青霉素酰化酶性质及应用研究
摘要
本论文研究了有机溶剂、温度、pH值对工业上广泛应用的青霉素酰化酶活性的影响,优化反应条件,如反应介质、温度、pH值、底物浓度等因素,研究了青霉素酰化酶在催化合成阿莫西林中的应用。主要进行了如下工作:
1.根据LogP规律筛选出12种有机溶剂,考察了它们对青霉素酰化酶活性的影响。在pH值等于7的缓冲溶液和有机溶剂各占50%(V/V)的体系中,通过测定酶在此体系中活性的变化,选出对酶活性影响较小的有机溶剂。发现丙三醇、乙二醇、二甲基亚砜等有机溶剂对酶活性影响较小;研究了温度及pH值对青霉素酰化酶活性及稳定性的影响,发现在20℃~55℃的范围内,随着温度升高,酶的活力增大,但酶的稳定性逐渐降低,综合得出温度为40℃时酶的活性及稳定性较好;pH值在6~8的范围内变化时,酶的活力先升高后下降,pH值等于7.5时,活力达到最大,而酶稳定性却随着pH值增大而下降,综合得出pH值为6.5或7时酶的活性及稳定性较好。
2.从对羟基苯甘氨酸邓钾盐出发制备了对羟基苯甘氨酸甲酯(p-OH-PGME),红外和熔点分析表明,制备的产物纯度较高。测定了对羟基苯甘氨酸甲酯(p-OH-PGME)在乙二醇,1,4-丁二醇,环己烷,正丁醇,丙三醇,叔丁醇等几种溶剂中的表观溶解度,发现对羟基苯甘氨酸甲酯(p-OH-PGME)在正丁醇,乙二醇等溶剂中的表观溶解度较好,底物的溶解性好在酶催化合成β-内酰胺抗生素反应中有利于向合成方向进行。
3.优化了青霉素酰化酶催化合成阿莫西林的反应条件,分别考察了温度、pH值、底物浓度以及不同反应介质时,6-APA的转化率和对羟基苯甘氨酸甲酯(p-OH-PGME)的水解率,得出较理想的反应条件为:乙二醇-缓冲溶液体系、温度为20℃、溶液pH值为6.5、底物6-APA浓度为75 mmol/L、原料浓度配比:6-APA: p-OH-PGME为1:2。
The effect of organic solvent, temperature and pH value on the activity of the industrial widely used penicillin G acylase(PGA) were studied in this thesis. The reaction conditions, including the reaction medium, temperature, pH value and the concentration of substrate, were optimized and used in the synthesis of amoxicillion with PGA as the catalytist. The following works have been done:
1. Some organic solvents were selected according to the law of logP for the further investigation about their effect on the activity of PGA. The preferable organic solvent which has slightly effect on the activity of PGA was chosen out by monitoring the change of the activity of PGA. Glycerin, ethylene glycol and dimethyl sulphoxide(DMSO) showed slightly effect on the activity of PGA. The effects of temperature and pH value on the activity and stability of PGA were also investigated. It was found that the activity of PGA increased and the stability of PGA decreased as the temperature increased from 20℃to 55℃. The most suitable temperature was 40℃. The activity of PGA increased firstly and then decreased when the pH changed from 6 to 8. However, the stability of PGA decreased as the pH value increasing. The activity and stability of PGA reached to the best point at pH 6.5 or 7.
2. The Methyl p-hydroxyphenylglycinate (p-OH-PGME) was synthesized from the p-hydroxyphenylglycinate dane salt. The IR and melting point analysis were used to characterized the synthesized p-OH-PGME. The apparent solubility of p-OH-PGME in some solvents was measured. The n-butyl alcohol and ethylene glycol are better solvents for p-OH-PGME. The better solubility of substrate is propitious to the synthesis ofβ-lactam antibiotic.
3. The reaction conditions in the synthesis of amoxicillion using PGA as catalyst were optimized. The conversion ratio of 6-APA and hydrolytic ratio of p-OH-PGME are investigated under different conditions. As a result, the favorable reaction conditions including the reaction medium, temperature, pH value of solution, concentration of 6-APA , the ratio of 6-APA and p-OH-PGME are ethylene glycol-buffer solution, 20℃, 6.5, 75 mmol/L, 1:2, respectively.
1.根据LogP规律筛选出12种有机溶剂,考察了它们对青霉素酰化酶活性的影响。在pH值等于7的缓冲溶液和有机溶剂各占50%(V/V)的体系中,通过测定酶在此体系中活性的变化,选出对酶活性影响较小的有机溶剂。发现丙三醇、乙二醇、二甲基亚砜等有机溶剂对酶活性影响较小;研究了温度及pH值对青霉素酰化酶活性及稳定性的影响,发现在20℃~55℃的范围内,随着温度升高,酶的活力增大,但酶的稳定性逐渐降低,综合得出温度为40℃时酶的活性及稳定性较好;pH值在6~8的范围内变化时,酶的活力先升高后下降,pH值等于7.5时,活力达到最大,而酶稳定性却随着pH值增大而下降,综合得出pH值为6.5或7时酶的活性及稳定性较好。
2.从对羟基苯甘氨酸邓钾盐出发制备了对羟基苯甘氨酸甲酯(p-OH-PGME),红外和熔点分析表明,制备的产物纯度较高。测定了对羟基苯甘氨酸甲酯(p-OH-PGME)在乙二醇,1,4-丁二醇,环己烷,正丁醇,丙三醇,叔丁醇等几种溶剂中的表观溶解度,发现对羟基苯甘氨酸甲酯(p-OH-PGME)在正丁醇,乙二醇等溶剂中的表观溶解度较好,底物的溶解性好在酶催化合成β-内酰胺抗生素反应中有利于向合成方向进行。
3.优化了青霉素酰化酶催化合成阿莫西林的反应条件,分别考察了温度、pH值、底物浓度以及不同反应介质时,6-APA的转化率和对羟基苯甘氨酸甲酯(p-OH-PGME)的水解率,得出较理想的反应条件为:乙二醇-缓冲溶液体系、温度为20℃、溶液pH值为6.5、底物6-APA浓度为75 mmol/L、原料浓度配比:6-APA: p-OH-PGME为1:2。
The effect of organic solvent, temperature and pH value on the activity of the industrial widely used penicillin G acylase(PGA) were studied in this thesis. The reaction conditions, including the reaction medium, temperature, pH value and the concentration of substrate, were optimized and used in the synthesis of amoxicillion with PGA as the catalytist. The following works have been done:
1. Some organic solvents were selected according to the law of logP for the further investigation about their effect on the activity of PGA. The preferable organic solvent which has slightly effect on the activity of PGA was chosen out by monitoring the change of the activity of PGA. Glycerin, ethylene glycol and dimethyl sulphoxide(DMSO) showed slightly effect on the activity of PGA. The effects of temperature and pH value on the activity and stability of PGA were also investigated. It was found that the activity of PGA increased and the stability of PGA decreased as the temperature increased from 20℃to 55℃. The most suitable temperature was 40℃. The activity of PGA increased firstly and then decreased when the pH changed from 6 to 8. However, the stability of PGA decreased as the pH value increasing. The activity and stability of PGA reached to the best point at pH 6.5 or 7.
2. The Methyl p-hydroxyphenylglycinate (p-OH-PGME) was synthesized from the p-hydroxyphenylglycinate dane salt. The IR and melting point analysis were used to characterized the synthesized p-OH-PGME. The apparent solubility of p-OH-PGME in some solvents was measured. The n-butyl alcohol and ethylene glycol are better solvents for p-OH-PGME. The better solubility of substrate is propitious to the synthesis ofβ-lactam antibiotic.
3. The reaction conditions in the synthesis of amoxicillion using PGA as catalyst were optimized. The conversion ratio of 6-APA and hydrolytic ratio of p-OH-PGME are investigated under different conditions. As a result, the favorable reaction conditions including the reaction medium, temperature, pH value of solution, concentration of 6-APA , the ratio of 6-APA and p-OH-PGME are ethylene glycol-buffer solution, 20℃, 6.5, 75 mmol/L, 1:2, respectively.
