 4-chloro-4,4-difluoro-3-hydroxybutanoate.J Mol Catal A Chem.1996,107:405-407.
[57]Yasohara Y.,.Kizaki N,Hasegawa J,Takahashi S,Wada M,Kataoka M,Shimizu S.Synthesis of optically active ethyl 4-chloro-3-hydroxybutanoate by microbial reduction.Appl Microbiol Biotechnol.1999,51:847-851.
[58]杨忠华.水相中酵母细胞催化前手性羰基不对称还原合成手性醇的研究.浙江大学博士学位论文,2005,1.
[59]王盛,制备重要他汀类药物中间体的研究.浙江大学硕士学位论文,2006,6.
[60]Stewart J D.Organic transformations catalyzed by engineered yeast cells and related systems.Curr Opin Biotech.2000,11:363-368.
[61]Yamamoto H,Kawada N,Matsuyama A,Kobayashi Y.Cloning and expression in Escherichia coli of a gene coding for a secondary alcohol dehydrogenase from Candida parapsilosis.Biosci Biotechnol Biochem.1999,63(6):1 051-1 055.
[62]Yasohara Y,Kizaki N,Hasegawa J,Wada M,Kataoka M,Shimizu S.Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae,involved in tereoselective reduction of ethyl 4-chloro-3-oxobutanoate.Biosci Biotechnol Biochem.2000,64(7):1 430-1 436.
[63]Rodringuez S,Kayser M M,Stewart J D.Improving the stereoselectivity of baker's yeast reductions by genetic engineering.Org Lett.1999,1(8):1 153-1 155.
[64]Sikkema J,Weber F J,Heipieper H J,Debont J.Cellular toxicity of lipophilic compounds:mechanisms,implications,and adaptations.Biocatalysis.1994,10:113-122.
[65]Inoue A,Horikoshi K.Estimation of solvent tolerance of bacteria by solvent parameter logP.J Ferment Bileng.1991,3:194-196.
[66]Vermun 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.1993,42:745-758.
[67]Qun J,Yao S J,Mei L.Tolerance of immobilize baker's yeast in organic solvents.Enzyme Micro.Tech.2002,30:721-725.
[68]Medson C,Smallridge A J,Trewhella M A.Baker's yeast activity in an organic solvent system.J mol Catal B Enzym.2001,11:897-903.
[69]Nakamura K,Kondo S,Nakajima N,Ohno Atsuyoshi.Mechanistic study for stereochemical control of microbial reduction of α-keto esters in an organic solvent.Tetrahedron.1995,51:687-694.
[70]何军邀,孙志浩,韩丽,钱嘉南.水/有机溶剂两相体系微生物催化不对称还原制备(S)-4-氯-3-羟基丁酸乙酯.高校化学工程学报,2007,21(2):310-315.
[71]He J Y,Sun Z H,Ruan W Q,Xu Y.Biocatalytic synthesis of ethyl (S)-4-chloro-3-hydroxy-butanoate in an aqueous-organic solvent biphasic system using Aureobasidium pullulans CGMCC 1244.Process Biochem.2006,41:244-249.
[72]Anderson B A,Hansen M M,Harkness A R,Henry C L,Vicenzi J T,Zmijewski M J.Application of a Practical Biocatalytic Reduction to an Enantioselective Synthesis of the 5H-2,3-Benzodiazepine LY300164.J Am Chem Soc.1995,117(49):12 358-12 359.
[73]Vicenzi J T,Zmijewski M J,Reinhard M R,Landen B E,Muth W L,Marler P G.Large-Scale Stereoselective Enzymatic Ketone Reduction with in situ Product Removal via Polymeric Adsorbent Resins.Enzyme Microb Technol.,1997,20(7):494-499.
[74]欧志敏.微生物法还原羰基化合物制备手性中间体的研究,浙江大学博士学位论文,2003,4.
[75]王新,李培军,巩宗强,张海荣.固定化细胞技术的研究与进展.农业环境保护.2001,20(2): 120-122.
[76]Hasegawa Y,Adachi S,Matsuno R.Effect of the molar ratio of an energy source to the substrate on yeast-mediated production of 2-chloro-α-methylbenzyl alcohol.J Biosci Bioeng.2000,89(4):329-333.
