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A reactor based on an enzymatic biofuel cell (an EBFC reactor) was constructed to simultaneously generate electricity and chemical products from biomass.

We have determined the crystal structure of thermoglobin (AaTgb) from a hyperthermophilic bacterium Aquifex aeolicus. Tyrosine and glutamine at the B10 and E7 position, respectively, are conserved ...

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) 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., 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.

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.

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.

Rationale and Objectives Severe calcifications of the coronary arteries are still a major challenge in coronary computed tomography (CT) angiography (CCTA). Subtraction CCTA using a 320-detector row CT scanner has recently been introduced for patients with severe calcifications. However, the conventional subtraction CCTA method requires a long breath-holding time of approximately 20–40 seconds. This is a major problem in clinical practice because many patients may not be able to perform such a long breath-hold. We explored a modified subtraction CCTA method with a short breath-holding time to overcome this problem. Materials and Methods This study was approved by our institutional review board, and all patients gave written informed consent. A total of 12 patients with a coronary calcium score of >400 were enrolled in this study. All patients were unable to hold their breath for more than 20 seconds. Modified subtraction CCTA was performed using the bolus-tracking method. The acquisition protocol was adjusted so that the mask scan was acquired 10 seconds after the postcontrast scan during a single breath-hold. The subtraction image was obtained by subtracting the mask image data from the postcontrast image data. The breath-holding times were recorded. Enhancement of the coronary arteries in the subtraction images was assessed. Subjective image quality was evaluated in a total of 32 segments using a 4-point scale. Results The mean breath-holding time was 12.8 ± 0.8 seconds (range, 12–14 seconds). The average CT number in the coronary arteries was 288.6 ± 80.5 Hounsfield units (HU) in the subtraction images. Average image quality was significantly increased from 2.1 ± 0.9 with conventional CCTA to 3.1 ± 0.7 with subtraction CCTA ( P P = 0.001). Conclusions This preliminary study has shown that our modified subtraction CCTA method allows the breath-holding time to be shortened to

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.

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.

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.

In 2014, the first fungal pyrroloquinoline-quinone (PQQ)-dependent enzyme was discovered as a pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH). This discovery laid the foundation for a new Auxiliary Activities (AA) family, AA12, in the Carbohydrate-Active enZymes (CAZy) database and revealed a novel enzymatic activity potentially involved in biomass conversion. This review summarizes recent progress made in research on this fungal oxidoreductase and related enzymes. CcPDH consists of the catalytic PQQ-binding AA12 domain, an N-terminal cytochrome b AA8 domain, and a C-terminal family 1 carbohydrate-binding module (CBM1). CcPDH oxidizes 2-keto-d-glucose (d-glucosone), l-fucose, and rare sugars such as d-arabinose and l-galactose, and can activate lytic polysaccharide monooxygenases (LPMOs). Bioinformatic studies suggest a widespread occurrence of quinoproteins in eukaryotes as well as prokaryotes.

A multi-enzyme electrode composed of FAD-dependent and NAD-dependent enzymes was fabricated using a poly-ruthenium complex (PAHA–Ru), which has two 1,10-phenanthroline-5,6-dione molecules as ligands. PAHA–Ru was used to immobilize FAD-dependent glucose dehydrogenase (FAD–GDH) onto an electrode and to examine PAHA–Ru containing the quinone moieties as an electron mediator. In cyclic voltammetry measurements of the FAD–GDH modified electrode in the presence of D-glucose, a catalytic current was obtained, which indicated electron transfer from FAD–GDH to PAHA–Ru. Our previous study has reported that PAHA–Ru with the quinone ligands also works as a mediator for NADH oxidation on an NAD-dependent alcohol dehydrogenase (NAD–ADH) modified electrode. Hence, FAD–GDH and NAD–ADH were co-immobilized with PAHA–Ru to make a multi-enzyme electrode. Using this multi-enzyme electrode as an anode, catalytic currents were observed in D-glucose solution, ethanol solution, and a mixed D-glucose and ethanol solution. The catalytic current in the mixed solution was greater than the currents obtained in the single substrate solutions, indicating bioelectrocatalysis reactions by the two enzymes and the single mediator in the mixed solution. Thus, we demonstrated that PAHA–Ru modified electrode enables selection of enzymes and their substrates from a wider range for enzymatic biofuel cells. Keywords: Redox polymer mediator, 1,10-Phenanthroline-5,6-dione, Flavin adenine dinucleotide dependent-glucose dehydrogenase, Nicotinamide adenine dinucleotide-dependent alcohol dehydrogenase, Multi-enzyme electrode, Entrapping immobilization method

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.

