Fungal FAD-dependent glucose dehydrogenases concerning high activity, affinity, and thermostability for maltose-insensitive blood glucose sensor

Abstract

Six FAD-dependent glucose dehydrogenase (FAD-GDH) genes, for blood glucose sensors, from the genomic DNAs of Aspergillus species were identified by degenerated PCR screening. The enzymatic properties of the gene products expressed by Pichia pastoris were characterized. Investigation of substrate selectivity to fourteen saccharides revealed that these enzymes were maltose-insensitive FAD-GDHs. A.bisporus, A.terreus var. aureus, and A. phoenicis FAD-GDHs showed high substrate selectivity; they exhibited reactivity to only one or two saccharides. A. phoenicis FAD-GDH specifically exhibited insensitivity to d-xylose. Furthermore, six FAD-GDHs exhibited superior parameter values, such as maximum velocity (Vmax), Michaelis-Menten constant (Km), and apparent transition midpoint temperature (Tm). A. phoenicis and A. terreus var. aureus FAD-GDHs exhibited high activity, with Vmax values of (8.66 ± 0.17) × 104 U/μmol and (1.14 ± 0.03) × 105 U/μmol, respectively. A. brunneo-uniseriatus, A.carneus, and A. malignus FAD-GDHs exhibited very high affinity for glucose, with Km values between 0.41 and 0.96 mM. A. bisporus FAD-GDH exhibited high thermostability, with a Tm value of 72.5 °C. Therefore, we propose that the FAD-GDHs will be practical in blood glucose sensors and in other bioelectrochemical devices exhibiting high substrate selectivity, high current output, high sensitivity, and/or high stability to storage and use.