[77]Milagre H,Milagre C,Moran P,Santana M H,Rodrigues J.Reduction of ethyl benzoylformate mediated by Saccharomyces cerevisiae entrapped in alginated fibers with double gel layers in a continuously operated reactor.Enzyme Microb Tech.2005,37:121-125.
[78]Wendhausen R,Moran P,Joekes I,Rodrigues A.Coutinuous process for large-scale preparation of chiral alcohols with baker's yeast immobilized on chrysotile fibers.J Mol Catal B Enzym.1998,5:69-73.
[79]王洪祚,刘世勇.酶和细胞的固定化[J].化学通报,1997,(2):22-27.
[80]Vassilev N,Vassileva M,Fenice M,et al.Immobilized cell technology applied in solubilization of insoluble inorganic(rock)phosphates and P plant acquisition.Bioresource Tech-nol.,2001,79:263-271.
[81]李冀新,张超,高虹.固定化细胞技术应用研究进展.化学与生物工程,2006,23(6):5-7
[82]杨文英,董学畅.细胞固定化及其在工业中的应用.云南民族学院学报(自然科学版),2001,10(3):406-410.
[83]Junter GA,Coquet L,Vilain S,et al.Immobilized-cell physiology:current data and the potentialities of proteomics.Enzyme Microb Tech.,2002,31:201-212.
[84]Jacobson BS,Branton D.Plasma membrane:Rapid Isolation and Exposure of the Cytoplasmic Surface by Use of Positively Charged Beads[J].Science,1977,195:302-304.
[85]Arkles BC,Miller AS,Brinigar WS.Whole cell and cell organelle immobilizationon siliceous surfaces.Midl.Macromol.Monogr.,1980,(7):363-375.
[86]Nagadomi H,Kitamura T,Watanabe M,et al.Simultaneous removal of chemical oxygen demand(COD),phosphate,nitrate and H_2S in the synthetic sewage wastewater using porous ceramic immobilized photosynthetic bacteria.Biotechnol Lett.,2000,22(17):1 369-1 374.
[87]Bonin P,Rontani JF,Bordenave L.Metabolic differences between attached andfree living marine bacteria:inadequacy of liquid cultures for describing in situbacterial activity.FEMS Microbiol Lett,2001,194:111-119.
[88]肖美燕.徐尔尼,陈志文.包埋法固定化细胞技术的研究进展[J].食品科学,2003,24(4):158-161.
[89]Zeng CM,Zhang YX,Lu LL,et al.Immobilization of human red cells in gel particles for chromatographic activity studies of the glucose transporter Glutl.Biochimica et Biophysica Acta,1997,1325(1):91-98.
[90]白凤武.无载体固定化细胞的研究进展[J].生物工程进展,2000,20(2):32-36.
[91]Manseld J,Schellenberger A,Rimbach J.Application of polystyrene-bound invertase to continuous sucrose hydrolysis on pilot scale[J].Biotechnol Bioeng.,1992,40(9):997-1 003.
[92]杨秀山,李军,田沈,等.固定化甲烷八叠球茵及处理高浓度有机废水的研究[J].环境科学学报,2003,23(2):282-284.
[93]宋秀兰,田建民,康静文.固定化胶质红环茵在好氧条件下降分解吲哚的研究[J].环境科学学报,2001,21(4):510-512.
[94]李超敏,韩梅,张良,等.细胞固定化技术-海藻酸钠包埋法的研究进展[J].安徽农业 科学,2006,34(7):1 281-1 282,1 284.
[95]徐鑫煤,陈英文,沈树宝.壳聚糖固定化酶和细胞研究新进展[J].化工科技,2006,14(1):54-57.
[96]Chang CC,Tseng SK Immobilization of Alcaligenes eutrophus using PVA cross-linked with sodium nitrate[J].Biotechnology Teehniques,1998,12(12):865-868.
[97]Mallouchos A,Reppa P,Aggelis G,et al.Grape skins as a natural support for yeast immobilization[J].Biotech Lett.,2002,24(16):1 331-1 335.