The purpose of this study was to explore the feasibility of subtraction coronary computed tomography angiography (CCTA) by second-generation 320-detector row CT in patients with severe coronary artery calcification using invasive coronary angiography (ICA) as the gold standard. This study was approved by the institutional board, and all subjects provided written consent. Twenty patients with calcium scores of >400 underwent conventional CCTA and subtraction CCTA followed by ICA. A total of 82 segments were evaluated for image quality using a 4-point scale and the presence of significant (>50 %) luminal stenosis by two independent readers. The average image quality was 2.3 ± 0.8 with conventional CCTA and 3.2 ± 0.6 with subtraction CCTA (P

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.

To explore the feasibility and diagnostic accuracy of modified subtraction coronary CT angiography (CCTA) with short breath-holding time in patients who have limited breath-hold capability and severe coronary artery calcification.11 patients with a coronary calcium score400 underwent CCTA using a modified subtraction protocol. All patients were unable to hold their breath for more than 20 s. Subjective image quality using a four-point scale and the presence of significant (50%) luminal stenosis were assessed for each calcified or stented segment on both conventional CCTA and modified subtraction CCTA images and compared with invasive coronary angiography (ICA) as the gold standard.The mean breath-holding time was 13.0 ± 0.9 s. A total of 35 calcified or stented coronary segments were evaluated. The average image quality was increased from 2.1 ± 0.9 with conventional CCTA to 3.1 ± 0.7 with subtraction CCTA (p 0.001). The segment-based diagnostic accuracy for detecting significant stenosis according to ICA revealed an area under the receiver-operating characteristic curve of 0.722 for conventional CCTA and 0.892 for subtraction CCTA (p = 0.036).Modified subtraction CCTA allows the breath-holding time to be shortened to15 s. As compared with conventional CCTA, modified subtraction CCTA showed improvement in image quality and diagnostic accuracy in patients with limited breath-hold capability and severe calcification.Modified subtraction CCTA can improve the diagnostic accuracy in patients with a high calcium score and patients who are unable to perform long breath-holds.

Severe calcifications of the coronary arteries are still a major challenge in coronary computed tomography (CT) angiography (CCTA). Subtraction CCTA using a 320-detector row CT scanner has recently been introduced for patients with severe calcifications. However, the conventional subtraction CCTA method requires a long breath-holding time of approximately 20-40 seconds. This is a major problem in clinical practice because many patients may not be able to perform such a long breath-hold. We explored a modified subtraction CCTA method with a short breath-holding time to overcome this problem.This study was approved by our institutional review board, and all patients gave written informed consent. A total of 12 patients with a coronary calcium score of400 were enrolled in this study. All patients were unable to hold their breath for more than 20 seconds. Modified subtraction CCTA was performed using the bolus-tracking method. The acquisition protocol was adjusted so that the mask scan was acquired 10 seconds after the postcontrast scan during a single breath-hold. The subtraction image was obtained by subtracting the mask image data from the postcontrast image data. The breath-holding times were recorded. Enhancement of the coronary arteries in the subtraction images was assessed. Subjective image quality was evaluated in a total of 32 segments using a 4-point scale.The mean breath-holding time was 12.8 ± 0.8 seconds (range, 12-14 seconds). The average CT number in the coronary arteries was 288.6 ± 80.5 Hounsfield units (HU) in the subtraction images. Average image quality was significantly increased from 2.1 ± 0.9 with conventional CCTA to 3.1 ± 0.7 with subtraction CCTA (P 0.001). With subtraction CCTA, the number of non-diagnostic segments was significantly reduced from 53% to 19% (P = 0.001).This preliminary study has shown that our modified subtraction CCTA method allows the breath-holding time to be shortened to15 seconds. This may substantially improve the success rate of subtraction CCTA by reducing artifacts and allowing this technique to be applied to patients who are unable to perform a long breath-hold.

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.

