Cloning, Expression And Characterization Of A Glucose Dehydrogenase From Bacillus Sp. G3 In Escherichia Coli

Glucose dehydrogenase (GlcDH, EC 1.1.1.47), a member of the short-chain family of alcohol dehydrogenase, consists of four identical subunits (30KD), it catalyzes the oxidation ofβ-D-glucose to D-glucono-δ-lactone in the presence of cofactor NAD~+ or NADP~+. It has been reported that the GlcDH plays an important role in spore germination, which is a marker enzyme synthesized at stage of sporulation. In recent years, GlcDH were widely studied and applied in many fields, i.e. in biofuel cells, clinical tests, and as a catalyst for coenzyme regeneration in large-scale chiral synthesis. At present, it was extracted from the mammal liver or the fermentation products of Bacillus species primarily, so the source is restricted. Domestic GlcDH rely mainly on imports. In this paper, we carried out the experiment to isolate the GlcDH gene from Bacillus sp.G3, expressed it in E.coli BL21 (DE3), as well as the purification and characterization of this enzyme. The results of this research were reported as following.(1) The gene coding for GlcDH was isolated from Bacillus sp. G3. The GlcDH-G3 gene (accession no. GQ40283) has an open reading frame of 786 bp and codes for a polypeptide of 261 residues with a molecular mass of 28 kDa predicted by the gene sequence.(2) The ORF of GlcDH-G3 was successfully cloned and ligated with the pET-28a (+). The recombined plasmids pET-28a-gdh was transformed into BL21(DE3) competent cells, after IPTG induction, the GlcDH-G3 successfully expressed in heterologous host strain E.coli BL21 (DE3).(3) The GlcDH-G3 was purified to homogeneity by Ni-NTA-resin and a clear single band was observed on SDS-PAGE. After being purified 20-fold, the specific activity of the enzyme was 371.9 U/mg.(4) The properties of the GlcDH-G3 were studied. The enzyme was optimally active at 40℃and a pH 9.0, and displayed broad substrate specificity. The catalytic efficiency of the rGlcDH-G3 would be improved 4 times when NADP~+ was used as cofactor instead of NAD~+. GlcDH-G3 was stable in the acidic region at 25℃and below 40℃at pH 8.0.