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Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has several advantages in metabolomics. It is a highly sensitive, high-throughput, and low sample-consuming (within 1 μL) technique compared to other analytical platforms based on LC-MS, GC-MS or CE-MS. In the present study, high-throughput and non-targeted metabolomic technique using MALDI-MS was developed for the rapid analysis of cellular metabolites. Either detection limit or linearity between concentrations of several standards and peak intensities was examined, indicating the detection limit lower than 10 fmol/well and high linearity at low concentrations. To verify the validity of the method, metabolites from human leukemia cells were analyzed. Only 2,500 cells (0.5 μL of 5 × 106 cells/mL) were applied onto the sample plate. Low-molecular weight metabolites were analyzed in negative ion mode. Up to 150 metabolite peaks were detected from the single analysis (200 shots/sample) within 90 s. For multivariate analysis of human acute lymphoblastc leukemia Jurkat cells against drug-treatment, three anticancer drugs (methotrexate, 5-fluorouracil, and epigallocatechin-3-O-gallate) were utilized. Prior to MS analysis, an inhibitory effect of these drugs on cancerous cell growth was clearly observed. Principal component analysis on metabolites showed a clear observation of independent clusters for cells treated with these anticancer drugs. Furthermore, several metabolites involved in nucleotide synthesis were found to contribute to the separation of each cluster. These data suggest that the high-throughput MALDI-MS-based metabolomic technique proposed in the present study can be utilized for the drug screening and validation of drug efficacy and safety.

Manganese peroxidase (MnP) oxidized 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(4-(hydroxymethyl)-2-methoxyphenoxy) -1,3-dihydroxypropane (I) in the presence of MnII and H2O2 to yield 1-(3,5-dimethoxy-4-hydroxyphenyl)- 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-1-oxo-3-hydroxypropane (II), 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-hydroxypropanal (V), syringaldehyde (VI), vanillyl alcohol (VII), and vanillin (VIII). MnP oxidized II to yield 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), vanillyl alcohol (VII), vanillin (VIII), syringic acid (IX), and 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-hydroxypropanoic acid (X). A chemically prepared MnIII-malonate complex catalyzed the same reactions. Oxidation of I and II in H2(18)O under argon resulted in incorporation of one atom of 18O into the quinone III and into the hydroquinone IV. Incorporation of one atom of oxygen from H2(18)O into syringic acid (IX) and the phenoxypropanoic acid X was also observed in the oxidation of II. These results are explained by mechanisms involving the initial one-electron oxidation of I or II by enzyme-generated MnIII to produce a phenoxy radical. This intermediate is further oxidized by MnIII to a cyclohexadienyl cation. Loss of a proton, followed by rearrangement of the quinone methide intermediate, yields the C alpha-oxo dimer II as the major product from substrate I. Alternatively, cyclohexadienyl cations are attacked by water. Subsequent alkyl-phenyl cleavage yields the hydroquinone IV and the phenoxypropanal V from I, and IV and the phenoxypropanoic acid X from II, respectively. The initial phenoxy radical also can undergo C alpha-C beta bond cleavage, yielding syringaldehyde (VI) and a C6-C2-ether radical from I and syringic acid (IX) and the same C6-C2-ether radical from II. The C6-C2-ether radical is scavenged by O2 or further oxidized by MnIII, subsequently leading to release of vanillyl alcohol (VII). VII and IV are oxidized to vanillin (VIII) and the quinone III, respectively.

Polyelectrolyte titration using a fluorescent indicator was applied to determine the electrolytic charges in 10 whiskies at various pH levels. Tannic acid was also titrated to compare the relation between pH and charge because the polyelectrolytes in whisky were postulated to be polyphenols, mainly tannic acid, which are gradually extracted from a wood cask over the years. Below pH 7, the pH-charge curves for whisky were analogous to those for tannic acid, indicating that carboxyl groups in tannic acid were fairly stable on maturation of the whisky. The charge of the tannic acid increased with increasing pH, whereas that of whisky had a tendency to level off. The degree of leveling off was higher for whiskies with longer maturation. These phenomena are believed to be due to the oxidation of pyrogallol and catechol moieties in tannic acid, which occurs during storage of the whisky. There was a clear tendency for the whiskies with higher prices to have higher charges, that is, a higher tannic acid content.

