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. 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.

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. 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.

Crystal Structure of the Catalytic and Cytochrome b 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 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 ß-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.

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). Because hexaric acids have four secondary hydroxy groups that are stereochemically diverse and two carboxyl groups, various applications of these acids have been studied. Conventionally, hexaric acids have been produced mainly by nitric acid oxidation of aldohexose, but full-scale commercialization has not been realized; there are many problems regarding yield, safety, environmental burden, etc. In recent years, therefore, improvements in hexaric acid production by nitric acid oxidation have been made, while new production methods, including biocatalytic methods, are actively being studied. In this paper, we summarize these production methods in addition to research on the application of hexaric 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. The distribution ratio into tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate decreased with decreasing isoelectric point of cytochrome c. Furthermore, cytochrome c possessing a low isoelectric point showed different distribution ratio depending on surface hydrophobicity. Taken together, these findings indicate that isoelectric point and surface hydrophobicity of cytochrome c are important factors controlling the distribution behavior in temperature sensitive biphasic systems.

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., respectively. Tetrabutylphosphonium hydroxide ([P4444]OH) aqueous solution was found to exhibit a highest solubility, specifically 40 wt% of SL and 3.0 wt% of KL. The superior solubility of OHAS is comprehended to be due to weak interactions between OH anions and phosphonium cations, and hence OH anions interact effectively with lignin. Epoxidation of SL was achieved by simply adding epichlorohydrin to [P4444]OH aq. dissolving SL. Films of epoxidized SL were prepared by thermal curing with the aid of a crosslinking agent, and the films were found to possess high thermal stability of >250 °C and excellent ductility. The thermal and mechanical properties were controllable by the concentration of [P4444]Cl as an additive.

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. 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.

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 (M1,2O2 TFSI) and N-ethoxyethyl-N-methylpiperidinium bis(trifluoromethanesulfonyl)imide (P1,2O2 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. Higher conducting properties of the piperidinium-based electrolyte resulted in enhanced cycling performances when tested with LiFePO4 (LFP) cathode in lithium cells. When mixing the P1,2O2 TFSI/LiTFSI electrolyte with a tailored alkyl carbonate mixture, the cycling performance of both Li and Li-ion cells greatly improved, with prolonged cyclability delivering very stable capacity values, as high as the theoretical one in the case of Li/LFP cell configurations.

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 0.85 ± 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.

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. 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.

Biofuel cell backpacked insect and its application to wireless sensing.

This study investigated an enzymatic biofuel cell (BFC) which can be backpacked by cockroaches. The BFC generates electric power from trehalose in insect hemolymph by the trehalase and glucose dehydrogenase (GDH) reaction systems which dehydrogenate ß-glucose obtained by hydrolyzing trehalose. First, an insect-mountable BFC (imBFC) was designed and fabricated with a 3D printer. The electrochemical reaction of anode-modified poly-L-lysine, vitamin K3, diaphorase, nicotinamide adenine dinucleotide, GDH and poly(sodium 4-styrenesulfonate) in the imBFC was evaluated and an oxidation current of 1.18 mAcm(-2) (at +0.6 V vs. Ag|AgCl) was observed. Then, the performance of the imBFC was evaluated and a maximum power output of 333 µW (285 µW cm(-)(2)) (at 0.5 V) was obtained. Furthermore, driving of both an LED device and a wireless temperature and humidity sensor device were powered by the imBFC. These results indicate that the imBFC has sufficient potential as a battery for novel ubiquitous robots such as insect cyborgs.

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 1.1.99.18) 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 (kcat/Km) 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.

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. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (RRMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.

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. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, 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 CaCl2 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 (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

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 (ß 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 0.4 and ß values of approximately 0.9 were suitable for dissolution of cyanobacteria. In particular, 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][MeO(H)PO2]) was found to dissolve cyanobacteria components, but not PHB. Thus, we verified that PHB produced in cyanobacteria could be separated and recovered by simple filtering after dissolution of cyanobacteria in [C2mim][MeO(H)PO2]. Using this technique, more than 98 % of PHB was obtained on the filter as residues separated from cyanobacteria. Furthermore, [C2mim][MeO(H)PO2] maintained the ability to dissolve cyanobacteria after a simple recycling procedure.

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. 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.

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. Under these conditions, the Michaelis constant for hydrogen peroxide (KmH2O2 ) was decreased. The turnover rate (kcat ) of ethylbenzene hydroxylation was increased and followed an S-shaped curve, with an increase in the pH value. The apparent acid dissociation constant (pKa (app) ) of the kcat was 7.0, which suggests that the rate-limiting step of this reaction is deprotonation of the Fe(III) -H2 O2 complex. By introducing a double mutation around the proximal heme ligand, the peroxygenase activity was increased over a wide pH range, and was dramatically increased at pH 5. The spectroscopic properties of this F310A/A320Q mutant indicated that the Lewis acidity of the heme was increased by this mutation. Kinetic investigations showed that the increase in the Lewis acidity of the heme facilitates the reaction rate of the rate-limiting step of peroxygenase reactions and decreases the KmH2O2 value. Differences in the pH dependence of the kcat value between wild-type P450st and the mutant suggest that the rate-limiting step switches to protonation of the ferric-hydroperoxo species (compound 0) under alkaline conditions.

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 62.8 wt% by adding N,N,N-tripentyl-4-sulfonyl-1-butanephosphonium-type ZI (P555C4S). In addition, the maximum water content decreased from 62.8 wt% to 34.1 wt% by increasing KH2PO4/K2HPO4 salt content in upper aqueous phosphate buffer phase. Horse heart cytochrome c (cyt.c) was dissolved selectively in IL phase by improving the water content of IL phase, and spectroscopic analysis revealed that the dissolved cyt.c retained its higher ordered structure. Furthermore, cyt. c dissolved in IL phase was re-extracted again from IL phase to aqueous phase by increasing the concentration of inorganic salts of the buffer solution.