Search

A copper-electrodeposited gold electrode that can quantitatively detect creatinine without being affected by urine components and can use collected urine as it is produced. In this study, the effect of interfering compounds was eliminated, and the linear range was expanded by increasing the concentration of Nafion covering the electrodes. Furthermore, by extending the electrodeposition time, the linear range was further expanded, and it was possible to measure concentrations up to 12.3 mM (M = mol dm−3), which is equivalent to the creatinine concentration range in the urine of healthy individuals.

Pyrroloquinoline quinone–dependent and flavin adenine dinucleotide–dependent enzymes catalyze the oxidation of various compounds. These enzymes are large molecules, and the embedding of active sites in the insulating portion of the molecule generally make direct bioelectrocatalysis difficult. Dehydrogenases with a built-in electron transfer domain are capable of direct electron transfer (DET) to an electrode. Attempts have also been made to realize DET by artificially producing fusion proteins in which protein engineering is fully exploited to connect electron transfer domains. Furthermore, the reports of the DET of enzymes without an electron transfer domain to an electrode have started to appear. This review summarizes recent reports on fundamental findings on DET and applications using DET-enzyme electrodes.

Hexarates are platform chemicals. Methods to produce d-glucarate and d-mannarate are desirable because these hexarates can be gained by oxidation of the corresponding hexuronates, which are abundantly found in algae and plants as the units of polyuronates. Oxidative production of the hexarates can be combined with a reductive reaction to coproduce electricity. An enzymatic biofuel cell is a device that enables this coproduction. To construct the cell, it is necessary to find enzymes that catalyze platform chemical production and are also suitable as anode catalysts. Here, we show the production of d-glucarate and d-mannarate from d-glucuronate and d-mannuronate, with both reactions catalyzed by pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase, in addition to the production of d-glucarate from l-guluronate by the PQQ domain of pyranose dehydrogenase from Coprinopsis cinerea. The enzymes are suitable as anode catalysts in biofuel cells that coproduce these hexarates and electricity.

Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase is one of the extensively studied sugar-oxidizing enzymes used as a biocatalyst for biosensors and biofuel cells. A novel pyranose dehydrogenase (CcPDH) derived from the basidiomycete Coprinopsis cinerea is the first discovered eukaryotic PQQ-dependent enzyme. This enzyme carries a b-type cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH); thus, CcPDH is a quinohemoprotein. CcPDH catalyzes the oxidation of various aldose sugars and shows significant activity toward the reverse-chair conformation of pyranoses. Interdomain electron transfer occurs in CcPDH similar to CDH, from the PQQ cofactor in the catalytic domain to the heme b in the cytochrome domain. This enzyme is able to direct electrical communication with electrodes, without artificial electron mediators, thus allowing direct electron transfer (DET)-type bioelectrocatalysis. In this review, we briefly describe recent progress in research on the biochemical discovery of CcPDH and the development of (bio)electrochemical applications (an amperometric biosensor) based on DET reactions.

Quantitative routine detection of fucose, which is a cancer marker, in urine is effective for the preliminary screening of cancer. Amperometric biosensing methods have the advantage of being simple, rapid, and precise for urinalysis. However, coexisting electroactive interferences such as ascorbic acid (AA), dopamine (DA), and uric acid (UA) prevent accurate measurements. In this work, an amperometric L-fucose biosensor unaffected by interferences was developed and utilizes direct electron transfer type bioelectrocatalysis of pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH). The isolated PQQ domain from CcPDH was immobilized on gold nanoparticle (AuNP)-modified electrodes, which obtained a catalytic current at a lower potential than the oxidation potential of the interfering compounds. Applying an operating potential of −0.1 V vs. Ag|AgCl (3 M NaCl) enabled the detection of L-fucose while completely eliminating the oxidation of AA, DA, and UA on the electrodes. The increase in the specific area of the electrodes by increasing the AuNP drop-casting time resulted in an improvement in the sensor performance. The biosensor exhibited a linear range for L-fucose detection between 0.1 mM and 1 mM (R2 = 0.9996), including a cut-off value, the sensitivity was 3.12 ± 0.05 μA mM−1 cm−2, and the detection limit was 13.6 μM at a signal-to-noise ratio of three. The biosensor can be used to quantify the concentration of L-fucose at physiological levels and does not require urine preprocessing, making it applicable to practical use for point-of-care testing with urine.

