Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.
We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea.
Inose
Ken
K
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-machi, Tokyo Koganei 184-8588, Japan.
Fujikawa
Masako
M
Yamazaki
Tomohiko
T
Kojima
Katsuhiro
K
Sode
Koji
K
D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013696:Temperature
D011994:Recombinant Proteins; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase