红纹黄单胞菌α-氨基酸酯水解酶的分离纯化及酶学性质

【目的】从红纹黄单胞菌中分离纯化了胞内α-氨基酸酯水解酶(AEH),并进行了酶学性质研究。【方法】采用乙酸丁酯破碎细胞,并相继用磷酸钙凝胶沉淀、硫酸铵分级沉淀、DEAE Sephadex A-50阴离子交换处理、CM Cellulose 52离子交换层析和Sephadex G-200凝胶过滤层析纯化得到了电泳纯α-氨基酸酯水解酶,并研究了此酶的酶学性质。【结果】SDS-PAGE显示α-氨基酸酯水解酶的亚基分子量为70 kDa。酶促合成头孢克洛的最适pH为6.8,最适温度为42℃,在pH5.0-8.0和35℃以下,酶保持了良好的稳定性。Mn2+和Ca2+对酶活有一定的促进作用,Cu2+、Fe2+及高浓度的丙酮对酶活有强的抑制作用。AEH催化D-苯甘氨酸甲酯、D-对羟基苯甘氨酸甲酯和头孢克洛水解反应的kcat/Km分别为123.7±3.7 mmol-1.s-1.L、2.9±0.6 mmol-1.s-1.L和101.3±2.1 mmol-1.s-1.L,AEH对D-苯甘氨酸甲酯的催化效率最高。AEH催化双底物反应的机制为乒乓机制,催化合成头孢克洛的kcat为547.3±38.2 s-1。【结论】有关红纹黄单胞菌α-氨基酸酯水解酶的酶学性质研究相对较少,本文的研究将为该酶催化合成β-内酰胺类抗生素的工业化应用提供重要参数。

Purification and characterization of alpha-amino acid ester hydrolase from Xanthomonas rubrillineans

[Objective] The intracellular α-amino acid ester hydrolase(AEH) from Xanthomonas rubrillineans was purified and characterized.[Methods] AEH was extracted by butyl acetate,and then purified to electrophoretic homogeneity by calcium phosphate gel precipitation,ammonium sulfate fraction precipitation,anion exchange with DEAE Sephadex A-50,cation exchange with CM cellulose 52,and Sephadex G 200 column chromatography.[Results] The subunit molecular mass of AEH was 70 kDa by SDS-PAGE.The optimal reaction pH for cefaclor synthesis was 6.8,and optimal temperature was 42 ℃.The enzyme was stable between pH 5.0 and 8.0,and at 35 ℃.The enzyme activity was enhanced by Mn2+ and Ca2+,however was strongly inhabited by Cu2+,Fe2+ and high concentration of acetone.The kinetic parameters that the enzyme hydrolyzed D-Phenylglycine methyl ester,D-Hydroxyphenylglycine methyl ester and cefaclor were determined,and the values of kcat/Km were 123.7±3.7,2.9±0.6 and 101.3±2.1 mmol-1·s-1·L respectively.The kcat/Km values indicated that the enzyme hydrolyzed D-Phenylglycine methyl ester more efficiently than other substrates.The mechanism of enzymatic reaction with bi-substrates by AEH is Ping-Pong kinetics,and the kcat value that the enzyme catalyzed the synthesis of cefaclor is 547.3±38.2 s-1.[Conclusion] The studies of AEH from Xanthomonas rubrillineans were rare,and our research may provide an important basis for industrial application of AEH for beta-lactam antibiotics synthesis.