Purification and Characterization of Uricase Enzyme Produced by Gliomastix gueg

Purification and Characterization of Uricase Enzyme Produced by  Gliomastix gueg

By

*Atalla, M. Mabrouk, *Eman R. Hamed,**Farag, M. M and *Nehad E. Ahmed

From

*Chemistry of Microbial Products Department, National Research Center (NRC), Dokki, Egypt

**Depatment of Biochemistry, Faculty of Agriculture, Cairo University, Giza, Egypt

Abstract

Uricase enzyme was produced and purified from Gliomastix gueg (NRC 1A) fermentation using ammonium sulphate selective precipitation (70% saturation) followed by ion exchange chromatography on DEAE- cellulose column. Results showed that pure enzyme produced during ammonium sulphate 70% saturation proved to be high uricolytic activity (146.84 U/ml) and specific activity (157.13 U/mg protein). Uricase produced from Gliomastix gueg (NRC 1A) was bound to the ion exchange column chromatography at pH 8.5 and eluted with linear NaCl (0-1M). Most of the uricolytic activity was found in the second protein peak in the 0.5 - 0.6M NaCl gradient. The enzyme was purified almost 33.2- fold with a specific activity 428.70U/mg protein. The molecular weight of the purified uricase was estimated by SDS-poly-acrylamide gel electrophoresis technique (60kDa). The maximum purified uricase activity was detected at 35ºc and pH 9.0, the enzyme was stable at 35ºc at different times tested. Maximum increase of uricase activity was obtained in the presence of Ca2+ ions reaching 387.05U/ml followed by Mn2+ (162.67U/ml). Inhibitors such as P.CMBA and cystine reduced the uricase activity by 15.26 and 12.50 U/ml, respectively. The effect of substrate concentration as well as enzyme concentration was also investigated. Results demonstrated that, 10µg of uric acid substrate and 100 µl (231U/ml) of the purified enzyme per reaction was enough to be used in the reaction mixture when incubated for 20 minutes. Results showed that the rate of uric acid consumption in the reaction mixture by purified uricase enzyme increase with increasing the incubation time.

Full text: Purification and Characterization of Uricase Enzyme Produced by Gliomastix gueg