pH-热再生型两水相体系中固定化青霉素酰化酶相转移催化头孢菌素G裂解为7-ADCA
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摘要
两水相体系由于聚合物回收的困难,在一定程度上阻碍了两水相萃取技术的应用。本课题使用温敏型可回收聚合物PNB及pH敏感型聚合物PADB组成两水相系统进行头孢菌素G的裂解反应,可以解除酶的底物,产物抑制,提高转化率,两水相系统的回收可大大减少成本及环境压力.
此两水相体系应用于青霉素酰化酶催化裂解头孢菌素G生成7-氨基-3-去乙酰氧基头孢烷酸(7-ADCA)。考察了几种常见的无机盐LiCl, NaCl, KCl, KSCN, Li2SO4, (NH4) 2SO4, Na2SO4, K2SO4, Na3PO4, K3PO4, NaC104对7-ADCA、苯乙酸和头孢菌素G分配系数的影响。发现整个体系在30mM (NH4) 2SO4的存在下,底物和产物达到较好的分配,7-ADCA、苯乙酸和头孢菌素G的分配系数为分别为3.56,1.57和5.34。在此体系中进行固定化青霉素酰化酶的催化反应,底物与产物富集在上相,固定化酶分配在下相,底物头孢菌素G从上相扩散到下相经酶裂解生成产物7-ADCA口苯乙酸转移到上相,从而解除底物和产物抑制,提高酶催化效率。最终抗生素中间体7-ADCA的得率为93.00%。
The main problem of aqueous two-phase systems is that they can not be recycled efficiently, which causes a high cost and pollution of the environment. Scientists are now focusing on a recycling aqueous two-phase system. In this study, a recycling pH-sensitive polymer and thermo-sensitive polymer forming ATPs were used to carry on an enzymatic reaction. The aqueous two-phase systems were used in the bioconversion reaction of penicillin G. The substrate and products inhibitions are removed, due to their different partitions between the top phase and bottom phase. The yield of 7-ADCA can be improved obviously. It was found that partition coefficients of 7-ADCA, PAA and cephalosporin G could reach 3.56,1.57 and 5.34 in the systems, respectively. The rate of conversion of the penicillin could reach 98.01%, while the yield of the reaction could be 93.00%.
此两水相体系应用于青霉素酰化酶催化裂解头孢菌素G生成7-氨基-3-去乙酰氧基头孢烷酸(7-ADCA)。考察了几种常见的无机盐LiCl, NaCl, KCl, KSCN, Li2SO4, (NH4) 2SO4, Na2SO4, K2SO4, Na3PO4, K3PO4, NaC104对7-ADCA、苯乙酸和头孢菌素G分配系数的影响。发现整个体系在30mM (NH4) 2SO4的存在下,底物和产物达到较好的分配,7-ADCA、苯乙酸和头孢菌素G的分配系数为分别为3.56,1.57和5.34。在此体系中进行固定化青霉素酰化酶的催化反应,底物与产物富集在上相,固定化酶分配在下相,底物头孢菌素G从上相扩散到下相经酶裂解生成产物7-ADCA口苯乙酸转移到上相,从而解除底物和产物抑制,提高酶催化效率。最终抗生素中间体7-ADCA的得率为93.00%。
The main problem of aqueous two-phase systems is that they can not be recycled efficiently, which causes a high cost and pollution of the environment. Scientists are now focusing on a recycling aqueous two-phase system. In this study, a recycling pH-sensitive polymer and thermo-sensitive polymer forming ATPs were used to carry on an enzymatic reaction. The aqueous two-phase systems were used in the bioconversion reaction of penicillin G. The substrate and products inhibitions are removed, due to their different partitions between the top phase and bottom phase. The yield of 7-ADCA can be improved obviously. It was found that partition coefficients of 7-ADCA, PAA and cephalosporin G could reach 3.56,1.57 and 5.34 in the systems, respectively. The rate of conversion of the penicillin could reach 98.01%, while the yield of the reaction could be 93.00%.
