Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli |
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| Institution: | 1. Faculty of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa 3200003, Israel;2. Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 3200003, Israel;1. Department of Industrial Technology and Innovation, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan;2. Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan;3. Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA;4. Life Science Research Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan;5. Ultizyme International Ltd., 1-13-16, Minami, Meguro, Tokyo 152-0013, Japan;6. Diabetes Research Institute, Mills-Peninsula Medical Center, 100 South San Mateo Drive, San Mateo, CA 94401, USA |
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| Abstract: | We have cloned a 1620-nucleotide gene encoding the catalytic subunit (α subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the α subunit. The deduced primary structure of the α 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 α subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 °C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the α subunit encoding an 18-kDa peptide, designated as γ subunit. The deduced primary structure of γ subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea. |
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