红纹黄单胞菌α-氨基酸酯水解酶的分离纯化及酶学性质

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

摘要:

摘要:【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±3.7mmol－1·s－1·L、2.9±0.6mmol－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。

Abstract:

Abstract:［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．