引文
[1]Wolfgang Riedl, Thomas Raiser. Membrane-supported extraction of biomolecules with aqueous two-phase systems. Desalination,2008,224(1-3):160-167
[2]Kula M R.,Kroner K H.Liquid-Liquid Seperation in Enzyme Isolation Process.Proc.Int.Ferment.Symp.5th,1976,52
[3]刘茉娥,陈欢林.新型分离技术基础第二版[M].浙江大学出版社.1999,83-89
[4]Bronstedt H N Z.Z phys Chemistry.1931.A155-343
[5]Gerson D F.Biochim.Biophys.Acta.1980,602:269-280
[6]Forciniti,D,Hall,C,K,Kula,M R.Influence of polymer weight and temperature on phase composition in aqueous two-phase system.Fluid phase Equil,1991,61:243-2 62
[7]Forciniti,D,Hall,C,K,Kula,M-R.Protein partitioning at the isoelectric point:Influeence of polymer molecular weight and concentration and protein size.Biotechnolo-gy and Bioengineering,1991,38:986-994
[8]Albertsson,P,A.partition of cell particles and macromolecules.Wiley,New York.1986
[9]Albertsson P, A, Sasakawa, s, Walter, H.Cross partition and isoelectric point of proteins. Nature 1970.228,1329-1330
[10]Eiteman,M,A.Partitioning of charged solytes in PEG/K3PO4 aqueous two-phase system.Separation science Technology.1994,29:685-700
[11]Eiteman,M,A.Predicting partition coefficients of multi-charged solutes in aqueous two-phase system,Journal of Chromatography A.668:21-30
[12]Brooks D E.,Sharp K A.,Fisher D.,Theeoretical Aspects of partitioning in Aqueous Two-phase Systems.ed.Harry Walter,Brooks D E. and Fisher D.Orlando:1985.Academic Press.1985,Chaper2
[13]Albertsson,P,A.Separation of particles and macromolecules by phase partition, Endeavour new series.1977,1:69-74
[14]Cascone,O,Andrews,B,A,Asenjo,J,A partitioning and purification of thaumatin in aqueous two-phase system,Enz Microb,Technol.1991,13:629-635
[15]Kula, R.L. Extraction of biopolymers.1985 451-457
[16]Brian C. Tripp, Eric W. Kaler,Stanley I. Sandler Alex Kopatsis Effect of ionic strength and ionic species on partitioning behavior of hydrophobic and hydrophilic polystyrene latex beads in aqueous two-phase polymer systems. Colloids and Surfaces B:Biointerfaces.1996,6:151-164
[17]Kula M R.Comprehensive Biotechnology vol.2.Eds.Humphrey A.and Cooney C.L.Oxford:Pergamon Press.1985.451-471
[18]Hustedt H.,Kroner K H.,Kula M R.Partitioning in Aqueous Two-Phase Systems.H.Walter,D.E.Brooks and D.Fisher(eds).Orlando:Academic Press.1985.534
[19]Hassan A, Muallem A, Mohamed I.M.W, Asrof A.S.K,Synthesis and solytion properties of a new ionic polymer and its behavior in aqueous two-phase polymer systems polymer.2002,43:1041-1050
[20]W.J.Zhou,M.J.Kurth,sunthesis of a novel pH-responding polymer with pendant barbituric acid moieties.polymer.2001,42(1):345-349.
[21]Patrickios C.S,Hertler W.R, and Hatton T.a,phase behavior of random and ABC triblock methacrylic polyampholytes with poly(viny alcohol)in wateneffect of pHamd salt, Fluid phade Equilibria.1995,108:243-245.
[22]Modlin R F, Alred P A, Tjerneld F.J Chromatogr.1994,668:229-236
[23]Josefine Persson, Hans-Olof Johansson, Folke Tjerneld. Purification of protein and recycling of polymers in a new aqueous two-phase using two thermoseparating polymers.Journal of Chromatography A.1999,4:31-48
[24]Persson J, Johansson H.O, Galaev I, Mattiasson B, Tjerneld F. Aqueous polymer two-phase systems formed by new thermoseparating polymers, Bioseparation.2000, 9:105-116
[25]Mos van Berlo, Karelch A M, Luyben, Luak A M, Van der wielen. Poly(ethylene glycol)-salt aqueous two-phase system with easily recyclable volatile salts.Journal of Chromatography B.1998,711:61-68
[26]Taboada M E, Graber T A, Asenjo J A, Andrews B A. Drowing-out crystallization of sodium sulfate using aqueous two-phase systems.J. Chromatograph.B.2000, 743:101-105
[27]CN.Patent.1687162
[28]A.Grece,M-R.Kula,J.Chem.Techn.Biotechnol.50(1991)27-41
[29]罗晖,李强.童忆舟,等.酶法生产7-氨基头孢烷酸的研究进展[J].现代工业.2002,22(12)：18-22.
