PEG-重组酵母尿酸酶结合物的基本特性研究  

Study on General Properties of PEGylated Recombinant Uricase

作　　者：赵兴秀[1] 何义国[1] 姚兴川[2] 杜林方[2] 孟延发[2] 

机构地区：[1]四川理工学院,自贡643000 [2]四川大学生命科学学院,成都610064

出　　处：《中国生物工程杂志》2009年第11期23-28,共6页China Biotechnology

基　　金：四川省教育厅资助项目(08ZB043)

摘　　要：重组Candida utilis尿酸酶由含PET-Uricase表达质粒的重组E.coli JM109(DE3)经乳糖诱导表达,菌体破碎后依次经过硫酸铵沉淀、阴离子交换层析和凝胶过滤层析可以获得纯度95%的重组尿酸酶。还原性SDS-PAGE和HPLC测得其亚基表观分子量和天然分子量分别约为33kDa和130kDa。获得的纯酶与20kDa(mPEG)2-Lys-NHS在特定的条件下反应合成PEG-重组酵母尿酸酶结合物,考察了重组酵母尿酸酶PEG化前后的基本性质,结果显示PEG化尿酸酶的最适pH为7.5,较修饰前下降了1个pH单位,酸碱稳定范围与修饰前类似,都在pH6～10范围内稳定;修饰前后最适温度均为40℃,重组酵母尿酸酶的热稳定性和抗蛋白酶水解能力较PEG修饰前有较大提高;PEG化尿酸酶可保留修饰前酶活力的87.5%;在最适条件下,PEG-尿酸酶结合物的Km为3.57×10-5mol/L,而修饰前测得的Km为3.91×10-5mol/L。研究结果为深入探讨PEG化尿酸酶的结构与功能奠定了基础。Induced by lactose, the recombinant Candida utilis uricase was expressed in the E. coli JM109 （DE3） that carried the PET-uricase expression plasmid. The crude extraction was processed by the following purification procedures including ammonium sulfate precipitation, anion-exchange chromatography and gel filtration chromatography and the recombinant uricase with 95% purity could be obtained. Using HPLC and reduced SDS-PAGE, the native molecular weight of this enzyme and the apparent molecular weight of its subunit were determinied as 130 kDa and 33 kDa. The （mPEG）2-Lys-NHS of 20 kDa was used to modify the purified uricase and the study on zymological properties of uricase before and after modification was carried on. The results showed that the optimum pH of PEG modified uricase and native uricase were pH 7.5 and pH 8.5 respectively and both of them had good pH-stability from pH 6 to 10; both of the two had the same optimum temperature of 40℃, but the thermal stability and the ability of antitrypsin hydrolysis of the pegylated enzyme were much better than those of native enzyme ; the modified uricase retained 87.5% of the enzymatic activity of native uricase; under each optimum condition, the determined Km of uricase before and after pegylation were 3.91 × 10 ^-5 mol/L and 3.57 × 10^ -5 mol/L respectively. These findings may provide some information for deep investigation of structure and function of pegylated uricase.

关 键 词：CANDIDA utilis 尿酸酶 PEG化尿酸酶 蛋白质化学修饰 

分 类 号：Q544[生物学—生物化学]