引文
[1] Sakaguchi K, Murao S, Anew enzyme, penicillin-amidase. J Agril Chem Soc Japan, 1950,23:411-416
[2] Vadamme E J, Enzyme involved in P-lactam antibiotic biosynthesis, Advanced in Applied Microbilogy. 1977, 21:89-123
[3] Vadamme E J, Voets J P, Microbial penicillin acylase, Advanced in Applied Microbilogy, 1974, 17:311-369
[4] Kostadinov M, Nilolov A, Tsoneva.N, et al, New tetrazolc-l-acid and easters for enzymatic synthesis of cefazolin, Appl Biocherm Biotechnol,1992,33:177-182
[5] Hamilton-Miller J M T, penicillin acylase, Bacteriological Reviews, 1966, 30:761-771
[6] Abbott B J, Preparation of pharmaceutical compounds by immobilized enzyme and cells, Advanced in Applied Microbiology, 1976, 20:203-257
[7]高波，朱广山，付学奇等，不同介孔材料固定青霉素酰化酶的稳定性研究，高等学校化学学报，2005，26 (10)：1852～1854
[8] Thomas RM, Barends H Y, Bauke W D, Three-dimensional structures of enzymes useful for P-lactam antibiotic production, Current Opinion in Biotechnology 2004, 15:356-363
[9] Duggleby H J, Tolley S P, Hill C P, et al, Penicillin acylase has a single-amino-acid catalytic center, Nature 1995, 373:264-268.
[10] Done S H, Brannigan J A, Moody PCE, et al, Ligand-induced conformational change in penicillin acylase, J Mol Biol 1998, 284:463-475.
[11] Kazan D, Influence of Polyhydric Compounds on the pH Stability of Penicillin G Acylase Obtained from a Mutant of Escherichia coli ATCC 1105 Process Biochem, 1996, 31(7): 691-697
[12] Kazan D, Stabilization of Penicillin G Acylase Against pH by Chemical Cross-Linking, Process Biochem, 1996, 31(2):135~140
[13] Dilek K, Effect of dextran polymers on the stability of soluble Escherichia Coli penicillin G acylase, J Chem Technol Biotech, 1999, 74:1157-1164
[14] Azevedo A M, Kinetic and stability studies of penicillin acylase in reversed micelles, J Chem Technol Biotech, 1999, 74: 1110-1116
[15] Chauhan S, Nichkawade A, Iyengar M, et al, Chainia penicillin v acylase:straincharacterisics, enzyme immobilization, and kinetic studies, Curr Microbiol, 1998, 37(3):186~191
[16]韩辉，徐冠珠，颗粒状固定化青霉素酰化酶的研究，微生物学报，2001，41(2)：204～207
[17]鲜海军，王祯祥，以聚丙烯纤维为载体制备固定化青霉素G酰化酶的研究，微生物学报，2001，41(4)：475～479
[18]徐冠珠，韩辉，王祯祥等，固定化PGA水解头孢菌素C制备7一ADCA的研究，微生物学报，1997，37(3)：190～193
[19]徐冠珠，王祯样，朱丽钊等，固定化青霉素酰化酶的研究，微生物学报，1992，32(31：212～215
[20]崔福绵，石家骥，白小东，酶法合成头孢环己二烯，微生物学报，1998，38(4)：300～303
[21]石家骥，崔福绵，青霉素G酰化酶在Y-氧化铝上的吸附交联固定化，中国抗生素杂志，2001，26(5)：334～336
[22]陈晖，韩辉，徐冠珠，聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究，微生物学报，2002，42(1)：76～80
[23] Woodward RB, Recent advances in the chemistry of P-lactam antibiotics, Spec Publ ChemSoc, 1977, 28:167-180
[24] Blechert S, Synthesis of new P-lactam antibiotics, Nachr Chem Tech Lab, 1979,27(3): 127-128
[25] Suga K I, Sorai T, Shioya S, Hydrolysis of penicillin by combination of immobilized penicillin acylase and electrodialysis. Microb Util Renewable Resour, 1993, 8: 93-104
[26] Haber F M, Cephalosporins and penicillins, Chemistry and biology, 1975, 12:314-320
[27] Park C B, Lee S B, Ryu D Y, Stabilisation of penicillin V acylase fromStreptomyces lavendulae by covalent immobilisation. J.Mol .Cataly .B: Enzyme, 2000, 9: 275-281
[28] Cole M, Deacylation of acylaminocompounds other than penicillin by thecell-bound penicillin acylase of Escherichia coli, Biochem.J. 1969,115: 741-746
[29] Becketta A, Process for the preparation of 7-amino and 7-substituted amino -desacetoxy cephalosporins from the corresponding 6-substituted amino -penicillin sulphoxides, Biochem Soc Trans, 1991, 19: 443-446
[30] Ariens E J, Wuis E W, Stereo selectivity of bioactive xenobiotics, A pre -Pasteur atitude in medicinal chemistry, pharmacokinetics and clinical pharmacology ,Biochem Pharmacol, 1988,37 :9~18
[31] Zmijewski M J, Briggs B S, hompson AR, Enantioselective acylation of aP-lactam intermediate in the synthesis of Loracarbef using penicillin G amidase Tetrahedron Lett, 1991, 32:1621-1622