Introduction A pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH) is an eukaryotic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase consisting three-domains structure [1]. The PQQ and heme b cofactors are located in the 45 kDa and 21 kDa domains, respectively, and a small C-terminal domain is family 1-type carbohydrate-binding module, which these domains are connected by a proline-rich linker region. CcPDH shows the oxidation activity toward monosaccharides in a 1C4 chair conformation such as D-glucosone and L-fucose. The N-terminal cytochrome domain is a 6-coordinated low-spin heme b with Met/His ligands that enables direct electrical contact between the enzyme and the electrode [2]. Electrons are transferred from reduced PQQ in catalytic domain to heme b in cytochrome domain. The previous study reported that intact CcPDH is capable of direct electron transfer (DET)-based bioelectrocatalysis on a glassy carbon (GC) electrode [3]. The catalytic currents were observed at a lower potential than the redox potential of heme b in the cytochrome domain, suggesting that both the PQQ and the cytochrome domains in CcPDH are able to DET with the GC electrode. In present study, to examine DET reaction of the PQQ domain, an isolated PQQ domain was expressed in Pichia expression system. Here, we demonstrated the direct bioelectrocatalysis for the isolated PQQ domain from C. cinerea pyranose dehydrogenase. The bioelectrocatalytic current density of 1.6 mAcm-2 was achieved on a preformed 2-mercaptoethanol (ME) self-assembled monolayer (SAM) modified gold nanoparticles (AuNPs) electrode under optimized conditions. Experimental The direct bioelectrocatalysis of DHPDH was obtained by using glassy carbon (GC) electrode modified with the enzyme as working electrode. An aliquot of 30 μL of 1 μM enzyme solution containing 10 μM PQQ and 1 mM CaCl2 was dropped on the entire surface of the GC electrode and then dried at room temperature. AuNPs-modified and unmodified polycrystalline Au electrodes were used for the direct bioelectrocatalysis of DHPDH. 1 μL of the concentrated AuNPs was dropped onto the surface of the Au electrode, and then air-dried. To prepare electrodes with immobilized DHPDH, AuNPs-modified and unmodified polycrystalline Au electrodes were immersed in 20 mM aqueous solution of 2-mercaptoethanol for 1 h at room temperature (ME-AuNPs electrode and ME-Au electrode). Then they were immersed in 1 μM holo-DHPDH in a 50 mM sodium acetate buffer solution (pH 6.0) at 4 ℃ for over 15 h. Cyclic voltammetry measurements were performed in 50 mM various pH buffer with 100 mM L-fucose. A conventional three-electrode cell was used, in which a platinum wire served as the counter electrode and Ag/AgCl (3 M NaCl) served as the reference electrode. Results and Discussion Initially, a clear catalytic current of L-fucose oxidation by the DHPDH on the GC electrode was observed starting at same potential of intact CcPDH. It is demonstrated that the DHPDH can also directly transfer electrons to the electrodes. The catalytic current density of DHPDH (0.9 μA cm-2, at 0.35 V) was an order of magnitude smaller than that of intact CcPDH. To improve the direct bioelectrocatalysis of DHPDH, a thiol-based SAM coated Au electrodes were studied. Accordingly, the optimal SAM for DHPDH was found to be shorter alkyl chain length and hydroxide-functionalized thiols. The catalytic current density of 11 μA cm-2 (at 0.35 V) was obtained when using ME-Au electrode. DHPDH immobilized on ME-Au electrode showed optimum at pH 6.0 and a value of K m = 35.1 mM, respectively. In order to obtain higher catalytic currents, furthermore, AuNPs were cast on the surface of Au electrode. Increasing the number of AuNP casts from 3 to 15 times on led to increasing catalytic current densities from 125 μA cm-2 to 240 μA cm-2 at 0.35 V. Additionally, we had considered the effects of the electrolyte (ion conductivity) and temperature dependence for catalytic current by AuNPs-Au electrode at the casting cycle three times. The catalytic current at 50 °C was approximately 5-times higher than that at 20 °C. Upon increasing acetate buffer concentration from 50 mM to 500 mM, the current density was enhanced to as 1.7-fold. Figure 1 shows the cyclic voltammogram of L-fucose oxidation on DHPDH-modified ME-AuNPs electrode under optimized conditions of 50 °C, pH 6, 500 mM acetate buffer, and the AuNPs casting cycle 15 times. Finally, the catalytic current reached to a steady-state value of 1.6 mA cm-2. References [1] H. Matsumura et al., PloS One 9, e104851 (2014). [2] K. Takeda et al., PloS One 10, e0115722 (2015). [3] K. Takeda et al., submitted . Figure 1

