Thermostable FAD-dependent Glucose Dehydrogenases from Thermophilic Filamentous Fungus <i>Thermoascus aurantiacus</i>

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 FADGDHimmobilized 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.

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase

“…The other issue of this report is substrate specificity of the novel fungal FAD-GDH, which has much less catalytic activity to xylose (monosaccharide) compared with the conventionally used FAD-GDH. 34,35 Therefore, we demonstrate that our DET electrode avoids an overlapped bias of the current toward glucose due to xylose.…”

“…The novel FAD-GDH was produced by the species of a eukaryote fungus and was purified with our procedure. 34,35…”

“…Glycosylation by fungus stabilized FAD-GDH against heat and pH was demonstrated previously. 2,34,35 Therefore, in addition to being able to be produced efficiently, our novel glycosylated FAD-GDH is stable in harsh conditions.…”

Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors

“…Even with structural and chemical information about the active center pocket obtained by crystal structure analysis of the enzyme, it would be difficult to predict the suitable mediator to use. Although there is an increasing number of reports on the discovery and recombinant production of new FAD-GDHs from various fungi [20,21,22,23,24,25,26], it is not realistic from the viewpoint of time and cost to clarify the mediator structure for all these newly discovered enzymes. Therefore, in this study, we propose a strategy for determining the optimum mediator for FAD-GDHs by screening a set of organic redox mediators via their bimolecular rate constants for the enzyme reaction.…”