产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究

从产气肠杆菌 (Enterobacteraerogenes)突变株EAM Z1中分离出一种具有较高转移酶活性的尿苷磷酸化酶 (UPase)。经测定这种Upase的分子量为 1 2 .8× 1 0 4,亚基分子量为 4 .3×1 0 4,由 3个同型亚基组成。N端氨基酸序列为 :MRMVDLIATKRDGGE。等电点为 4 .46。对尿苷的Km为 0 .2 9mmol L。酶反应的最适pH为 7.8,最适温度为 50℃。该酶能磷酸化尿苷、胸苷、5 氟尿苷、2′ 脱氧 5 氟尿苷及尿嘧啶 β D 阿拉伯呋喃糖 ,且具有较高的转移酶活性 ,能将尿苷和 5 氟尿嘧啶转化成 5 氟尿苷 (一种抗癌药物的中间体 ) ,其转化率为 47%。该酶的这些特性对于酶法合成核苷类抗肿瘤药物和抗病毒药物是十分有用的。

Purification and Characterization of a Uridine Phosphorylase from Enterobacter aerogenes EAM-Z1

A uridine phosphorylase(UPase) was isolation from Enterobacter aerogenes EAM-Z1 and purified by means of ammonium sulfate precipitation, DEAE-cellulose, Phenyl-Sepharose, DEAE-Sepharose, FPLC ion exchange, and Sephacryl S-200 column chromatography. The purified UPase showed homogeneity on the native polacrylamide gel electrophoresis. The UPase is a trimer of 43 kD subunits. Fifteen residues from the amino terminal end of UPase were identified as MRMVDLIATKRDGGE. The isoelectric point was pH 4.46. Michaelis constant for uridine was 0.29 mmol/L. The UPase has a maximal activity at a pH value of 7.8 and 50 degrees C. The UPase could catalyses the phosphorolysis of uridine, thymidine, 5-Fluorouridine, 5-Fluoro-2'-deoxyuridine, uracil-beta-D-arbinofuranoside, and could also catalyse the synthesis of 5-Fluorouridine, a better prodrug form of the anticancer drug 5-fluorouracil, from 5-fluorouracil and uridine, and 47% uridine was converted to 5-Fluoro-uridine.