An amperometric biosensor of L-fucose in urine for the first screening test of cancer.

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.

Discovery of a novel quinohemoprotein from a eukaryote and its application in electrochemical devices.

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.

Crystal Structure of the Catalytic and Cytochrome Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase.

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 (PDH) 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 domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database.

Production of Hexaric Acids from Biomass.

Sugar acids obtained by aldohexose oxidation of both the terminal aldehyde group and the hydroxy group at the other end to carboxyl groups are called hexaric acids (i.e., six-carbon aldaric acids).

Effects of charge balance and hydrophobicity of the surface of cytochrome c on the distribution behaviour in an ionic liquid/buffer biphasic system.

Factors contributing to the different distribution behaviour of cytochrome c were investigated in a biphasic tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer system, which shows a lower critical solution temperature. To change charge balance and hydrophobicity of cytochrome c, surface modification with a few modifier molecules was applied. Surface charge and hydrophobicity affected the distribution behavior of chemically modified cytochrome c in the tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer biphasic system.

Preparation of epoxy resins derived from lignin solubilized in tetrabutylphosphonium hydroxide aqueous solutions.

Organic onium hydroxide aqueous solutions (OHAS) are demonstrated to be potential solvents for the dissolution of lignin and its epoxidation. A series of OHAS has been assessed in terms of the solubility of soda lignin (SL) and Klason lignin (KL), which are moderately and rarely soluble in NaOH aq. soln.

Hydrated ionic liquids enable both solubilisation and refolding of aggregated concanavalin A.

The dissolution and refolding of aggregated concanavalin A have been achieved, in hydrated ionic liquids containing a limited number of water molecules. Both ammonium and phosphonium salts were examined to find a suitable hydrophobicity of ions and water content for refolding. Recovery of sugar recognition was confirmed as a proof of refolding.

Fungal PQQ-dependent dehydrogenases and their potential in biocatalysis.

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.

New Ether-functionalized Morpholinium- and Piperidinium-based Ionic Liquids as Electrolyte Components in Lithium and Lithium-Ion Batteries.

Here, two ionic liquids, N-ethoxyethyl-N-methylmorpholinium bis(trifluoromethanesulfonyl)imide (M TFSI) and N-ethoxyethyl-N-methylpiperidinium bis(trifluoromethanesulfonyl)imide (P TFSI) were synthesized and compared. Fundamental relevant properties, such as thermal and electrochemical stability, density, and ionic conductivity were analyzed to evaluate the effects caused by the presence of the ether bond in the side chain and/or in the organic cation ring. Upon lithium salt addition, two electrolytes suitable for lithium batteries applications were found.

A method of expression for an oxygen-tolerant group III alcohol dehydrogenase from Pyrococcus horikoshii OT3.

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.

pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase.

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.

Characterization of a novel PQQ-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea classified into auxiliary activities family 12 in carbohydrate-active enzymes.

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.

Real-time dynamic adsorption processes of cytochrome c on an electrode observed through electrochemical high-speed atomic force microscopy.

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry.

A novel pyrroloquinoline quinone-dependent 2-keto-D-glucose dehydrogenase from Pseudomonas aureofaciens.

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.

A simple recovery process for biodegradable plastics accumulated in cyanobacteria treated with ionic liquids.

Here, we proposed a simple recovery process for poly(3-hydroxybutyrate) (PHB) accumulated in cyanobacteria by using ionic liquids (ILs), which dissolve cyanobacteria but not PHB. First, we investigated the effects of IL polarity on hydrogen-bonding receipt ability (β value) and hydrogen-bonding donating ability (α value) and evaluated the subsequent dissolution of cyanobacteria. We found that ILs having α values higher than approximately 0.

Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase.

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.

Discovery of a eukaryotic pyrroloquinoline quinone-dependent oxidoreductase belonging to a new auxiliary activity family in the database of carbohydrate-active enzymes.

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.

Identification of the rate-limiting step of the peroxygenase reactions catalyzed by the thermophilic cytochrome P450 from Sulfolobus tokodaii strain 7.

Cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st) is a thermophilic cytochrome P450 that shows high tolerance of harsh conditions and is capable of catalyzing some peroxygenase reactions. Here, we investigated the pH dependence of the peroxygenase reactions catalyzed by wild-type P450st and a mutant in which the residues located close to the proximal heme ligand are mutated. Both hydrogen peroxide-driven ethylbenzene hydroxylation and styrene epoxidation by wild-type P450st were found to be activated in weak acidic and weak basic solutions.

Introduction of hydrophilic groups onto the ortho-position of benzoate anions induced phase separation of the corresponding ionic liquids with water.

Ionic liquids (ILs) composed of tetrabutylphosphonium cations and benzoate anion derivatives with hydrophilic hydroxyl or carboxyl groups introduced onto the ortho-position were found to be less hydrophilic and showed phase separation with water, whereas unmodified benzoate-type IL was freely miscible with water.

The two-step electrochemical oxidation of alcohols using a novel recombinant PQQ alcohol dehydrogenase as a catalyst for a bioanode.

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.

Design of phosphonium-type zwitterion as an additive to improve saturated water content of phase-separated ionic liquid from aqueous phase toward reversible extraction of proteins.

We designed phosphonium-type zwitterion (ZI) to control the saturated water content of separated ionic liquid (IL) phase in the hydrophobic IL/water biphasic systems. The saturated water content of separated IL phase, 1-butyl-3-methyimidazolium bis(trifluoromethanesulfonyl)imide, was considerably improved from 0.4 wt% to 62.

Ionic liquids showing phase separation with water prepared by mixing hydrophilic and polar amino acid ionic liquids.

Both water soluble lysine- and aspartic acid-based amino acid ionic liquids (AAILs) were mixed to prepare polar AAILs showing phase separation with water.

Direct dissolution of wet and saliferous marine microalgae by polar ionic liquids without heating.

We successfully dissolved wet and saliferous microalgae (WSM) in polar ionic liquids (ILs) under mild conditions. The Kamlet-Taft parameters, especially β for the ILs, were good predictors of the ability to dissolve WSM. 1-Ethyl-3-methylimidazolium methylphosphate ([C2mim][MeO(H)PO(2)]) was the IL that best dissolved WSM without heating.

Spectroscopic characterization of the acid-alkaline transition of a thermophilic cytochrome P450.

The spectroscopic properties of thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st) were investigated in acidic and basic solutions. The resting form of ferric-P450st in weakly-acidic and neutral solutions contained a thiolate/H₂O coordinated low-spin heme. Below pH 1.