Thermostable FAD-dependent Glucose Dehydrogenases from Thermophilic Filamentous Fungus Thermoascus aurantiacus

Abstract

We newly identified FAD-dependent glucose dehydrogenase (FADGDH) gene homologs from thermophilic filamentous fungus Thermoascus aurantiacus. The gene homologs were cloned from two strains of Th. aurantiacus, NBRC 6766 and NBRC 9748. Recombinant FADGDHs of the two strains were prepared by using Escherichia coli and Pichia pastoris as hosts. Absorption spectra and enzymatic characterization clearly showed that these enzymes contained oxidized FAD as a coenzyme and exhibited glucose dehydrogenase activity. Analysis of thermal stability revealed that the denaturation midpoints (T_m) of these FADGDHs were at least above 71.4°C. The results showed that these FADGDHs exhibited remarkably high thermal stability. We also performed bioelectrochemical experiments using unglycosylated and glycosylated FADGDHs of Th. aurantiacus NBRC 6766, which exhibited higher affinities for glucose than those of Th. aurantiacus NBRC 9748 FADGDHs. Th. aurantiacus NBRC 6766 FADGDH-immobilized electrodes effectively showed current responses to glucose. Therefore, these thermostable FADGDHs could be applied to a bioelectro-catalyst for glucose oxidation with long-term storage and continuous use.