To replace fossil resources with biomass, a lot of conversion methods have been studied. Most of each biomass-conversion usually correspond to one specific purpose, such as to produce chemicals, fuels, or energy. However, when a production of chemicals is through one or more oxidation reactions, co-production of electricity is possible through a conversion on an enzymatic bioanode in a biofuel cell. The simultaneous production will reduce the energy required for producing chemicals. According to the coproduction concept, here we show a production of meso-galactaric acid which is considered a platform chemical. meso-Galactaric acid can be obtained from C1 aldehyde oxidation of d-galacturonic acid, which exists in large quantities as pectin in food process residue. d-Galacturonic acid oxidation catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and subsequent meso-galactaric acid production was confirmed for the first time by NMR measurements. PQQ-GDH is a useful catalyst for in vitro production, especially for electrosynthesis, because it requires neither the expensive cofactor nor O2. Hence, PQQ-GDH was fixed on an electrode to fabricate the PQQ-GDH electrode. The catalytic current from d-galacturonic acid oxidation with the electrode was confirmed in the electrochemical experiments to show the simultaneous production of meso-galactaric acid and the electric current.

A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of l-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters.

Bioelectrocatalysis is the use of biomaterials as catalysts for redox reactions combined with electrode reactions. Electrodes modified with enzymes have been extensively studied in the field of bioelectrochemistry. Since electron transfer between the enzyme and the electrode is the most important phenomenon, the discussion about it has dominated the study of bioelectrocatalysis. Direct electron transfer (DET) between oxidoreductases and electrodes is important not only for understanding the fundamental features of redox proteins, but also for developing mediator-free bioelectronics devices. Among the enzymes with various coenzymes, the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases are promising biocatalysts for both biosensors and biofuel cells. However, a limited number of studies have reported DET between PQQ in the enzyme and the electrode. We discovered a fungal PQQ-dependent pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH) as the first example of a eukaryotic PQQ-dependent enzyme [1-3]. This enzyme comprises three domains: an N-terminal cytochrome b domain, a central PQQ dependent catalytic domain (PQQ domain), and a C-terminal family 1 carbohydrate-binding module (CBM1), and thus, it is regarded as quinohemoprotein. A previous study demonstrated that full-length CcPDH exhibited the direct bioelectrocatalysis based on DET on a glassy carbon (GC) electrode [4]. The catalytic currents were observed at a lower potential than the redox potential of heme b in the cytochrome domain, suggesting that both PQQ in the catalytic domain and heme b in the cytochrome domain are capable of DET. In the present study, the PQQ domain (containing only the PQQ cofactor) which was prepared through genetic engineering, was studied in DET with various electrodes. A 2-mercaptoethanol self-assembled monolayer (SAM)-coated polycrystalline gold electrode was found to be superior for the DET of PQQ in the domain, and the catalytic current density was higher than that of the bare GC electrode. The catalytic current density has increased 10 times. The amount of immobilized PQQ domain was determined to be 8.2 ± 0.4 pmol/cm2 by using a 27 MHz quartz-crystal microbalance (QCM), suggesting an approximate protein monolayer formation on the SAM modified gold surface. To improve current density, gold nanoparticles (AuNPs) were modified on top of polycrystalline gold electrodes. Importantly, a high catalytic current density of 1.6 mA/cm2 for the oxidation of L-fucose was achieved under optimized conditions. These results show highly efficient DET to PQQ in the active site of the fungal PQQ-dependent dehydrogenase. Thus, the PQQ-domain/SAM/AuNP coated polycrystalline gold electrode could serve as a highly sensitive biosensor and a highly active sugar oxidizing anode for a biofuel cell. Cyclic voltammetry using a AuNP modified electrode in the absence of substrate showed two pairs of redox peaks with midpoint potentials of E 1 = -45 mV and E 2 = -216 mV, indicating the redox reaction of the bound PQQ in the catalytic domain is composed of two one-electron steps via a semiquinone radical state. This was confirmed by the detection of an EPR signal with a g-value of 2.0065, which was assigned to a PQQ semiquinone radical. The details of DET of the PQQ domain will be discussed. [1] H. Matsumura, et al.,PLoS One, 9 (2014) e104851. [2] K. Takeda, et al., PLoS One, 10 (2015) e0115722. [3] K. Takeda, et al., Curr Opin Chem Biol, 49 (2019) 113-121. [4] K. Takeda, et al., Biochem Biophys Res Commun, 477 (2016) 369-373.

