关键词: 变形假单胞菌, 2-酮基葡萄糖酸差向异构酶, kguE基因, 克隆, 表达, 生物信息学

Abstract: The complete nucleotide sequence of the gene (kguE) encoding 2-ketogluconate epimerase was cloned by touchdown polymerase chain reaction (TD-PCR) from an industrial 2-ketogluconic acid producer, Pseudomonas plecoglossicida JUIM01. A recombinant vector was constructed by ligating the restriction enzymes digested products of the target gene to pET-28a(+) vector and then transferred into the expression host E. coli BL21(DE3). A specific fusion protein with molecular weight of about 30.5 ku was expressed in the recombinant strain E. coli BL21(DE3)/pET-28a(+)-kguE after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction with a positive Western-blot result. Bioinformatic analysis showed that the protein was predicted to be a hydrophilic protein with molecular weight of 28.5 ku located in the cytoplasm, sharing 78% amino acid sequence identity with the 2-ketogluconate epimerase from P. putida. The predicted secondary structure consisted of 40.62% of α-helix, 17.19% of extended strand and 42.19% of random coil. This study is expected to provide a basis for further elucidating the function of 2-ketogluconate epimerase in P. plecoglossicida.

Key words: Pseudomonas plecoglossicide, 2-ketegluconate epimerase (KguE), kguE gene, cloning, expression, bioinformatics