%0 Journal Article
%T Purification and characterization of alpha-amino acid ester hydrolase from Xanthomonas rubrillineans<br>红纹黄单胞菌α-氨基酸酯水解酶的分离纯化及酶学性质
%A Feng Qu
%A Baxian Yi
%A Lijuan Ye
%A <br>屈凤
%A 易八贤
%A 叶丽娟
%J 微生物学报
%D 2012
%I 
%X 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.
%K α-amino acid ester hydrolase
%K beta-lactam antibiotics
%K purification
%K enzyme characterization
%K Ping-Pong kinetics<br>α-氨基酸酯水解酶
%K β-内酰胺类抗生素
%K 分离纯化
%K 酶学性质
%K 乒乓机制
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A3C6BA55AB623B90FA9104CFFC826F3C&aid=FDB09CD97DEFC4B69303286647FE1BDC&yid=99E9153A83D4CB11&vid=286FB2D22CF8D013&iid=94C357A881DFC066&sid=E6A0A363CA4FC5CF&eid=3EABEBD973E45554&journal_id=0001-6209&journal_name=微生物学报&referenced_num=0&reference_num=0