微杆菌属ZZJ4-1菌株的耐热尿酸氧化酶基因的克隆及重组酶性质

Cloning and characterization of a thermostable urate oxidase from Microbacterium sp. strain ZZJ4-1

为了研究微杆菌Microbacterium sp.ZZJ4-1菌株的耐热尿酸氧化酶(Uox)的性质,克隆其基因(uox),得到1个894 bp的开放阅读框。该基因与多数已报道的uox无明显同源性,仅与球形节杆菌Arthrobacterglobiformis的uox有72%的同源性。将基因插入质粒pET-15b构成pET-15b-uox表达载体,转化至Escherichiacoli BL21(DE3)中诱导表达。对重组Uox的主要理化性质研究表明:该酶由大小约为35 kDa的亚基组成;其最佳反应温度和pH分别为30℃和7.5;在65℃以下和pH 8.5～11.0范围内稳定;以尿酸为底物的Km值为0.22 mmol/L;Ag+、Zn2+、Cu2+和SDS均能完全抑制酶活,Tween 20、Tween 80和Triton X-100对酶活有一定的促进作用。该重组酶的耐热性是目前报道的重组Uox中最好的,这一特性有利于其在诊断治疗中的开发应用。

In order to characterize a thermostable urate oxidase （Uox） from Mi its gene （uox）. The open reading frame of uox contained 894 base pairs and Alignment of gene sequences indicated there was no obvious identity with the m lbund with uox from Arthrobacter globiformis. We inserted the gene into the pl crobacterium sp. strain ZZJ4-1, we cloned encoded a protein with 297 amino acids. ost reported uox and that 72% identity was asmid pET-15b to construct an expression vector pET-15b-uox and got it induced expression in Escherichia coli BL21 （DE3）. After the purification of the recombinant Uox by the His＇Bind column, we studied some properties of it. It was composed of subunits with a molecular mass of about 35 kDa. The optimal temperature and pH was 30 ~C and pH 7.5. It was stable below 65 ~C and from pH 8.5 to 11.0. The Km value was 0.22 mmol/L with the uric acid as the substrate. Ag~, Zn2＋, Cu2＋ and SDS could totally inhibit its activity while Tween 20, Tween 80 and Triton X-100 had a slight promotion effect. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox. Based on this property, it would be very useful in the application.