Screening Of Strain Producing Uricase, Extracting, Optimizing Of Fermentation Conditions And Characteristics Of Uricase

The thesis has included four parts: (1) screening of strain which could producing uricase; (2)optimizing fermentation conditions of producing uricase by Torula utilis Henneb 2. 281;(3)studying an extracting of uricase from Torula utilis Henneb 2. 281 ;(4)purif ication of uricase from Torula utilis Henneb 2.281 and studying of its properties. The achievements made are as follows:1. A strain Torula utilis Henneb 2. 281, which could produce uricase, was screened from 19 clones. The result of fermentation kinetics showed that uricase was produced synchronously with cell growth in liquid fermentation. Uricase activity was 0. 85 U/ml.2. Uricase-forming conditions of Torula utilis Henneb 2. 281 was investigated. The results were obtained as follow: Sucrose was the best carbon sources and proteose-peptone and yeast extract were the best nitrogen sources for enzyme formation. Uric acid, xanthine and guanine were found to be effective inducers for enzyme formation. Uric acid, thesubstrate of the uricase, was the best inducer. In addition, we had studied the effects of some metal ion on the enzyme activiy. The best conditions for liquid fermentation was found to be :pH 6.0, 28℃, the proper inoculation amount was 10%(v/v). The components of medium was 3%(w/v)sucrose, 2% proteose-peptone, 0. 5%yeast extract, 0. 05%(NH4)2SO4, 0. 05%uric acid, 0. 05%KCl. When the strain was grown in 250 ml conic flask containing 50 ml of the medium mentioned above on the rotary shaker (200r/min) at 28℃ for 23h. Uricase activity reached 11U/ml.3. The combination of ultrasonic and Triton X-100 and β -ME made it easy to release Uricase from Torula utilis Henneb 2.281 quickly when the pH of cell suspending mixture was 8. 5. The results of our researches were obtained as follow : the cell suspending mixture obtained the highest enzyme activity (22. 4U/ml), after being treated with by 5%(v/v)Triton X-100 and 5% β-ME and ultrasonic(30 min). The enzyme activity obtained by this method was obviously higher than by single treating method, e. g. ultrasonic (10.9U/ml) or Triton X-100 and β -ME (14.7U/ml).4. Uricase had been purified to electrophoretic homogeneity from liquid cultured cells of the Torula utilis Henneb 2.281 through the following separation procedure which including fractionation of 30~75% saturation of ( NH4 ) 2SO4, Phenyl Sepharose CL-4B chromatography and Xanthine-Agaro-se affinity chromatography. The specific activities of pure products reached 210.5 U/mg, and the yield of enzyme activity is 18.4% with a 176.9 purification factor. The purified uricase migrated as a single protein band on reduced SDS-PAGE. The purity analyzed by HPLC is above 95%. Native molecular masses was about 127kDa, measured by HPLC on TSK-Gel G2000SWXL. Reduced SDS-PAGE demonstrated the molecular weight of 33kDa. So, the uricase was consists of four identical subunits. IEF-PAGE identified the PI of uricase is 6. 7~7.5. The enzyme was found to have goodpH stability from 7~12 and thermal stability. Dynamic analysis showed that the optimum pH value for the enzyme is 8. 5. The optimum temperature is about 40 ℃. The Michaelis-menten constant (Km) of purified Uricase is 29. 4 μ mol/L at pH 8.5 and 40℃. In addition, we had studied the effects of some reagents on the enzyme activity.