[32] Brtnik F, Barth T, JoSt K, Synthesis and application of methionin S-tert-butylsulfonium peptides, Collect Czech Chem Commun, 1981,46 :1983-1987
[33] Waldmann H, Bohm G, Schmid H, Maximum cell productibity by repeated fed- batch culture for constant yield case.Syn.Lett, 1994,33 :116-123
[34] Hermann P, Anti-Endothelial Cell Antibodies in Takayasu Arteritis, Biomed Biochem.ACTA, 1991, 5:19-31
[35] Shaw S Y, Shyu J C ,Hsieh Y W , Enzymatic synthesis of cephalothin by Penicillin G acylase Enzyme, Microb Technol.2000, 26 :142-151
[36] Fernandez L R, Cristina M R, Jose M G, Dynamic reaction design of enzymic biotransformations in organic media: equilibrium- controlled synthesis of antibiotics by penicillin G acylase Biotechnol Appl Biochem, 1996, 24:129-143
[37] Diender M B, Straathof A J, Does V T, et al, Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction, Enzyme Microb Technol, 2000, 27(8):576~582
[38] Fernandez L R, Cristina M R, Jose M G, The use of stabilized penicillin acylase derivatives improves the design of kinetically controlled synthesis, J Mol Catal A: Chem, 1995, 101:91-97
[39] Arroyo M, Torres-Guzman R, Castillon M , Carmen Acebal Prediction ofpenicillin V acylase stability in water-organic co-solvent monophasic system as a function of Solvent composition.Enzyme Microb Technol, 2000, 7(1,2) : 122-126
[40] Femadez L R, Rosell C M, Guisan J M , Enzyme reaction engineering: Synthesis of antibiotic catalysed by stabilized penicillin G acylase in the presence of organic solvents, Enzyme Microb Technol, 1991,13(11) :898~905
[41] Diender M B, Straathof A J, Heijnen J J, Feasibility of the thermodynamically controlled synthesis of amoxicillin, Journal of Molecular Catalysis B:Enzymatic 1998,15:249-253
[42] Andre's I, Adrian F, Kinetically controlled synthesis of ampicillin withimmobilized penicillin acylase in the presence of organic cosolvents, Journal of Molecular Catalysis B: Enzymatic 2001,11:587-595
[43] Min G K, Sun B L, Penicillin acylase-catalyzed synthesis of pivaampicillin: Effect of reaction variables and organic cosolvents, Journal of Molecular Catalysis B: Enzyme, 1996, 1:71-80
[44] Rosell C M, Terreni M, Fernandez-Lafuente R , et al, A criterion for the selection of Monophasic solvents for enzymatic synthesis , Enzyme Microb Technol, 1998,23:64-69
[45] Carolina A, Mauri cio T, Valentina M, Effect of cosolvent and pH on thekinetically controlled synthesis of cephalexin with immobilised penicillin acylase, Process Biochemistry, 2002 ,38: 351-360
[46] Marcelo P A, Andrea L O, Raquel L C , Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates, Journal of Molecular Catalysis B: Enzymatic 2005 ,33:81-86
[47] YANG L, WEI D Z, XUE P ,et al, Kinetically Controlled synthesis of Cefaclor using Penicillin G Acylase Jouronl of molecular catalysis, 2003,17(2):81~87
[48] Yen T C , Christopher T, Seto D , Trivalent r-Ketocarboxylic Acids as Inhibitors of Protein Tyrosine Phosphatases, J Med Chem, 2002, 45: 3946-3952
[2] Vadamme E J, Enzyme involved in P-lactam antibiotic biosynthesis, Advanced in Applied Microbilogy. 1977, 21:89-123
[3] Vadamme E J, Voets J P, Microbial penicillin acylase, Advanced in Applied Microbilogy, 1974, 17:311-369
[4] Kostadinov M, Nilolov A, Tsoneva.N, et al, New tetrazolc-l-acid and easters for enzymatic synthesis of cefazolin, Appl Biocherm Biotechnol,1992,33:177-182
[5] Hamilton-Miller J M T, penicillin acylase, Bacteriological Reviews, 1966, 30:761-771
[6] Abbott B J, Preparation of pharmaceutical compounds by immobilized enzyme and cells, Advanced in Applied Microbiology, 1976, 20:203-257
[7]高波，朱广山，付学奇等，不同介孔材料固定青霉素酰化酶的稳定性研究，高等学校化学学报，2005，26 (10)：1852～1854
[8] Thomas RM, Barends H Y, Bauke W D, Three-dimensional structures of enzymes useful for P-lactam antibiotic production, Current Opinion in Biotechnology 2004, 15:356-363
[9] Duggleby H J, Tolley S P, Hill C P, et al, Penicillin acylase has a single-amino-acid catalytic center, Nature 1995, 373:264-268.
