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Updated: Apr 17, 2026
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Published on: October 4, 2017
Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond
Genki Sakai1, Katsuhiro Kojima, Kazushige Mori
1Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Nakamachi, Koganei, Tokyo, Japan.
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.
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