[30]李永刚,刘东志,张伟,等.从青霉索G生产7-氨基脱乙酰氧基头孢烷酸[J].中国医药工业杂志.2000,31(18)：376-380
[31]熊振平,李明纭.酶工艺在半合成β-内酰胺抗生索的研究[J].药学通报.1985,20(8)：453-460.
[32]Barton JW. Protective groups in organic chemistry,[M].New York:Plenum press,1973, Chapter 2.
[33]Mangia AA. mild and convenient synthesis of penicillin and cephalosporin sulfoxides[J].Synthesis,1978,5:361.
[34]Cooper RD, Demarco PV, Cheng JC, et al.Structural studies on penicillin derivatives I: The configuration of phenoxymethyl penicillin sulfoxide[J].JACS,1969,91:1408.
[35]Harrison CR, Hodge P. Oxidation of some penicillins and other sulphides by use of a polymer-supported peroxy acid[J]. J Chem Soc,1976,21:2252.
[36]Barton DH, Comer F, Greig DG, et al.Transformations of penicillin[J]. J Chem Soc(C), 1971,21:3540.
[37]Casara PJ, Jung M, Metcalf BW, etal. Acetylenic derivatives of a-amino acid[P]. US Patent:4260823,1981-04-07.
[38]Spary DO. Conversion of penicillin to cephalosporin via a double sulfoxide rearrangement[J]. JACS,1970,92:5006.
[39]Demain AL, Kohsaka M, Acellular synthesis of cephalosporins [P]. US Patent:4178210, 1979-12-11.
[40]肖正华,张梦军,王云霞,等.青霉索酰化酶固定化及应用[J].国外医药抗生索分册.2004,25(4)：156-187.
[41]广东工学院精细化工教研室.精细化工基本技术及其应用[M].广东：1995.8,54-57.
[42]沈同,王镜岩.生物化学[M].北京高等教育出版杜.1990.12,253—255.
[43]Erarslan A. The hydrolysis of cephalosporin G by free and immobilized penicillin G acylase from a mutant of Escherichia coli ATCC 11105. Process Biochem.1998:28:311
[44]史兰香,赵地顺,徐志栋,等,头孢菌素G酶裂解制备7-ADCA的研究[J].华西药学杂志.2000,15(2)：105-106.
[45]金科铭,曹学君,固定化青霉素酰化酶在光-pH敏感可回两水相中裂解青霉素G为6-APA.中国生物工程杂志.2007,27(10)：53-58
[46]Mateo C, Abian O. Fernandez-Lorente G, elal. Epoxysepabeads:A novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment[J]. Biotechnol Prog,2002,18(3):629-634.
[47]Pasini D. Filippini M, Pianetti I, etal. Efficient biocatalytic cleavage and recovery of organic substrates supported on soluble polymers [J]. Adv Synth Catal,2007,349(6): 971-978
[48]He J. Li X F. Evans D G, etal. A new support for the immobilization of penicillin acylase [J]. J Mol Catal B:Enzym,2000,11(1):45-53.
[49]Kallenberg A I,Rantwijk F V. Sheldon R A. Immobilization of penicillin G acylase: The key to optimum performance [J]. Adv Synth Catal,2005,347(7-8):905-926.
[50]薛屏,卢冠忠,郭杨龙,等.高分子载体材料对青霉素酰化酶的固定化作用[J].功 能高分子学报.2005,18(2)：340-346.
[51]Van R J L, Schroen C G P H, Tramper J, et al. Biocatalysts:Measurement,modelling and design of heterogeneity[J]. Biotechnol Adv,2007,25(2):137-147.