[10] Done S H, Brannigan J A, Moody PCE, et al, Ligand-induced conformational change in penicillin acylase, J Mol Biol 1998, 284:463-475.
[11] Kazan D, Influence of Polyhydric Compounds on the pH Stability of Penicillin G Acylase Obtained from a Mutant of Escherichia coli ATCC 1105 Process Biochem, 1996, 31(7): 691-697
[12] Kazan D, Stabilization of Penicillin G Acylase Against pH by Chemical Cross-Linking, Process Biochem, 1996, 31(2):135~140
[13] Dilek K, Effect of dextran polymers on the stability of soluble Escherichia Coli penicillin G acylase, J Chem Technol Biotech, 1999, 74:1157-1164
[14] Azevedo A M, Kinetic and stability studies of penicillin acylase in reversed micelles, J Chem Technol Biotech, 1999, 74: 1110-1116
[15] Chauhan S, Nichkawade A, Iyengar M, et al, Chainia penicillin v acylase:straincharacterisics, enzyme immobilization, and kinetic studies, Curr Microbiol, 1998, 37(3):186~191
[16]韩辉，徐冠珠，颗粒状固定化青霉素酰化酶的研究，微生物学报，2001，41(2)：204～207
[17]鲜海军，王祯祥，以聚丙烯纤维为载体制备固定化青霉素G酰化酶的研究，微生物学报，2001，41(4)：475～479
[18]徐冠珠，韩辉，王祯祥等，固定化PGA水解头孢菌素C制备7一ADCA的研究，微生物学报，1997，37(3)：190～193
[19]徐冠珠，王祯样，朱丽钊等，固定化青霉素酰化酶的研究，微生物学报，1992，32(31：212～215
[20]崔福绵，石家骥，白小东，酶法合成头孢环己二烯，微生物学报，1998，38(4)：300～303
[21]石家骥，崔福绵，青霉素G酰化酶在Y-氧化铝上的吸附交联固定化，中国抗生素杂志，2001，26(5)：334～336
[22]陈晖，韩辉，徐冠珠，聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究，微生物学报，2002，42(1)：76～80
[23] Woodward RB, Recent advances in the chemistry of P-lactam antibiotics, Spec Publ ChemSoc, 1977, 28:167-180
[24] Blechert S, Synthesis of new P-lactam antibiotics, Nachr Chem Tech Lab, 1979,27(3): 127-128
[25] Suga K I, Sorai T, Shioya S, Hydrolysis of penicillin by combination of immobilized penicillin acylase and electrodialysis. Microb Util Renewable Resour, 1993, 8: 93-104
[26] Haber F M, Cephalosporins and penicillins, Chemistry and biology, 1975, 12:314-320
[27] Park C B, Lee S B, Ryu D Y, Stabilisation of penicillin V acylase fromStreptomyces lavendulae by covalent immobilisation. J.Mol .Cataly .B: Enzyme, 2000, 9: 275-281
[28] Cole M, Deacylation of acylaminocompounds other than penicillin by thecell-bound penicillin acylase of Escherichia coli, Biochem.J. 1969,115: 741-746
[29] Becketta A, Process for the preparation of 7-amino and 7-substituted amino -desacetoxy cephalosporins from the corresponding 6-substituted amino -penicillin sulphoxides, Biochem Soc Trans, 1991, 19: 443-446
[30] Ariens E J, Wuis E W, Stereo selectivity of bioactive xenobiotics, A pre -Pasteur atitude in medicinal chemistry, pharmacokinetics and clinical pharmacology ,Biochem Pharmacol, 1988,37 :9~18
[31] Zmijewski M J, Briggs B S, hompson AR, Enantioselective acylation of aP-lactam intermediate in the synthesis of Loracarbef using penicillin G amidase Tetrahedron Lett, 1991, 32:1621-1622
[32] Brtnik F, Barth T, JoSt K, Synthesis and application of methionin S-tert-butylsulfonium peptides, Collect Czech Chem Commun, 1981,46 :1983-1987
