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. 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 A degrees 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), K-m = 35 mM, and catalytic efficiency (k (cat)/K-m) = 22 s(-1) mM(-1)) were almost identical to those of the wild-type enzyme (k (cat) = 780 s(-1), K-m = 35 mM, k (cat)/K-m = 22 s(-1) mM(-1)). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type. 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.