枯草杆菌ylnF基因编码蛋白产物性质的研究

The Identification of the Product Encoded by the ylnF Gene from Bacillus subtilis

将枯草杆菌ylnF基因经PCR扩增,酶切后插入表达质粒pET15b中,转化大肠杆菌BL21(DE3),诱导表达,金属螯合亲和层析一步纯化,酶活性测定。结果表明:ylnF基因编码产物是依赖于NAD+的前咕啉-2氧化酶,一个西罗血红素生物合成末端的酶。SDS-PAGE显示YlnF亚基分子量约22kD,全酶分子量约25kD,显示酶为单体酶。将Asp102定点突变Ala102,酶活丧失,表明Asp102在催化中起重要作用。

The ylnF gene from Bacillus subtilis, amplified by PCR, was excised with Nde Ⅰ and BamH Ⅰ, and inserted into the vector pET15b with the same manner, given the constructed vector pYF, which encoded the His6-tagged YlnF. The vector pYF was transformed into Escherichia coli strain BL21 （DE3）. The recombinant ceils were cultured, induced and disrupted. The His6-tagged YlnF was purified to almost homogeneous by Ni-NTA affinity chromatography. The enzymatic assay revealed that the YlnF protein has the activity of precorrin-2 oxidase, an enzyme involved in the terminal biosynthetic pathway of siroheme. The relative molecular weight of UROD holoenzyme was estimated about 25 kD using Superdex 75 size exclusion and that of UROD subunit was about 22 kD, as shown by SDS/PAGE. Site-directed mutation indicated that Aspire played the vital role in the catalysis.