产朊假丝酵母尿酸酶与苛求芽孢杆菌尿酸酶特性的比较

Comparison between Candida utilis uricase and Bacillus fastidious uricase

目的探讨产朊假丝酵母尿酸酶与苛求芽孢杆菌胞内尿酸酶的特性。方法复苏、培养新购进菌株以制备尿酸酶,利用硫酸铵分级沉淀法、DEAE纤维素52层析和制备PAGE纯化尿酸酶,测定纯化的尿酸酶的活力、表征,并分析其亚基构成。结果两种尿酸酶分子均为同四聚体,产朊假丝酵母尿酸酶的单肽链分子量和天然酶分子量分别为33.0 ku和134.0 ku,其最适pH值接近8.8,在pH7.4时可保留50%以上活性,该酶的米氏常数(Km)为(32.8±3.1)μmol/L(n=10),黄嘌呤的抑制常数为(4.8±0.2)μmol/L(n=3);而苛求芽孢杆菌胞内尿酸酶则分别为35.7 ku和151.0 ku,最适pH大于9.0,pH7.4时酶活性保留不到30%,其Km为(204±14)μmol/L(n=8),黄嘌呤的抑制常数为(41±7)μmol/L(n=5)。结论该研究对构建杂合药用尿酸酶以治疗高尿酸血症相关疾病具有一定的理论意义。

Objective To identify the characteristics of Candida utilis uricase and that of Bacillus fastidious intracellular uricase.Methods The new strains were cultivated to prepare the uricase.Then the uricase was purified by ammonium sulfate fractionation precipitation,DEAE-cellulose 52 chromatography and preparative PAGE.The activity,characterization and subunit constitution were determined with the purified uricase.ResultsThe two uricases were the iso-tetramer.The single peptide chain molecular weight and total molecular weight of Candida utilis uricase were 33.0 ku and 134.0 ku individually,the optimum pH was close to 8.8 and more than 50% activity was reserved at pH 7.4,the Michaelis-Menten constant was（32.8±3.1） μmol/L（n=10） and the inhibition constant of xanthine was（4.8±0.2） μmol/L（n=3） for this uricase.For Bacillus fastidious intracellular uricase,its single peptide chain molecular weight and total molecular weight were 35.7 ku and 151.0 ku,the optimum pH was over 9.0 and less than 30% activity left at pH 7.4,the Michaelis-Menten constant and the inhibition constant of xanthine were（204±14）μmol/L（n=8） and（41±7）μmol/L（n=5） individually for it.Conclusion Through the research,a significant theoretical basis was funded for constructing hybrid medicinal uricase to treat diseases associated with hyperuricemia.