[98]王克明.复合栽体固定化细胞红曲色素发酵条件的研究[J].中国酿造,2005,(6):9-12.
[1]Kurbanoglu E B,Zilbeyaz K,Kurbanoglu N I,Kilic H.Enantioselective reduction of substituted acetophenones by Aspergillus niger.Tetrahedron Asymmetry.2007,18:1 159-1 162.
[2]Forni A,Moretti I,Prati F,Torre G.Microbial synthesis of(+)and(-)methyl 4-chioro-4,4-difluoro-3-hydroxybutanoate.J Mol Catal A Chem.1996,107:405-407.
[3]Nakamura K,Inoue Y,Mastsuda T,Ohno A.Microbial deracemization of 1-arylethanol.Tetrahedron lett.1995,36:6 263-6 266.
[4]Hasegawa Y,Adachi S,Matsuno R.Effect of the molar ratio of an energy source to the substrate on yeast-mediated production of a-Chloro- -methylbenzyl alcohol.J Biosci Bioeng.2000,4:329-333.
[5]Bucciarelli M,Forni A,Moretti I,Torre G.Asymmetric reduction of trifluoromethyl and methyl ketones by yeast;an improved method.Synthese.1983,897-899.
[6]Itoh N,Mizuguchi N,Mabuchi M.Production of chiral alcohol by enantioselective reduction with NADH-dependent phenylacetaldehyde reductase from Corynebacterium strain,ST-10.J Mol Catal B Eenzym.1999,6:41-50.
[7]Utsukihara T,Misumi O,Kato N,Kuroiwa T,Horiuchi C A.Reduction of various ketones by red algae.Tetrahedron Asymmetry.2006,17:1 179-1 185.
[8]Andrade L H,Comasseto J V,Rodrigues D F,Pellizari V H,Porto A M.Enantioselective reduction of ortho-substituted acetophenones by bacterial strains isolated from medium enriched with biphenyl or diesel fuel.J Mol Catal B Enzym.2005,33:73-79.
[9]Xie Qing,Wu Jina-ping,Xu Gang,Yang Li-rong,"Asymmetric reduction of ortho-chloroacetophenone with Candida pseudotropicalis 104",Biotechnology Progress,2006,22(5):1 301-1 304。
[10]S.G.Cull,J.D.Holbrey,V.Vargas-Mora,et al.,Biotechnol.Bioeng.,2000,69,227-233.
[11]J.Howarth,P.James and J.F.Dai,Tetrahedron Letters,2001,42,7 517-7 519.
[12]解晴.酵母催化不对称还原2'-氯苯乙酮及相关酶的克隆表达.浙江大学博士论文,2008.1.
[13]Lon DH,Zong MH,Zhang YY,et al.Eficient synthesis of optically active organosilyl alcohol via asymmetric reduction of acyl silane with immobilized yeast[J].Enzyme Microb Technol,2004,35(2-3):190-196.
[1]肖美燕,徐尔尼,陈志文.包埋法固定化细胞技术的研究进展[J].食品科学,2003,24(4):158-161.
[2]王新,李培军,巩宗强.固定化细胞技术的研究与进展[J].农业环境保护,2001,20(2):120-122.
[3]刘阳,曹军卫,翟超.米曲霉菌体细胞固定化及DL-蛋氨酸的光学拆分[J].武汉大学学报(自然科学版),Vol.46 No.6,Dec.2000,769-772.
[4]黄仁杰.海藻酸钙凝胶小球的含量测定及溶出考察[J].海峡药学,2003,14(3):29-31.
[5]郑志勇,赵占军.利用固定化细胞发酵生产2一酮基一D一葡萄糖酸的研究.山西农业大学学报(自然科学版),2003,23(2):158-160.
[6]王蓓.海藻酸盐固定化北京丙酸杆菌丙酸发酵的研究[J].生物工程学报,1992,8(2):178-183.
[2]Welton T.Room-Temperature Ionic Liquids:solvents for synthesis and catalysis[J].Chem Rev.,1999,99(8):2071-2084.
[3]Koch V R,Miller L L,Osteryoung R A.Electroinitiated friedel-crafts transalkylations in a Room Temperature Molten-Salt medium[J].J.Ame.Chem.Soc.,1976,98(18):5 277-5 281.
