The flavoenzymes flavin adenine dinucleotidedependent glucose dehydrogenase (FAD-GDH) and oxidase (FAD-GOx) do not undergo direct electron transfer (DET) at conventional electrodes, because the flavin adenine dinucleotide (FAD) cofactor is buried deeply (∼1.4 nm) below the protein surface. We present a mediator-less DET between oxygeninsensitive FAD-GDH and single-walled carbon nanotubes (SWCNTs). A glucose-concentration-dependent current (GCDC) is observed at the electrode with the combination of glycosylated FAD-GDH and debundled SWCNTs; the GCDC, because of an increase in the polarized potential during potential sweep voltammetry, increases steeply (+0.1 V of onset, 1.2 mA cm −2 at +0.6 V 48 mM glucose) without the appearance of the FAD redox peak at −0.45 V. In the control experiment, the GCDC is not observed at the counterpart with either bundled SWCNTs or debundled multiwalled carbon nanotubes (MWCNTs). In the control experiment, the GCDC is observed at an analogous electrode based on oxygen-sensitive FAD-GOx with all CNT types (bundled SWCNTs, debundled SWCNTs, and debundled MWCNTs) in the presence of oxygen because oxygen acts as a natural and mobile mediator. Therefore, observation of the GCDC at the electrode with oxygen-insensitive FAD-GDH and debundled SWCNTs provides evidence of mediator-less DET, even though oxygen is present. Details of the DET are discussed with respect to the recently reported crystallographic model of FAD-GDH. The three-dimensional globular FAD-GDH molecule is 4.5 nm × 5.6 nm × 7.8 nm, which is larger than the 1.2 nm diameter of an individual SWCNT and smaller than the 10 nm diameter of an individual MWCNT and the 1 μm size of a SWCNT bundle. Only individual SWCNTs can be plugged into the groove of FAD-GDH, which is close to and within 1.0 nm of FAD, while maintaining their catalytic activity. Images obtained using transmission electron and atomic force microscopies support the stated configuration of FAD-GDH molecules and debundled SWCNTs. We demonstrate that DET can be explained by quantum tunneling theory. Electrochemical experiments with various FAD-GDHs suggest that (i) DET with debundling SWCNT can be applied to any type of FAD-GDH, (ii) the electrode with various types of FAD-GDH implements superior functions (compared to an analogous electrode with FAD-GOx and nicotineamide adenine dinucleotide-GDH), and (iii) glycan chains present on FAD-GDH prevent denaturation when the SWCNT is close to FAD.

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T ) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.

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Flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) was identified and cloned from thermophilic filamentous fungi Talaromyces emersonii using the homology cloning method. A direct electron transfer bioanode composed of T. emersonii FAD-GDH and a single-walled carbon nanotube was produced. Enzymes from thermophilic microorganisms generally have low activity at ambient temperature; however, the T. emersonii FAD-GDH bioanode exhibits a large anodic current due to the enzymatic reaction (1 mA cm–2) at ambient temperature. Furthermore, the T. emersonii FAD-GDH bioanode worked at 70 °C for 12 h. This is the first report of a bioanode with a glucose-catalyzing enzyme from a thermophilic microorganism that has potential for biosensor and biofuel cell applications. In addition, we demonstrate how the glycoforms of T. emersonii FAD-GDHs expressed by various hosts influence the electrochemical properties of the bioanode.

Co-chaperonin GroES from Escherichia coli works with chaperonin GroEL to mediate the folding reactions of various proteins. However, under specific conditions, i.e. the completely disordered state in guanidine hydrochloride, this molecular chaperone forms amyloid fibrils similar to those observed in various neurodegenerative diseases. Thus, this is a good model system to understand the amyloid fibril formation mechanism of intrinsically disordered proteins. Here, we identified a critical intermediate of GroES in the early stages of this fibril formation using NMR and mass spectroscopy measurements. A covalent rearrangement of the polypeptide bond at Asn 45 -Gly 46 and/or Asn 51 -Gly 52 that eventually yield ␤-aspartic acids via deamidation of asparagine was observed to precede fibril formation. Mutation of these asparagines to alanines resulted in delayed nucleus formation. Our results indicate that peptide bond rearrangement at Asn-Gly enhances the formation of GroES amyloid fibrils. The finding provides a novel insight into the structural process of amyloid fibril formation from a disordered state, which may be applicable to intrinsically disordered proteins in general.Intrinsically disordered proteins are commonly defined as proteins that do not adopt a well defined structure in solution (1), and they can fold into ordered structures only upon binding to their cellular targets (2). They are abundant in eukaryotic proteins and play a significant role in biological functions associated with signaling and regulation events (3). Some intrinsically disordered proteins, exemplified by ␣-synuclein (4 -6) and poly(Q) (7-9) proteins, are capable of forming insoluble aggregates referred to as amyloid fibrils. Understanding the detailed mechanisms in which intrinsically disordered proteins self-assemble into amyloid fibrils is a very important issue because they associate closely with amyloid-related degenerative diseases (10).The prevalent model to explain the mechanisms of amyloid fibril formation in vitro is nucleation-dependent fibril formation (11). Fibril formation kinetics consists of two phases, that is, nucleation and extension, as traced with Thioflavin-T-binding fluorescence or turbidity of incubated samples. Nucleus formation requires a series of associations between protein monomers that are thermodynamically unfavorable, and this step represents the rate-limiting step in amyloid fibril formation. Once the nucleus has been formed, the further addition of monomers becomes thermodynamically favorable, resulting in a rapid extension of amyloid fibrils (12, 13). It has generally been assumed until recently that the cytotoxicity in amyloidrelated degenerative diseases was due to mature amyloid fibrils, but now attention has shifted to various intermediate species formed during nucleation, such as an oligomeric but soluble state (14 -16) or even some monomeric states (17, 18). Therefore, details regarding the structural characteristics of these species that appear in the early stage of the fibril formation are ve...