title: Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond.
abstract: OBJECTIVE
  To improve the stability of E. coli-produced non-glycosylated fungal FAD-glucose dehydrogenase induced a disulfide bond by site-directed mutagenesis based on structural comparisons with glucose oxidases.
  
  
  RESULTS
  The FAD-glucose dehydrogenase (GDH) mutant Val149Cys/Gly190Cys, which was constructed based on a comparison with the three dimensional structure of glucose oxidase, showed a 110 min half-life of thermal inactivation at 45 °C, which is 13-fold greater than that of the wild-type enzyme. The considerable increase in thermal stability was further supported by Eyring plot analysis. The kinetic parameters of Val149Cys/Gly190Cys (k cat = 760 s(-1), Km = 35 mM, and catalytic efficiency (k cat/Km) = 22 s(-1 )mM(-1)) were almost identical to those of the wild-type enzyme (k cat = 780 s(-1), Km = 35 mM, k cat/Km = 22 s(-1 )mM(-1)). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type.
  
  
  CONCLUSIONS
  The constructed mutant, Val149Cys/Gly190Cys, had significantly increased structural stability without changing the catalytic activity and kinetic parameters of FAD-GDH, including its characteristic substrate specificity.
authors: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Nakamachi, Koganei, Tokyo, Japan.
authors: Genki
authors: G
authors: Sakai
authors: Katsuhiro
authors: K
authors: Kojima
authors: Kazushige
authors: K
authors: Mori
authors: Yosuke
authors: Y
authors: Oonishi
authors: Koji
authors: K
authors: Sode
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D042843:Glucose 1-Dehydrogenase