[4]Wilkes J S,Zaworotko M J.Air and water stable l-ethyl-3-methylimidazolium based ionic liquids[J].J.Chem.Soc.Chem.Commun.,1992,(13):965-967.
[5]李汝雄,王建基.化工进展,2002,21(1),43.
[6]王明慧,吴坚平,杨立荣.有机化学,2005,25(4),364.
[7]Anthony J L,Maginn E J,Brennecke J F.Solution thermodynamics of imidazolium-based ionic liquids and water[J].J.Phys.Chem.B,2001,105(44):10 942-10 949.
[8]Ellis B,Keim W,Wassercheid P.Linear dimerization of But-l-ene in biphasic mode using buffered chloro-aluminate ionic liquid solvents[J].Chem.Commun.,1999,377-381.
[9]F.van Rantwijk,R.M.Lau,R.A.Sheldon,Trends in Biotechnology,2003,21,131-138.
[10]U.Kragl,M.Eckstein,N.Kaftzik,Current Opinion in Biotechnology,2002,13,565-571.
[11]Magnusson D.K.,Bodey J.and Adams D.F.,J.Sol.Chem.,1984,13(2),583-587.
[12]Cull S.G.,et al.,J.Biotechnol.Bioeng.,2000,69(2),227-229.
[13]Erbeldinger M.,Mesiano A.J.,Biotechnol.Progr.,2000,16,1 129-1 131.
[14]Lozano P.,et al.,Biotechnol.Bioeng.,2001,75,563-569.
[15]Eckstein M.,Sesing M.,Kragl U.,Adlercreutz P.,Biotechnol.Lett.,2002,24,867-872.
[16]Laszlo J.A.,Compton D.L.,Biotechnol.Bioeng.,2001,75,181-186.
[17]Zhao H.,Malhotra S.V.,Biotechnol.Lett.,2002,24,1 257-1 260.
[18]Park S.,Kazlanskas R.J.,Org.Chem.,2001,66(25),8 395-8 401.
[19]Schofer S.H.,Kaftzik N.,Wasserscheid P.,Kragl U.,Chem.Commun.,2001,425-426.
[20]Itoh T.,Akasak E.,Kudo K.,Shirakami S.,Chem.Lett..,2001,262-263.
[21]Nara S.J.,Mohile S.S.,Harjani J.R.,Naik P.U.,Mol.Catal.B,2004,28,39-43.
[22]Lau R.M.,et al.,Org.Lett.,2000,26(2),4 189-4 191.
[23]Park S.,Viklund F.,Hult K.,Kazlauskas R.J.,Green Chem.,2003,5,715-719.
[24]Hinckley G.,Mozhaev V.,Budde C.,Biotechnol.Lett.,2002,24,2 083.
[25]Okrasa K.,et al.,Tetrahedron Asymmetry,2003,14,2 487-2 490.
[76]Howarth J,James P,Dai J.Immobilized baker's yeast reduction of ketones in an ionic liquid,[bmim]PF6 and water mix.Tetrahedron Lett.2001,42:7 517-7 519.
[27]Wen-Yong Lou,Min-Hua Zong,Thomas J.Smith.Use of ionic liquids to improve whole-cell biocatalytic asymmetric reduction of acetyltrimethylsilane for efficient synthesis of enantiopure (S)- 1-trimethylsilylethanol.Green Chem.,2006,8,147-155.
[28]Holger Pfruender,Ross Jones,Dirk Weuster-Botz.Water immiscible ionic liquids as solvents for whole cell biocatalysis.Journal of Biotechnology,2006(124):182-190.
[29]Beroza P,Suto M.I.Designing chiral libraries of drug discovery.DDV.2000,5(8):364.
[30]Oti-Amoako K,Vozeh S,Ha H R.The relative potency of major metabolites and enantiomers of propafenone in an experimental reperfusion arrhythmia mode.J Cardiovasc Pharm.1990,15(1):75-81.
[31]Agranat I,Caner H,Caldwell J.Putting chirality to work;the strategy of chiral switches.Nat Rev Drug Discov.2002,1(10):753-768.