In the present study, a high-throughput and nontargeted metabolomic technique using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was developed for the rapid analysis of cellular metabolites. Either the detection limit or the linearity between concentrations of several standards and peak intensities was examined, indicating a detection limit lower than 10 fmol/well with a high linearity at low concentrations. To verify the validity of this method, metabolites from human acute lymphoblastic leukemia Jurkat cells were analyzed. Only 2,500 cells suspended in PBS were directly dropped onto a stainless MALDI sample plate, followed by mixing with matrix on the sample plate. Up to 150 metabolite peaks were detected from a single analysis within 90 s. For multivariate analysis of Jurkat cells against drug-treatment, three anticancer drugs were utilized. Principal component analysis of metabolites showed clear independent clusters for cells treated with these anticancer drugs. Furthermore, several metabolites involved in nucleotide synthesis were found to contribute to the separation of each cluster. These data suggest that the high-throughput MALDI-MS-based metabolomic technique proposed in the present study can be utilized for drug screening and validation of drug efficacy and safety.

We explored the molecular diversity of cytochrome P450 genes in the filamentous fungus Aspergillus oryzae using bioinformatic and experimental approaches. Based on bioinformatic annotation, we found 155 putative genes of cytochromes P450 in the whole genome sequence; however, 13 of 155 appeared to be pseudogenes due to sequence deletions and/or inframe stop codon(s). There are 87 families of A. oryzae cytochromes P450 (AoCYPs), indicating considerable phylogenetic diversity. To characterize A. oryzae AoCYPs, we attempted to isolate cDNAs using RT-PCR and determined their transcriptional capabilities. To date, we have confirmed gene expression of 133 AoCYPs and cloned 121 AoCYPs as full-length cDNAs encoding a mature open reading frame. Using experimentally deduced sequences and intron-exon organization, we analyzed AoCYPs phylogenetically. We also identified intronic consensus sequences in AoCYPs genes. The experimentally validated exonic and intronic sequences will be a powerful advantage in identification and characterization of novel P450s from various ascomycetous fungi.

Paramagnetic (13)C and (15)N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide ((13)C(15)N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The (13)C and (15)N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The (13)C NMR shifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.

Denervation is known to induce skeletal muscle atrophy and fiber-type transitions, the molecular mechanisms of which are poorly understood. To investigate the effect of denervation on skeletal muscle, proteomic analysis was performed to compare denervated soleus muscle with normal soleus muscle. The muscles were fractionated to myofibrillar and sarcoplasmic fractions, which were analysed using two-dimensional gel electrophoresis (2-DE), followed by MALDI-TOF-MS. At least 30 differentially regulated proteins were identified in the sarcoplasmic fractions of normal and denervated soleus muscles. This group included metabolic enzymes, signaling molecules, chaperones, and contractile proteins. We also found two proteins, APOBEC-2 (RNA-editing enzyme) and Gamma-synuclein (breast cancer related protein), which have not been recognized as denervation-induced proteins to date. Our results might prove to be beneficial in elucidating the molecular mechanisms of denervation-induced muscle atrophy.

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.3kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3'-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo-p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.

Enzymatic glyco-modification of transparent cellulose films with lactose was achieved by nonaqueous biocatalysis, and rat hepatocyte attachment behavior to the lactose-modified cellulose films was investigated. Regenerated cellulose films were incubated with lactose using a surfactant-enveloped cellulase in lithium chloride/dimethylacetamide solvent at 37 degrees C for 24 h, and lactose molecules were successfully introduced to the cellulose films. The initial cell adhesion on the lactose-modified cellulose films was superior to the original cellulose film. In the absence of serum, hepatocytes were significantly attached only on the lactose-modified cellulose films. This process was markedly suppressed by the addition of free lactose as an inhibitor. These results suggest that such cell adhesion proceeded through a direct interaction between galactose residues on the cellulose films and asialoglycoprotein receptors on the rat liver cell surface. This novel approach for surface glyco-modification of a cellulose matrix and its biofunctional properties are expected to provide potential application as a bioactive scaffold for cell culture engineering.

7 páginas, 7 figuras, 2 tablas -- PAGS. nros. 208-214 Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca2+ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H2O2 and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (kcat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (Km) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP This work was supported by the Spanish Projects BIO2007-65890-C02-01 and BIO2008-01533, and the BIORENEW project of the European Union (Contract NMP2-CT-2006-026456). Y.M. and F.J.R.-D. acknowledge two EU project contracts, and M.M. acknowledges a FPI fellowship of the Spanish MICINN

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.

Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different 14C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of 14C-sidechain-labeled syringyl lignin, a 14C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of 14C-sidechain- and 14C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzoquinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium .

Cu nanoparticles (CuNPs) were successfully synthesized in situ on ZnO whiskers as a selective scaffold, which were supported in a microstructured paper matrix composed of inorganic fibers; as-prepared paper composites were easy to handle in practical use and demonstrated excellent catalytic performance in the methanol reforming process for effective hydrogen production.