Direct electron transfer (DET) between oxidoreductases and electrodes is crucial for understanding the fundamental features of redox proteins as well as developing mediator-free bioelectronics devices. Bioelectrocatalysis with pyrroloquinoline quinone (PQQ)-dependent dehydrogenases is promising for both biosensors and biofuel cells. However, a limited number of studies have reported DET between PQQ in the enzyme and the electrode. Here, the PQQ domain (containing only the PQQ cofactor) of the fungal PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea, was studied in DET with various electrodes. A 2-mercaptoethanol self-assembled monolayer (SAM)-coated polycrystalline gold electrode was found to give excellent DET for the PQQ domain, resulting in 10-times the catalytic current density compared to bare glassy carbon. The amount of immobilized PQQ domain was determined to be 8.2 ± 0.4 pmol/cm2 by using a 27 MHz quartz-crystal microbalance (QCM), suggesting an approximate protein monolayer formation on the SAM modified gold surface. To improve current density, gold nanoparticles (AuNPs) were modified on top of polycrystalline gold electrodes. Importantly, high catalytic current densities of 1.6 mA/cm2 for the oxidation of l -fucose were achieved under optimized conditions. Together, these results demonstrate highly efficient DET to PQQ in the active site of the fungal PQQ-dependent dehydrogenase. Thus, our PQQ-domain/SAM/AuNPs coated polycrystalline gold electrode could serve as an exquisitely sensitive biosensor and a highly active sugar oxidizing anode for biofuel cells.

Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo- and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade β-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer.IMPORTANCE Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens . The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl 2 , revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto- d -glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl 2 were added. 1 H nuclear magnetic resonance ( 1 H-NMR) analysis of reaction products revealed 2-keto- d -gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase ( Pa 2KGDH). Considering the high substrate specificity, the physiological function of Pa 2KGDH may be for production of 2KGA.

A bioanode for ethanol oxidation was prepared by immobilizing the recombinant pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase from Pseudomonas putida KT 2440 (PpADH) with polyion complex (PIC) and redox polymer. The PIC based on poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) was suitable for immobilizing PpADH on the electrode. PpADH was immobilized using only one redox polymer, aminoferrocene, which was attached to the PGA backbone (PGA-AmFc) on the electrode. The anodic current density at 0.6 V (vs. Ag/AgCl) was 22.6 μA·cm−2. However, when the number of the cycles was increased, the catalytic current drastically decreased. PpADH was immobilized using PGA-AmFc and PIC on the electrode. The anodic current density at 0.5 V (vs. Ag/AgCl) was 47.3 μA·cm−2, and the performance maintained 74% of the initial value after five cycles. This result indicated that the combination of PIC and PGA-AmFc was suitable for the immobilization of PpADH on the electrode. In addition, the long-term stability and catalytic current density were improved by using the large surface area afforded by the gold nanoparticles.

NAD(P)-dependent group III alcohol dehydrogenases (ADHs), well known as iron-activated enzymes, generally lose their activities under aerobic conditions due to their oxygen-sensitivities. In this paper, we expressed an extremely thermostable group III ADH from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PhADH) heterologously in Escherichia coli. When purified from a culture medium containing nickel, the recombinant PhADH (Ni-PhADH) contained 0.85 ± 0.01 g-atoms of nickel per subunit. Ni-PhADH retained high activity under aerobic conditions (9.80 U mg−1), while the enzyme expressed without adding nickel contained 0.46 ± 0.01 g-atoms of iron per subunit and showed little activity (0.27 U mg−1). In the presence of oxygen, the activity of the Fe2+-reconstituted PhADH prepared from the Ni-PhADH was gradually decreased, whereas the Ni2+-reconstituted PhADH maintained enzymatic activity. These results indicated that PhADH with bound nickel ion was stable in oxygen. The activity of the Ni2+-reconstituted PhADH prepared from the expression without adding nickel was significantly lower than that from the Ni-PhADH, suggesting that binding a nickel ion to PhADH in this expression system contributed to protecting against inactivation during the expression and purification processes. Unlike other thermophilic group III ADHs, Ni-PhADH showed high affinity for NAD(H) rather than NADP(H). Furthermore, it showed an unusually high k cat value toward aldehyde reduction. The activity of Ni-PhADH for butanal reduction was increased to 60.7 U mg−1 with increasing the temperature to 95 °C. These findings provide a new strategy to obtain oxygen-sensitive group III ADHs.

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.

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.

A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca(2+). PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The Km values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the kcat values were unaltered. For instance, the Km values at T=298.15K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02)mM and 5.8 (± 0.60)mM, respectively. The kcat values for ethanol and acetaldehyde were 24.8 (± 1.2) s(-1) and 31.1 (± 1.2) s(-1), respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10(-3) · C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10(-3) · C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH.

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.