Purification of a marine bacterial glucose dehydrogenase fromCytophaga marinoflava and its application for measurement of 1,5-anhydro-d-glucitol

A novel glucose dehydrogenase (GDH) from a marine bacteriumCytophaga marinoflava IFO 14170 was isolated from its membrane fraction. This GDH catalyzes the oxidation of a hydroxy group of glucose, but does not react in its C-l position. This enzyme is composed of a single peptide with a mol wt of 67,000. The GDH can react under high salinity. The optimum pH is around 8.0, showing a typical property of marine bacterial enzymes. Using this novel enzyme, an enzymatic determination of 1,5-anhydro-D-glucitol (1,5AG) utilizing 2,6-dichrolophenolindophenol (DCIP) and phenazine methosulfate (PMS) as electron mediators was caried out. A good linear correlation was observed from 0.5 mM to 4 mM of 1,5AG.     

Cloning and Characterization of Fructosamine-6-Kinase from Arthrobacter aurescens

Fructosamine-6-kinases (FN6Ks) that catalyze phosphorylation of glycated amino acids, i.e., fructosyl amino acids (FAs), have been shown as a potential recognition element for glycated protein detection. However, there are only two available FN6Ks: those from Escherichia coli which is specific for ε-fructosyl lysine (ε-FK) and Bacillus subtilis which recognizes both ε-FK and α-FA as substrates. In this study, we characterized an FN6K homologue isolated from Arthrobacter, some of whose species are reported to assimilate FA. The BLAST searches of Arthrobacter genomic database, using the bacterial FN6K primary structure information, revealed the presence of an FN6K homologue in Arthrobacter aurescens TC1 strain. Indeed, enzymatic assays confirmed that the putative FN6K from A. aurescens is an FN6K that is specific for ε-FK, although the primary sequence alignments showed similarity of A. aurescens FN6Ks with FN6Ks from B. subtilis and E. coli at the same level. In this study, we describe for the first time the presence of FN6K in Arthrobacter spp. and ε-FK-specific degradation pathway from Gram-positive bacteria, providing important information for the development of FA-recognizing molecules as well as for the FA assimilation system in bacteria.     

A methodology for investigating new nonprecious metal catalysts for PEM fuel cells

This paper reports an approach to investigate metal-chalcogen materials as catalysts for the oxygen reduction reaction (ORR) in proton exchange membrane (PEM) fuel cells. The methodology is illustrated with reference to Co-Se thin films prepared by magnetron sputtering onto a glassy-carbon substrate. Scanning Auger microscopy (SAM), X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) have been used, in parallel with electrochemical activity and stability measurements, to assess how the electrochemical performance relates to chemical composition. It is shown that Co-Se thin films with varying Se are active for oxygen reduction, although the open circuit potential (OCP) is lower than for Pt. A kinetically controlled process is observed in the potential range 0.5-0.7 V (vs reversible hydrogen electrode) for the thin-film catalysts studied. An initial exposure of the thin-film samples to an acid environment served as a pretreatment, which modified surface composition prior to activity measurements with the rotating disk electrode (RDE) method. Based on the SAM characterization before and after electrochemical tests, all surfaces demonstrating activity are dominated by chalcogen. XRD shows that the thin films have nanocrystalline character that is based on a Co(1-x)Se phase. Parallel studies on Co-Se powder supported on XC72R carbon show comparable OCP, Tafel region, and structural phase as for the thin-film model catalysts. A comparison for ORR activity has also been made between this Co-Se powder and a commercial Pt catalyst.     

An optical resolution of racemic organophosphorous esters by phosphotriesterase-catalyzing hydrolysis

Conclusion  We have shown that PTE chiral recognition is limited to organophosphorous esters having the chiral center on the phosphorous atoms, which is attacked with nucleophile, not having the chiral center on the other atoms of the leaving group. Therefore, PTE can be utilized for the synthesis of their chiral organophosphorous esters.     

Tuning of Electronic Properties of Single‐Walled Carbon Nanotubes under Homogenous Conditions

Reversible and non‐bonding interaction between SWNTs and ODCB is observed from the analyses of visible near‐infrared absorption data and Raman spectroscopies (see spectra). The solvent effect on SWNTs effectively controls the electronic structure of SWNTs under homogeneous conditions.

     

Enzyme electrochemical preparation of a 3-keto derivative of 1,5-anhydro-d-glucitol using glucose-3-dehydrogenase

A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. α-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion of 1,5-anhydro-d-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol (3-ketoAG), which was identified by ¹³C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L·h), and the electrochemical yield of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%.     

Site-directed mutagenesis study on the thermal stability of a chemiric PQQ glucose dehydrogenase and its structural interpretation

We have previously reported that a chimeric pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH), E97A3, which was made up of 97% of Escherichia coli PQQGDH sequence and 3% of Acinetobacter calcoaceticus PQQGDH, showed increased thermal stability compared with both parental enzymes. Site-directed mutagenesis studies were carried out in order to investigate the role of amino-acid substitution at the C-terminal region, Ser 771, of a chimeric PQQGDHs on their thermal stability. A series of Ser 771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction governs the thermal stability of the chimeric enzymes. Comparison of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97 A3 acquired stability at the first step of denaturation, which is reversible, and where no significant secondary structure change was observed. These results suggested that the interaction between C-terminal and N-terminal regions may play a crucial role in maintaining the overall structure of β-propeller proteins.     

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphono-thioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the K _i value of each enantiomer toward electriceel acetylcholinesterase. R-EPN (K _i=6 μM) inhibited acetylcholinesterase much more effectively than S-EPN (K _i=52 μM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The K _is value for R-EPN decreased from 0.24 to 0.03 mM, and the V _max value increased from 0.25 to 0.60 U/mg of protein. V _max/K _is values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.     

Integrated biosensor for glucose and galactose

A glucose microsensor based on quinoprotein glucose dehydrogenase (GDH) and electron mediators is described. It is unaffected by the concentration of dissolved oxygen. Its calibration graph is linear below 10 mg dl ^?1. The insensitivity to oxygen arises because the concentration of the oxidized forms of the mediators is insufficient to oxidize the reduced form of GDH. An integrated microsensor for glucose and galactose based on GHD and galactose oxidase was constructed. Glucose and galactose concentrations were determined from the current increase due to oxidation of the mediators or the current decrease due to reduction of oxygen.