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. CONCLUSION: 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.
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. CONCLUSION: 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: Chi En Lin; Yuka Ito; Anna Deng; Jared Johns; Daniel Matloff; Curtiss B Cook; Koji Sode; Jeffrey T La Belle Journal: J Diabetes Sci Technol Date: 2018-04-18