[52]Arroyo M, Mata I, Acebal C, etal. Riotechnological applications of penicillin acylases:state-of-the-art[J]. Appl Microbiol Biotechnol,2003,60(5):507-514.
[53]Maria CAS, Zhao X S. Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst[J]. Catal Today,2004,93-95(1):293-299.
[54]薛屏.大孔GAM珠状聚合物固定化青霉素酰化酶催化性能的研究[J].宁夏大学学报(自然科学版).2005,26(2)：144-147.
[55]乌云高娃,卢冠忠.6-APA合成反应中固定化青霉素酰化酶载体及固定化研究进展[J].分子催化.2002,16(1)：75-80.
[56]姚汝华.宗敏华.超声波在生物工程中的应用[J].生物工程进展.1999,19(3)：45-48.
[57]杨根敏.固定化青霉素酰化酶动力学参数测定[J].华东化工学院报.1989,15(4)：412-418.
[58]张小飞,刘友全,莫章桦.固定化青霉素酰化酶制备7-ADCA的条件优化[J].中南林学院学报.2006,26(5)：113-116
[59]Kim Y J, Weigand W A. Experimental analysis of a product; inhibited fermentation in an Aqueous Two-Phase Systems. Applied Biochemistry and Biotechnology.1992, 34:419-430
[60]Chu I M.,Chang S L.,Wang S H.,Yang W Y. Biotechnol. Techniques.1990,4(2):143-146
[61]Yang W Y. and Chu I M. Biotechnol. Techniques.1990,4(3):191-194
[62]Guan Y X., Mei L H., Zhu Z Q. Biotechnol. Techniques.1994,8(7):491-496
[63]关怡新.用两水相分配技术提纯抗生素的基础与工艺研究：[浙江大学博士学位论文].杭州：浙江大学,1994.
[64]李勉.新型两水相系统的基础及工程研究：[浙江大学博士学位论文].杭州：浙江大学,1996.
[65]Yang W Y, Lin C D., Chu I M.,Lee C J. Biotechnol.Bioeng.1994.43:439-445
[2]Kula M R.,Kroner K H.Liquid-Liquid Seperation in Enzyme Isolation Process.Proc.Int.Ferment.Symp.5th,1976,52
[3]刘茉娥,陈欢林.新型分离技术基础第二版[M].浙江大学出版社.1999,83-89
[4]Bronstedt H N Z.Z phys Chemistry.1931.A155-343
[5]Gerson D F.Biochim.Biophys.Acta.1980,602:269-280
[6]Forciniti,D,Hall,C,K,Kula,M R.Influence of polymer weight and temperature on phase composition in aqueous two-phase system.Fluid phase Equil,1991,61:243-2 62
[7]Forciniti,D,Hall,C,K,Kula,M-R.Protein partitioning at the isoelectric point:Influeence of polymer molecular weight and concentration and protein size.Biotechnolo-gy and Bioengineering,1991,38:986-994
[8]Albertsson,P,A.partition of cell particles and macromolecules.Wiley,New York.1986
[9]Albertsson P, A, Sasakawa, s, Walter, H.Cross partition and isoelectric point of proteins. Nature 1970.228,1329-1330
[10]Eiteman,M,A.Partitioning of charged solytes in PEG/K3PO4 aqueous two-phase system.Separation science Technology.1994,29:685-700
[11]Eiteman,M,A.Predicting partition coefficients of multi-charged solutes in aqueous two-phase system,Journal of Chromatography A.668:21-30
[12]Brooks D E.,Sharp K A.,Fisher D.,Theeoretical Aspects of partitioning in Aqueous Two-phase Systems.ed.Harry Walter,Brooks D E. and Fisher D.Orlando:1985.Academic Press.1985,Chaper2
[13]Albertsson,P,A.Separation of particles and macromolecules by phase partition, Endeavour new series.1977,1:69-74
[14]Cascone,O,Andrews,B,A,Asenjo,J,A partitioning and purification of thaumatin in aqueous two-phase system,Enz Microb,Technol.1991,13:629-635
[15]Kula, R.L. Extraction of biopolymers.1985 451-457
[16]Brian C. Tripp, Eric W. Kaler,Stanley I. Sandler Alex Kopatsis Effect of ionic strength and ionic species on partitioning behavior of hydrophobic and hydrophilic polystyrene latex beads in aqueous two-phase polymer systems. Colloids and Surfaces B:Biointerfaces.1996,6:151-164
[17]Kula M R.Comprehensive Biotechnology vol.2.Eds.Humphrey A.and Cooney C.L.Oxford:Pergamon Press.1985.451-471
[18]Hustedt H.,Kroner K H.,Kula M R.Partitioning in Aqueous Two-Phase Systems.H.Walter,D.E.Brooks and D.Fisher(eds).Orlando:Academic Press.1985.534
[19]Hassan A, Muallem A, Mohamed I.M.W, Asrof A.S.K,Synthesis and solytion properties of a new ionic polymer and its behavior in aqueous two-phase polymer systems polymer.2002,43:1041-1050
[20]W.J.Zhou,M.J.Kurth,sunthesis of a novel pH-responding polymer with pendant barbituric acid moieties.polymer.2001,42(1):345-349.