[32]芮建中,吴锦芳,庞晓东.手性药物对映体选择性的药代动力学和药效学与临床合理用药.中国药理学通报,1998,14(1):14-18.
[33]Sanchez C,Bogeso K P,Ebert B.Escitalopram versus citalopram;the surprising role of the R-enantiomer.Psychopharmacology.2004,174(2):163-176.
[34]Strong M.FDA policy and regulation of stereojsomers;paradigm shift and the future of safer,more effective drugs.Food Drug Law J.1999,54(3):463-487.
[35]Rentsch KM.The importance of stereoseclecetive determination of drugs in the clinical laboratory.Biochem Biophys Methods.2002,54:1.
[36]舒之罡.制药领域是手性化合物的最大市场.医药中间体及其化工原料,2004,2:23-26.
[37]制药原料及中间体信息.2006,11:20-23.
[38]Cushman D W,Cheung M S,Sabo E F,Ondetti M A.Biochemistry.1977,16:5 484-5 491.
[39]Patel R N,Banerjee A,McNamee C G,Syarka L J.Appl Micrebiol Biotechnol.1993,40:241-245.
[40]Wada M,Kataoka M,Kawabata H.Purification and Characterization of NADPH-Dependent Carbonyl Reductase,Involved in Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate,from Candida magnoliae.Biosci Biotechnol Biochem.1998,62(2):280-285.
[41]Wei ZL,Li ZY,Lin GQ.Anti-Prelog microbial reduction of aryl α-halomethyl or α-hydroxymethyl ketones with Geotrichum sp.38.Tetrahedron.1998,54:13 059- 13 072.
[42]Srebrik M,Ramachandran P V,Brown H C.A Highly Enantioselective Synthesis of Both Optical Isomers of Tomoxetine,Fluoxetine,and Nisoxetine.J Org Chem.1988,53:2 916-2 920.
[43]Fronza G,Fuganti C,Grasselli P,Mele A.On the mode of bakers' yeast transformation of 3-chloropropiophenone and related ketones.Synthesis of(2S)-[2-2H]propiophenone,(R)-Fluoxetine,and(R)- and(S)-Fenfluramine.J Org Chem.1991,56:6 019-6 023.
[44]Corey E J,Reichard G A.Enantioselective and practical syntheses of R- and S-fluoxetines. Tetrahedron Lett.1989,30(39):5 207-5 210.
[45]Shimizu S,Hattori S,Hata H,Yamada H.Stereoselective enzymatic oxidation and reduction system for the production of D(-)-pantoyl lactone from a racemic mixture of pantoyl lactone.Enzyme Microb Technol.1987,9(7):411-416.
[46]Shimizu S,Hattori S,Hata H,Yamada H.One-step microbial conversion of a racemic mixture of pantoyl lactone to optically active D-(-)-pantoyi lactone.Appl Environ Microb.1987,3:519-522.
[47]Glanzer B I,Faber K,Griengi H.Microbial resolution of O-acetylpantoyl lactone.Enzyme Microb Technol.1988,10(11):689-690.
[48]王明慧,吴坚平,杨立荣,陈新志.相转移催化法合成2-氯-3-氯甲基-噻吩及噻康唑.应用化学,2006,23(1):106-108.
[49]黄蓓,杨立荣,吴坚平.手性拆分技术的工业应用,化工进展,2002,21(6):375-380.
[50]Nozaki H,Monut S,Noyori R.Asymmetric induction in carbeniod reaction by means of a dissymmetric copper chelate.Tetrahedron Lett.1966,7:5 239-5 244.
[51]Ohkuma T,Ooka H,Hashiguchi S.Practical enantioselective hydrogenation of aromatic ketones.J Am Chem Soc.1995,117(9):2 675-2 676.
[52]Noyori R,Tomino I,Tanimoto Y.Highly enantioselective reduction of aromatic ketones by binaphthol-modified lithium aluminum hydride reagents.J Am Chem Soc.1984,106(22):6 709-6716.
[53]廖永卫,陈卫平.手性唑硼烷催化酮的不对称还原反应.中国药物化学杂志,1994,4(3),202-208.
