关键词: 密苏里游动放线菌, 葡萄糖异构酶, 表达, Slowdown PCR

Abstract:

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.

Key words: Actinoplanes missouriensis, glucose isomerase, expression, slowdown PCR