Resonance Raman (RR) spectra of several compounds III of lignin peroxidase (LiP) have been measured at 90 K with Soret and visible excitation wavelengths. The samples include LiPIIIa (or oxyLiP) prepared by oxygenation of the ferrous enzyme, LiPIIIb generated by reaction of the native ferric enzyme with superoxide, LiPIIIc prepared from native LiP plus H2O2 followed by removal of excess peroxide with catalase, and LiPIII* made by addition of excess H2O2 to the native enzyme. The RR spectra of these four products appear to be similar and, thus, indicate that the environments of these hexacoordinate, low-spin ferriheme species must also be very similar. Nonetheless, the Soret absorption band of LiPIII* is red-shifted by 5 nm from the 414-nm maximum common to LiPIIIa, -b, and -c [Wariishi, H., & Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077]. Analysis of the iron-porphyrin vibrational frequencies indicates that the electronic structures for the various compounds III are consistent with an FeIIIO2 ?- formulation. The spectral changes observed between the oxygenated complex and the ferrous heme of lignin peroxidase are similar to those between oxymyoglobin and deoxymyoglobin. The contraction in the core sizes in compound III relative to the native peroxidase is analyzed and compared with that of other heme systems. EPR spectra confirm that the high-spin ferric form of the native enzyme, with an apparent g = 5.83, is converted into the EPR-silent LiPIII* upon addition of excess H2O2. Its magnetic behavior may be explained by antiferromagnetic coupling between the low-spin FeIII and the superoxide ligand. The Fe-O2 stretching vibration of the oxygenated peroxidase is observed at 563 cm-1 and shifts to 538 cm-1 with 18O isotope. The Fe-histidine stretching vibration is observed at 245 cm-1 in ferrous peroxidase and appears to shift to 276 cm-1 in the oxygenated complex. ? 1990 American Chemical Society.

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H{sub 2}O{sub 2}-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H{sub 2}O{sub 2} concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H{sub 2}O{sub 2} to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H{sub 2}O{sub 2} and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H{sub 2}O{sub 2}. These results support amore » mechanism whereby VA protects the enzyme from inactivation by H{sub 2}O{sub 2}, thus making more enzyme available for the oxidation of recalcitrant substrates, such as AA and probably polymeric lignin. The results do not support a mechanism whereby VA acts as a radical mediator in the LiP oxidation of AA and other monomethoxylated aromatics.« less

Stopped-flow techniques were used to investigate the kinetics of the reaction of lignin peroxidase compounds II and III (LiPII and LiPIII) with peroxides. Rate data were obtained from single-turnover experiments under pseudo-first-order conditions. LiPII reacts with H2O2 or peracetic acid (AcOOH) to form a modified LiPIII, designated as LiPIII*, via a biphasic reaction. During the first phase, LiPIII is formed as an intermediate. Kinetic analysis also indicates a LiPII-peroxide complex. The first-order dissociation rate constants for the reaction of LiPII with H2O2 and AcOOH are 7.9 +/- 0.5 and 4.9 +/- 0.6 s-1, respectively. The rate of the H2O2 reaction is approximately 500 times the rate of the comparable reaction with horseradish peroxidase, suggesting it is physiologically significant. The activation energy for the formation of LiPIII is 23 kJ mol-1. During the second phase, the intermediate LiPIII is converted to LiPIII*, confirmed by analyzing the reaction of exogenously prepared LiPIII with peroxides. The second-order rate constants for the reaction of LiPIII with H2O2 and AcOOH are (3.7 +/- 0.2) x 10(2) M-1 s-1 and (2.9 +/- 0.2) x 10(2) M-1 s-1, respectively. The conversion of LiPIII to LiPIII* is reversible; the first-order rate constant for the reverse reaction is approximately (6.6 +/- 0.6) x 10(-2) s-1. The rates of both LiPIII and LiPIII* formation decrease markedly above pH 4.0. The pH dependence of these reactions is controlled by a heme-linked ionizable group of pK alpha congruent to 4.2.