Introduction Enzymes, which are reproducible catalysts, can catalyze selective and environmentally friendly production of platform chemicals from biomass, in simple systems. When the platform chemical-production is through one or more oxidation reactions, the electrons obtained from the substrate are usually just dumped to electron accepters. However, when a biofuel cell anode is used as an electron acceptor, enzymatic, oxidative conversions on the anode can be combined with O2 reduction on the cathode to generate electricity. Hence, the simultaneous production of platform chemicals and electricity is possible through an oxidative conversion with enzymatic biofuel cells. D-Galacturonic acid is included in most land plants as the main component of pectin. Because it is a cheap raw material that can also be extracted in large quantities from food process residues, such as fruit peels and pulp of sugar beets and chicory, the conversion of pectin to fuels and chemicals has been studied. meso-Galactaric acid is a target compound of a pectin biorefinery, and is expected to be both a chelating agent and a precursor for polymers with various applications, a cross-linking agent, and other platform chemicals. Uronate dehydrogenase, uronate oxidase, and glucose oxidase are known to oxidize D-galacturonic acid to produce meso-galactaric acid, so far. However, these enzymes are not suitable for an electrode catalyst. In vitro use of uronate dehydrogenase requires an expensive NAD addition as its cofactor. The oxidases donate electrons to O2 to result in reducing the electric current. Glucose dehydrogenase (GDH) which binds pyrroloquinoline quinone (PQQ) tightly, but not covalently, as its cofactor has superior characteristics as an electrode catalyst. PQQ-GDH contains its cofactor and GDH catalysis does not use O2. To date, there have been no reports on uronic acid oxidation, including D-galactuornic acid, catalyzed by GDHs. However, we expected PQQ-GDH to catalyze the oxidation of D-galacturonic acid into meso-galactaric acid because PQQ-GDH is known to have the broad selectivity for substrates including D-galactose. Hence, we observed the oxidation reaction of D-galacturonic acid catalyzed by PQQ-GDH mainly through NMR spectrometry and constructed a PQQ-GDH bioanode to produce meso-galactaric acid. Experimental NMR measurements. Methylene green (MG: ca. 15 mmol), D-galacturonic acid Na salt (ca. 15 mmol), and 0.13 mg PQQ-GDH were added to 530 μL of deuterated phosphate buffer (pD 7.1). NMR measurements of the mixture were conducted 1 h and 2 days after the mixing at 500 MHz, holding the probe temperature constant at 20 °C. Electrode preparation and electrochemical measurements. A conventional three-electrode cell was used, in which a Pt wire served as the counter electrode and Ag/AgCl (3 M NaCl) served as the reference electrode. PolyMG (PMG) film was formed on the PFC electrode by 5 sets of continuous cyclic sweeps in 0.5 mM MG / 0.1 M phosphate buffer (pH 7.0). Then, 6 μL of 50 μg μL-1 PQQ-GDH / 0.1 M phosphate buffer (pH 7.0) was dropped onto the PMG-modified electrode. The cyclic voltammetry (CV) measurements of the PQQ-GDH-modified electrode were conducted in 0.1 M D-galacturonic acid Na salt / 0.1 M phosphate buffer (pH 7.0). Results & discussion The NMR spectra obtained from the samples with PQQ-GDH showed the production of D-galactaro-1,4-lactone and meso-galactaric acid 1 h and 2 day after reaction started, respectively. The spectra of control samples without PQQ-GDH showed no signal from products. These results clearly showed PQQ-GDH-catalyzed D-galacturonic acid oxidation. The delayed appearance of meso-galactaric acid in the spectra that followed the emergence of D-galactaro-1,4-lactone reflected the non-enzymatic equilibrium among these chemical species as previously reported. To construct an enzymatic anode that produces meso-galactaric acid by using an electrode as the electron acceptor, a PQQ-GDH-modified electrode was fabricated. A redox couple with a half-wave potential (E 1/2) of approximately -0.10 V was observed when CV measurements of the PQQ-GDH-modified electrodes were conducted in the control solution without D-galacturonic acid (Figure 1: broken line). In the D-galacturonic acid Na salt solution, an increase in the anodic current density was observed, which started from around E 1/2 (Figure 1: solid line). In contrast, no catalytic current was observed in the cyclic voltammogram of the PMG-modified electrodes without PQQ-GDH in the D-galacturonic acid Na salt solution. The voltammograms of the PMG-modified electrodes in the D-galacturonic acid Na salt solution and in the control solution without D-galacturonic acid were almost the same. These results demonstrated continuous D-galacturonic acid oxidation, which was catalyzed by PQQ-GDH, and the subsequent oxidation of PQQ-GDH by the electrode. We will also report a biofuel cell with this PQQ-GDH-modified anode, which will produce meso-galactaric acid and electricity simultaneously. Figure 1

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