[33] Waldmann H, Bohm G, Schmid H, Maximum cell productibity by repeated fed- batch culture for constant yield case.Syn.Lett, 1994,33 :116-123
[34] Hermann P, Anti-Endothelial Cell Antibodies in Takayasu Arteritis, Biomed Biochem.ACTA, 1991, 5:19-31
[35] Shaw S Y, Shyu J C ,Hsieh Y W , Enzymatic synthesis of cephalothin by Penicillin G acylase Enzyme, Microb Technol.2000, 26 :142-151
[36] Fernandez L R, Cristina M R, Jose M G, Dynamic reaction design of enzymic biotransformations in organic media: equilibrium- controlled synthesis of antibiotics by penicillin G acylase Biotechnol Appl Biochem, 1996, 24:129-143
[37] Diender M B, Straathof A J, Does V T, et al, Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction, Enzyme Microb Technol, 2000, 27(8):576~582
[38] Fernandez L R, Cristina M R, Jose M G, The use of stabilized penicillin acylase derivatives improves the design of kinetically controlled synthesis, J Mol Catal A: Chem, 1995, 101:91-97
[39] Arroyo M, Torres-Guzman R, Castillon M , Carmen Acebal Prediction ofpenicillin V acylase stability in water-organic co-solvent monophasic system as a function of Solvent composition.Enzyme Microb Technol, 2000, 7(1,2) : 122-126
[40] Femadez L R, Rosell C M, Guisan J M , Enzyme reaction engineering: Synthesis of antibiotic catalysed by stabilized penicillin G acylase in the presence of organic solvents, Enzyme Microb Technol, 1991,13(11) :898~905
[41] Diender M B, Straathof A J, Heijnen J J, Feasibility of the thermodynamically controlled synthesis of amoxicillin, Journal of Molecular Catalysis B:Enzymatic 1998,15:249-253
[42] Andre's I, Adrian F, Kinetically controlled synthesis of ampicillin withimmobilized penicillin acylase in the presence of organic cosolvents, Journal of Molecular Catalysis B: Enzymatic 2001,11:587-595
[43] Min G K, Sun B L, Penicillin acylase-catalyzed synthesis of pivaampicillin: Effect of reaction variables and organic cosolvents, Journal of Molecular Catalysis B: Enzyme, 1996, 1:71-80
[44] Rosell C M, Terreni M, Fernandez-Lafuente R , et al, A criterion for the selection of Monophasic solvents for enzymatic synthesis , Enzyme Microb Technol, 1998,23:64-69
[45] Carolina A, Mauri cio T, Valentina M, Effect of cosolvent and pH on thekinetically controlled synthesis of cephalexin with immobilised penicillin acylase, Process Biochemistry, 2002 ,38: 351-360
[46] Marcelo P A, Andrea L O, Raquel L C , Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates, Journal of Molecular Catalysis B: Enzymatic 2005 ,33:81-86
[47] YANG L, WEI D Z, XUE P ,et al, Kinetically Controlled synthesis of Cefaclor using Penicillin G Acylase Jouronl of molecular catalysis, 2003,17(2):81~87
[48] Yen T C , Christopher T, Seto D , Trivalent r-Ketocarboxylic Acids as Inhibitors of Protein Tyrosine Phosphatases, J Med Chem, 2002, 45: 3946-3952