title: Preparation of enzymes required for enzymatic quantification of 5-keto-D-gluconate and 2-keto-D-gluconate.
abstract: For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
authors.forename: Ittipon
authors.initials: I
authors.lastname: Saichana
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ano
authors.forename: Osao
authors.initials: O
authors.lastname: Adachi
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Matsushita
authors.forename: Hirohide
authors.initials: H
authors.lastname: Toyama
mesh_terms: D000595:Amino Acid Sequence; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D005942:Gluconates / Q000032:analysis* / Q000302:isolation & purification; D020576:Gluconobacter / Q000201:enzymology* / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014170:Transformation, Genetic
chemical_list: D005942:Gluconates; D005182:Flavin-Adenine Dinucleotide; C093583:5-ketogluconic acid; D002237:Carbohydrate Dehydrogenases; C030691:gluconic acid