[21]Patrickios C.S,Hertler W.R, and Hatton T.a,phase behavior of random and ABC triblock methacrylic polyampholytes with poly(viny alcohol)in wateneffect of pHamd salt, Fluid phade Equilibria.1995,108:243-245.
[22]Modlin R F, Alred P A, Tjerneld F.J Chromatogr.1994,668:229-236
[23]Josefine Persson, Hans-Olof Johansson, Folke Tjerneld. Purification of protein and recycling of polymers in a new aqueous two-phase using two thermoseparating polymers.Journal of Chromatography A.1999,4:31-48
[24]Persson J, Johansson H.O, Galaev I, Mattiasson B, Tjerneld F. Aqueous polymer two-phase systems formed by new thermoseparating polymers, Bioseparation.2000, 9:105-116
[25]Mos van Berlo, Karelch A M, Luyben, Luak A M, Van der wielen. Poly(ethylene glycol)-salt aqueous two-phase system with easily recyclable volatile salts.Journal of Chromatography B.1998,711:61-68
[26]Taboada M E, Graber T A, Asenjo J A, Andrews B A. Drowing-out crystallization of sodium sulfate using aqueous two-phase systems.J. Chromatograph.B.2000, 743:101-105
[27]CN.Patent.1687162
[28]A.Grece,M-R.Kula,J.Chem.Techn.Biotechnol.50(1991)27-41
[29]罗晖,李强.童忆舟,等.酶法生产7-氨基头孢烷酸的研究进展[J].现代工业.2002,22(12)：18-22.
[30]李永刚,刘东志,张伟,等.从青霉索G生产7-氨基脱乙酰氧基头孢烷酸[J].中国医药工业杂志.2000,31(18)：376-380
[31]熊振平,李明纭.酶工艺在半合成β-内酰胺抗生索的研究[J].药学通报.1985,20(8)：453-460.
[32]Barton JW. Protective groups in organic chemistry,[M].New York:Plenum press,1973, Chapter 2.
[33]Mangia AA. mild and convenient synthesis of penicillin and cephalosporin sulfoxides[J].Synthesis,1978,5:361.
[34]Cooper RD, Demarco PV, Cheng JC, et al.Structural studies on penicillin derivatives I: The configuration of phenoxymethyl penicillin sulfoxide[J].JACS,1969,91:1408.
[35]Harrison CR, Hodge P. Oxidation of some penicillins and other sulphides by use of a polymer-supported peroxy acid[J]. J Chem Soc,1976,21:2252.
[36]Barton DH, Comer F, Greig DG, et al.Transformations of penicillin[J]. J Chem Soc(C), 1971,21:3540.
[37]Casara PJ, Jung M, Metcalf BW, etal. Acetylenic derivatives of a-amino acid[P]. US Patent:4260823,1981-04-07.
[38]Spary DO. Conversion of penicillin to cephalosporin via a double sulfoxide rearrangement[J]. JACS,1970,92:5006.
[39]Demain AL, Kohsaka M, Acellular synthesis of cephalosporins [P]. US Patent:4178210, 1979-12-11.
