产朊圆酵母尿酸酶的分离纯化及其部分性质研究  

Study on Purification and Characterization of Uricase from Candida utilis

作　　者：辛渝[1] 潭晓晶[1] 杨波[1] 卢戌[1] 余彩霞[1] 许晓燕[1] 孟延发[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《四川大学学报（自然科学版）》2005年第6期1246-1251,共6页Journal of Sichuan University(Natural Science Edition)

摘　　要：产朊圆酵母Y0401经尿酸诱导培养,菌体采用超声波处理,依次经过DEAE-Sepharose离子交换层析、Phenyl Sepharose CL-4B疏水层析和Xanthine-agarose亲和层析,尿酸酶比活力达到18U/mg,纯化倍数为409倍,回收率为19.2%.纯化后的酶经还原、非还原SDS-PAGE分析为单一蛋白染色带,HPLC显示纯度为98%以上.用HPLC和Superdex 200分子筛层析两种方法测得该酶的相对分子质量为127kDa,在还原条件下进行SDS-PAGE,测得表观分子质量为33kDa,综上结果推测该酶是由分子量相同的4个亚基组成的四聚体蛋白.等电聚焦显示其等电点在6.8～7.5之间.该酶具有较强的酸碱稳定性和热稳定性.最适pH为8.5,最适温度为40.0℃.在最适pH和温度条件下测得该酶Km值为3.34×10-5 mol/L.对某些金属离子和有机物对酶活性的影响也进行了研究.The Candida utilis sp. Y0401 was cultivated with inducement of substrate including uric acid. Using the techniques of ultrasonic fragmentation and ammonium sulfate precipitation for crude extraction, uricase was purified to electrophoretic homogeneity from liquid cultured cells of the Candida utilis sp. Y0401 through the following separation procedures which including DEAE-Sepharose chromatography, Phenyl Sepharose CL- 4B chromatography, and Xanthine-agarose affinity chromatography. The specific activity of pure preparation reached 18.0U/mg, and the yield of enzyme activity is 19.2% with a 409-fold purification factor. The purified uricase migrated as a single protein band on reduced / non-reduced SDS-PAGE. The purity analyzed by HPLC is above 98 %. Native molecular masses was about 127.0kDa, measured by Superdex 200 chromatography. Molecular weight of Single peptide chain at Reduced SDS-PAGE conditions demonstrated 33.0 kDa. According to above findings the uricase consisted of four subunits with identical Molecular weight. Polyacrylamide gel isoelectric focusing identified the pI of uricase is 6.8--7.5. The enzyme was found to have good pH stability from 7-10 and thermal stability, with 8.5 as the optimum pH of enzyme activity and 40℃ as the optimum temperature. The Km of purified Uricase is 3.34 × 10^-5 mol/L at pH 8.5 and 40.0℃. Also, the effects of some metal ions and organic compounds on the enzyme activity were studied .

关 键 词：尿酸酶 产朊圆酵母 尿酸酶性质 

分 类 号：Q554[生物学—生物化学]