Site directed mutagenesis studies of FAD-dependent glucose dehydrogenase catalytic subunit of Burkholderia cepacia

• Published in: Biotechnology letters Vol. 30; no. 11; pp. 1967 - 1972
• Main Authors: Yamaoka, Hideaki, Yamashita, Yuki, Ferri, Stefano, Sode, Koji
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.11.2008; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Alanine - genetics; Amino Acid Sequence; Applied Microbiology; Asparagine - genetics; Base Sequence; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Burkholderia cepacia; Burkholderia cepacia - enzymology; Burkholderia cepacia - genetics; Burkholderia cepacia - metabolism; Catalytic Domain - genetics; Dehydrogenase; Diabetes; Enzymes; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Glucose; Glucose - metabolism; Glucose 1-Dehydrogenase - genetics; Glucose 1-Dehydrogenase - metabolism; Life Sciences; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed - methods; Mutation; Original Research Paper; Sequence Homology, Amino Acid; Substrate Specificity; Substrates; Glucose dehydrogenase; Glucose sensor; Diabetes; Substrate specificity; Enzyme; Diabetes mellitus; Site directed mutagenesis; Glucose 1-dehydrogenase; Subunit; Detector; Burkholderia cepacia; Bacteria; Oxidoreductases
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-008-9777-3

A FAD-dependent glucose dehydrogenase (FADGDH) mutant with narrow substrate specificity was constructed by site-directed mutagenesis. Several characteristics of FADGDH, such as high catalytic activity and high electron transfer ability, make this enzyme suitable for application to glucose sensors. However, for further applications, improvement of the broad substrate specificity is needed. In this paper, we mutated two residues, Asn475 and Ala472, which are located near the putative active site of the catalytic subunit of FADGDH and have been predicted from the alignment with the active site of glucose oxidase. Of the 38 mutants constructed, Ala472Phe and Asn475Asp were purified and their activities were analyzed. Both mutants showed a higher specificity toward glucose compared to the wild type enzyme.