[54]Wei Z L,Li Z Y,Lin G Q.anti-Prelog Microbial Reduction of Aryl α -Halomethyl or α-Hydroxymethyl Ketones with Geotrichum sp.38.Tetrahedron.1998,54:13 059-13 072.
[55]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):2 725-2 737.
[56]Forni A,Moretti I,Pratt F,Torre G.Microbial synthesis of(+)and(-)methyl 4-chloro-4,4-difluoro-3-hydroxybutanoate.J Mol Catal A Chem.1996,107:405-407.
[57]Yasohara Y.,.Kizaki N,Hasegawa J,Takahashi S,Wada M,Kataoka M,Shimizu S.Synthesis of optically active ethyl 4-chloro-3-hydroxybutanoate by microbial reduction.Appl Microbiol Biotechnol.1999,51:847-851.
[58]杨忠华.水相中酵母细胞催化前手性羰基不对称还原合成手性醇的研究.浙江大学博士学位论文,2005,1.
[59]王盛,制备重要他汀类药物中间体的研究.浙江大学硕士学位论文,2006,6.
[60]Stewart J D.Organic transformations catalyzed by engineered yeast cells and related systems.Curr Opin Biotech.2000,11:363-368.
[61]Yamamoto H,Kawada N,Matsuyama A,Kobayashi Y.Cloning and expression in Escherichia coli of a gene coding for a secondary alcohol dehydrogenase from Candida parapsilosis.Biosci Biotechnol Biochem.1999,63(6):1 051-1 055.
[62]Yasohara Y,Kizaki N,Hasegawa J,Wada M,Kataoka M,Shimizu S.Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae,involved in tereoselective reduction of ethyl 4-chloro-3-oxobutanoate.Biosci Biotechnol Biochem.2000,64(7):1 430-1 436.
[63]Rodringuez S,Kayser M M,Stewart J D.Improving the stereoselectivity of baker's yeast reductions by genetic engineering.Org Lett.1999,1(8):1 153-1 155.
[64]Sikkema J,Weber F J,Heipieper H J,Debont J.Cellular toxicity of lipophilic compounds:mechanisms,implications,and adaptations.Biocatalysis.1994,10:113-122.
[65]Inoue A,Horikoshi K.Estimation of solvent tolerance of bacteria by solvent parameter logP.J Ferment Bileng.1991,3:194-196.
[66]Vermun 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.1993,42:745-758.
[67]Qun J,Yao S J,Mei L.Tolerance of immobilize baker's yeast in organic solvents.Enzyme Micro.Tech.2002,30:721-725.
[68]Medson C,Smallridge A J,Trewhella M A.Baker's yeast activity in an organic solvent system.J mol Catal B Enzym.2001,11:897-903.
[69]Nakamura K,Kondo S,Nakajima N,Ohno Atsuyoshi.Mechanistic study for stereochemical control of microbial reduction of α-keto esters in an organic solvent.Tetrahedron.1995,51:687-694.
[70]何军邀,孙志浩,韩丽,钱嘉南.水/有机溶剂两相体系微生物催化不对称还原制备(S)-4-氯-3-羟基丁酸乙酯.高校化学工程学报,2007,21(2):310-315.
[71]He J Y,Sun Z H,Ruan W Q,Xu Y.Biocatalytic synthesis of ethyl (S)-4-chloro-3-hydroxy-butanoate in an aqueous-organic solvent biphasic system using Aureobasidium pullulans CGMCC 1244.Process Biochem.2006,41:244-249.
[72]Anderson B A,Hansen M M,Harkness A R,Henry C L,Vicenzi J T,Zmijewski M J.Application of a Practical Biocatalytic Reduction to an Enantioselective Synthesis of the 5H-2,3-Benzodiazepine LY300164.J Am Chem Soc.1995,117(49):12 358-12 359.
[73]Vicenzi J T,Zmijewski M J,Reinhard M R,Landen B E,Muth W L,Marler P G.Large-Scale Stereoselective Enzymatic Ketone Reduction with in situ Product Removal via Polymeric Adsorbent Resins.Enzyme Microb Technol.,1997,20(7):494-499.
[74]欧志敏.微生物法还原羰基化合物制备手性中间体的研究,浙江大学博士学位论文,2003,4.
[75]王新,李培军,巩宗强,张海荣.固定化细胞技术的研究与进展.农业环境保护.2001,20(2): 120-122.
[76]Hasegawa Y,Adachi S,Matsuno R.Effect of the molar ratio of an energy source to the substrate on yeast-mediated production of 2-chloro-α-methylbenzyl alcohol.J Biosci Bioeng.2000,89(4):329-333.
[77]Milagre H,Milagre C,Moran P,Santana M H,Rodrigues J.Reduction of ethyl benzoylformate mediated by Saccharomyces cerevisiae entrapped in alginated fibers with double gel layers in a continuously operated reactor.Enzyme Microb Tech.2005,37:121-125.
[78]Wendhausen R,Moran P,Joekes I,Rodrigues A.Coutinuous process for large-scale preparation of chiral alcohols with baker's yeast immobilized on chrysotile fibers.J Mol Catal B Enzym.1998,5:69-73.
[79]王洪祚,刘世勇.酶和细胞的固定化[J].化学通报,1997,(2):22-27.
[80]Vassilev N,Vassileva M,Fenice M,et al.Immobilized cell technology applied in solubilization of insoluble inorganic(rock)phosphates and P plant acquisition.Bioresource Tech-nol.,2001,79:263-271.
[81]李冀新,张超,高虹.固定化细胞技术应用研究进展.化学与生物工程,2006,23(6):5-7
[82]杨文英,董学畅.细胞固定化及其在工业中的应用.云南民族学院学报(自然科学版),2001,10(3):406-410.
[83]Junter GA,Coquet L,Vilain S,et al.Immobilized-cell physiology:current data and the potentialities of proteomics.Enzyme Microb Tech.,2002,31:201-212.
[84]Jacobson BS,Branton D.Plasma membrane:Rapid Isolation and Exposure of the Cytoplasmic Surface by Use of Positively Charged Beads[J].Science,1977,195:302-304.
[85]Arkles BC,Miller AS,Brinigar WS.Whole cell and cell organelle immobilizationon siliceous surfaces.Midl.Macromol.Monogr.,1980,(7):363-375.
[86]Nagadomi H,Kitamura T,Watanabe M,et al.Simultaneous removal of chemical oxygen demand(COD),phosphate,nitrate and H_2S in the synthetic sewage wastewater using porous ceramic immobilized photosynthetic bacteria.Biotechnol Lett.,2000,22(17):1 369-1 374.
[87]Bonin P,Rontani JF,Bordenave L.Metabolic differences between attached andfree living marine bacteria:inadequacy of liquid cultures for describing in situbacterial activity.FEMS Microbiol Lett,2001,194:111-119.
[88]肖美燕.徐尔尼,陈志文.包埋法固定化细胞技术的研究进展[J].食品科学,2003,24(4):158-161.
[89]Zeng CM,Zhang YX,Lu LL,et al.Immobilization of human red cells in gel particles for chromatographic activity studies of the glucose transporter Glutl.Biochimica et Biophysica Acta,1997,1325(1):91-98.
[90]白凤武.无载体固定化细胞的研究进展[J].生物工程进展,2000,20(2):32-36.
[91]Manseld J,Schellenberger A,Rimbach J.Application of polystyrene-bound invertase to continuous sucrose hydrolysis on pilot scale[J].Biotechnol Bioeng.,1992,40(9):997-1 003.
[92]杨秀山,李军,田沈,等.固定化甲烷八叠球茵及处理高浓度有机废水的研究[J].环境科学学报,2003,23(2):282-284.
[93]宋秀兰,田建民,康静文.固定化胶质红环茵在好氧条件下降分解吲哚的研究[J].环境科学学报,2001,21(4):510-512.
[94]李超敏,韩梅,张良,等.细胞固定化技术-海藻酸钠包埋法的研究进展[J].安徽农业 科学,2006,34(7):1 281-1 282,1 284.
[95]徐鑫煤,陈英文,沈树宝.壳聚糖固定化酶和细胞研究新进展[J].化工科技,2006,14(1):54-57.