[40]肖正华,张梦军,王云霞,等.青霉索酰化酶固定化及应用[J].国外医药抗生索分册.2004,25(4)：156-187.
[41]广东工学院精细化工教研室.精细化工基本技术及其应用[M].广东：1995.8,54-57.
[42]沈同,王镜岩.生物化学[M].北京高等教育出版杜.1990.12,253—255.
[43]Erarslan A. The hydrolysis of cephalosporin G by free and immobilized penicillin G acylase from a mutant of Escherichia coli ATCC 11105. Process Biochem.1998:28:311
[44]史兰香,赵地顺,徐志栋,等,头孢菌素G酶裂解制备7-ADCA的研究[J].华西药学杂志.2000,15(2)：105-106.
[45]金科铭,曹学君,固定化青霉素酰化酶在光-pH敏感可回两水相中裂解青霉素G为6-APA.中国生物工程杂志.2007,27(10)：53-58
[46]Mateo C, Abian O. Fernandez-Lorente G, elal. Epoxysepabeads:A novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment[J]. Biotechnol Prog,2002,18(3):629-634.
[47]Pasini D. Filippini M, Pianetti I, etal. Efficient biocatalytic cleavage and recovery of organic substrates supported on soluble polymers [J]. Adv Synth Catal,2007,349(6): 971-978
[48]He J. Li X F. Evans D G, etal. A new support for the immobilization of penicillin acylase [J]. J Mol Catal B:Enzym,2000,11(1):45-53.
[49]Kallenberg A I,Rantwijk F V. Sheldon R A. Immobilization of penicillin G acylase: The key to optimum performance [J]. Adv Synth Catal,2005,347(7-8):905-926.
[50]薛屏,卢冠忠,郭杨龙,等.高分子载体材料对青霉素酰化酶的固定化作用[J].功 能高分子学报.2005,18(2)：340-346.
[51]Van R J L, Schroen C G P H, Tramper J, et al. Biocatalysts:Measurement,modelling and design of heterogeneity[J]. Biotechnol Adv,2007,25(2):137-147.
[52]Arroyo M, Mata I, Acebal C, etal. Riotechnological applications of penicillin acylases:state-of-the-art[J]. Appl Microbiol Biotechnol,2003,60(5):507-514.
[53]Maria CAS, Zhao X S. Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst[J]. Catal Today,2004,93-95(1):293-299.
[54]薛屏.大孔GAM珠状聚合物固定化青霉素酰化酶催化性能的研究[J].宁夏大学学报(自然科学版).2005,26(2)：144-147.
[55]乌云高娃,卢冠忠.6-APA合成反应中固定化青霉素酰化酶载体及固定化研究进展[J].分子催化.2002,16(1)：75-80.
[56]姚汝华.宗敏华.超声波在生物工程中的应用[J].生物工程进展.1999,19(3)：45-48.
[57]杨根敏.固定化青霉素酰化酶动力学参数测定[J].华东化工学院报.1989,15(4)：412-418.
[58]张小飞,刘友全,莫章桦.固定化青霉素酰化酶制备7-ADCA的条件优化[J].中南林学院学报.2006,26(5)：113-116
[59]Kim Y J, Weigand W A. Experimental analysis of a product; inhibited fermentation in an Aqueous Two-Phase Systems. Applied Biochemistry and Biotechnology.1992, 34:419-430
[60]Chu I M.,Chang S L.,Wang S H.,Yang W Y. Biotechnol. Techniques.1990,4(2):143-146
[61]Yang W Y. and Chu I M. Biotechnol. Techniques.1990,4(3):191-194
[62]Guan Y X., Mei L H., Zhu Z Q. Biotechnol. Techniques.1994,8(7):491-496
[63]关怡新.用两水相分配技术提纯抗生素的基础与工艺研究：[浙江大学博士学位论文].杭州：浙江大学,1994.
[64]李勉.新型两水相系统的基础及工程研究：[浙江大学博士学位论文].杭州：浙江大学,1996.
[65]Yang W Y, Lin C D., Chu I M.,Lee C J. Biotechnol.Bioeng.1994.43:439-445
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