密苏里游动放线菌葡萄糖异构酶基因xylA的克隆及其表达条件的优化

为克隆、表达密苏里游动放线菌葡萄糖异构酶(GI)基因xylA，并对其诱导表达条件进行初步优化。采用Slowdown PCR方法克隆得到密苏里游动放线菌(Actinoplanes missouriensis)CICIM B0118(A)的葡萄糖异构酶基因xylA，构建pET-28a(+)-xylA 表达载体，并转化至E. coli BL21 (DE3)，经异丙基-β -D- 硫代半乳糖苷(IPTG)诱导表达，并对其表达产物进行SDS-PAGE 电泳。结果表明，融合蛋白分子质量约为45kD。在诱导时间9h、0.6mmol/L IPTG、30℃和OD600nm 值为0.8 的最适培养条件下，酶比活力最高达到62.42U/mL。

Cloning of Glucose Isomerase Gene from Actinoplanes missouriensis and Its Expression in Escherichia coli

The xylA gene encoded glucose isomerase (GI) was amplified from genomic DNA of A. missouriensis CICIM B0118 (A) by Slowdown PCR and subcloned into the expression vector pET-28a(+) to obtain an N-terminus His-tagged fusion expression plasmid pET-28a(+)-xylA. pET-28a(+)-xylA was then transformed into E. coli BL21 (DE3). The recombinant protein was actively expressed in E. coli BL21 (DE3) in the presence of isopropy-β-D-thiogalactoside (IPTG). SDS-PAGE analysis showed that the partially purified recombinant protein exhibited a major band with an apparent molecular weight of 45 kD in, which was consistent with the molecular weight calculated from the amino acid sequence. When the induction conditions were time 9h, IPTG 0.6mmol/L, temperature 30℃ and OD600nm 0.8, the enzyme activity of recombinant GI were 62.42 U/mL. These results can offer an experimental basis for the further investigation of directed evolution of GI into an artificial enzyme with the ability to isomerize lactose into lactulose.