Purification and Characterization of a Uridine Phosphorylase from Enterobacter aerogenes EAM-Z1
产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究

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.