[96]Chang CC,Tseng SK Immobilization of Alcaligenes eutrophus using PVA cross-linked with sodium nitrate[J].Biotechnology Teehniques,1998,12(12):865-868.
[97]Mallouchos A,Reppa P,Aggelis G,et al.Grape skins as a natural support for yeast immobilization[J].Biotech Lett.,2002,24(16):1 331-1 335.
[98]王克明.复合栽体固定化细胞红曲色素发酵条件的研究[J].中国酿造,2005,(6):9-12.
[1]Kurbanoglu E B,Zilbeyaz K,Kurbanoglu N I,Kilic H.Enantioselective reduction of substituted acetophenones by Aspergillus niger.Tetrahedron Asymmetry.2007,18:1 159-1 162.
[2]Forni A,Moretti I,Prati F,Torre G.Microbial synthesis of(+)and(-)methyl 4-chioro-4,4-difluoro-3-hydroxybutanoate.J Mol Catal A Chem.1996,107:405-407.
[3]Nakamura K,Inoue Y,Mastsuda T,Ohno A.Microbial deracemization of 1-arylethanol.Tetrahedron lett.1995,36:6 263-6 266.
[4]Hasegawa Y,Adachi S,Matsuno R.Effect of the molar ratio of an energy source to the substrate on yeast-mediated production of a-Chloro- -methylbenzyl alcohol.J Biosci Bioeng.2000,4:329-333.
[5]Bucciarelli M,Forni A,Moretti I,Torre G.Asymmetric reduction of trifluoromethyl and methyl ketones by yeast;an improved method.Synthese.1983,897-899.
[6]Itoh N,Mizuguchi N,Mabuchi M.Production of chiral alcohol by enantioselective reduction with NADH-dependent phenylacetaldehyde reductase from Corynebacterium strain,ST-10.J Mol Catal B Eenzym.1999,6:41-50.
[7]Utsukihara T,Misumi O,Kato N,Kuroiwa T,Horiuchi C A.Reduction of various ketones by red algae.Tetrahedron Asymmetry.2006,17:1 179-1 185.
[8]Andrade L H,Comasseto J V,Rodrigues D F,Pellizari V H,Porto A M.Enantioselective reduction of ortho-substituted acetophenones by bacterial strains isolated from medium enriched with biphenyl or diesel fuel.J Mol Catal B Enzym.2005,33:73-79.
[9]Xie Qing,Wu Jina-ping,Xu Gang,Yang Li-rong,"Asymmetric reduction of ortho-chloroacetophenone with Candida pseudotropicalis 104",Biotechnology Progress,2006,22(5):1 301-1 304。
[10]S.G.Cull,J.D.Holbrey,V.Vargas-Mora,et al.,Biotechnol.Bioeng.,2000,69,227-233.
[11]J.Howarth,P.James and J.F.Dai,Tetrahedron Letters,2001,42,7 517-7 519.
[12]解晴.酵母催化不对称还原2'-氯苯乙酮及相关酶的克隆表达.浙江大学博士论文,2008.1.
[13]Lon DH,Zong MH,Zhang YY,et al.Eficient synthesis of optically active organosilyl alcohol via asymmetric reduction of acyl silane with immobilized yeast[J].Enzyme Microb Technol,2004,35(2-3):190-196.
[1]肖美燕,徐尔尼,陈志文.包埋法固定化细胞技术的研究进展[J].食品科学,2003,24(4):158-161.
[2]王新,李培军,巩宗强.固定化细胞技术的研究与进展[J].农业环境保护,2001,20(2):120-122.
[3]刘阳,曹军卫,翟超.米曲霉菌体细胞固定化及DL-蛋氨酸的光学拆分[J].武汉大学学报(自然科学版),Vol.46 No.6,Dec.2000,769-772.
[4]黄仁杰.海藻酸钙凝胶小球的含量测定及溶出考察[J].海峡药学,2003,14(3):29-31.
[5]郑志勇,赵占军.利用固定化细胞发酵生产2一酮基一D一葡萄糖酸的研究.山西农业大学学报(自然科学版),2003,23(2):158-160.
[6]王蓓.海藻酸盐固定化北京丙酸杆菌丙酸发酵的研究[J].生物工程学报,1992,8(2):178-183.