We examined the direct electron transfer (DET) reaction of bilirubin oxidase (BOD) at three-dimensional gold nanoparticle (AuNP) electrodes in DET-type biofuel cells. The BOD-modified AuNP electrode, which does not have a thiol self-assembled monolayer, provided a current density as high as 5.2 mA cm⁻² at a 4 000 rpm electrode rotation rate. A remarkable improvement in the stability of catalytic currents was also achieved by utilizing AuNP electrodes. Current densities retained about 90% of their initial value after 48 h of continuous measurement. Moreover, we constructed a mediator-free and compartmentless fructose/O₂ biofuel cell based on DET-type bioelectrocatalysis via the BOD-cathode and the fructose dehydrogenase (FDH)-anode at pH 6.0. When carbon paper was used as the substrate upon which the electrode of the biofuel cell was constructed, the maximum current density of 2.6 mA cm⁻² and the maximum power density of 0.66 mW cm⁻² were achieved at 360 mV of the cell voltage in quiescent solution. Under stirring, a maximum current density of 4.9 mA cm⁻² and a maximum power density of 0.87 mW cm⁻² at an operating voltage of 300 mV were achieved.

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.

The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18) from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (k_cat/K_m) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the ¹C₄ chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.

Dissolution and refolding of aggregated concanavalin A were demonstrated in hydrated ionic liquids.