Lignin peroxidase compound III (LiPIII) was prepared via three procedures: (a) ferrous LiP + O2 (LiPIIIa), (b) ferric LiP + O2-. (LiPIIIb), and (c) LiP compound II + excess H2O2 followed by treatment with catalase (LiPIIIc). LiPIIIa, b, and c each have a Soret maximum at approximately 414 nm and visible bands at 543 and 578 nm. LiPIIIa, b, and c each slowly reverted to native ferric LiP, releasing stoichiometric amounts of O2-. in the process. Electronic absorption spectra of LiPIII reversion to the native enzyme displayed isosbestic points in the visible region at 470, 525, and 597 nm, suggesting a single-step reversion with no intermediates. The LiPIII reversion reactions obeyed first-order kinetics with rate constants of approximately 1.0 X 10(-3) s-1. In the presence of excess peroxide, at pH 3.0, native LiP, LiPII, and LiPIIIa, b, and c are all converted to a unique oxidized species (LiPIII*) with a spectrum displaying visible bands at 543 and 578 nm, but with a Soret maximum at 419 nm, red-shifted 5 nm from that of LiPIII. LiPIII* is bleached and inactivated in the presence of excess H2O2 via a biphasic process. The fast first phase of this bleaching reaction obeys second-order kinetics, with a rate constant of 1.7 X 10(1) M-1 s-1. Addition of veratryl alcohol to LiPIII* results in its rapid reversion to the native enzyme, via an apparent one-step reaction that obeys second-order kinetics with a rate constant of 3.5 X 10(1) M-1 s-1. Stoichiometric amounts of O2-. are released during this reaction. When this reaction was run under conditions that prevented further reactions, HPLC analysis of the products demonstrated that veratryl alcohol was not oxidized. These results suggest that the binding of veratryl alcohol to LiPIII* displaces O2-., thus returning the enzyme to its native state. In contrast, the addition of veratryl alcohol to LiPIII did not affect the rate of spontaneous reversion of LiPIII to the native enzyme.

Individual cellulose macromolecules were successfully visualized on a highly oriented pyrolytic graphite (HOPG) surface by tapping-mode atomic force microscopy under ambient condition. Monomolecular-level dispersion of cellulose chains was achieved through the momentary contact of dilute cellulose/cupri-ethylenediamine (Cu-ED) solution onto the HOPG substrate. Both concentrations of cellulose and Cu-ED provided critical impacts on the topographical images. Single cellulose chains with molecular height of ca. 0.55 nm could be observed under the optimal conditions, showing rigid molecular rods with a unique morphology of hexagonal regularity. It was strongly suggested that the cellulose chains were aligned along the HOPG crystal lattice through a specific attraction, possibly due to a CH-pi interaction between the axial plane of cellulose and the HOPG pi-conjugated system. These phenomena would imply the potential applications of an HOPG substrate for not only nano-level imaging, but also for molecular alignment of cellulose and other structural polysaccharides.

It was previously reported that an unique peroxidase isoenzyme, cationic cell-wall-bound peroxidase (CWPO-C), from poplar callus oxidizes sinapyl alcohol, ferrocytochrome c and synthetic lignin polymers, unlike other plant peroxidases. Here, the catalytic mechanism of CWPO-C was investigated using chemical modification and homology modeling. The simulated CWPO-C structure predicts that the entrance to the heme pocket of CWPO-C is the same size as those of other plant peroxidases, suggesting that ferrocytochrome c and synthetic lignin polymers cannot interact with the heme of CWPO-C. Since Trp and Tyr residues are redox-active, such residues located on the protein surface were predicted to be active sites for CWPO-C. Modification of CWPO-C Trp residues did not suppress its oxidation activities toward guaiacol and syringaldazine. On the other hand, modification of CWPO-C Tyr residues using tetranitromethane strongly suppressed its oxidation activities toward syringaldazine and 2,6-dimethoxyphenol by 90%, respectively, and also suppressed its guaiacol oxidation activity to a lesser extent. Ferrocytochrome c was not oxidized by Tyr-modified CWPO-C. These results indicate that the Tyr residues in CWPO-C mediate its oxidation of syringyl compounds and high-molecular-weight substrates. Homology modeling indicates that Tyr-177 and Tyr-74 are located near the heme and exposed on the protein surface of CWPO-C. These results suggest that Tyr residues on the protein surface are considered to be important for the oxidation activities of CWPO-C with a wide range of substrates, and potentially unique oxidation sites for the plant peroxidase family.

TiO2 powder-containing paper composites, called TiO2 paper, were prepared by a papermaking technique, and their photocatalytic efficiency was investigated. The TiO2 paper has a porous structure originating from the layered pulp fiber network, with TiO2 powders scattered on the fiber matrix. Under UV irradiation, the TiO2 paper decomposed gaseous acetaldehyde more effectively than powdery TiO2 and a pulp/TiO2 mixture not in paper form. Scanning electron microscopy and mercury intrusion analysis revealed that the TiO2 paper had characteristic unique voids ca. 10 μm in diameter, which might have contributed to the improved photocatalytic performance. TiO2 paper composites having different void structures were prepared by using beaten pulp fibers with different degrees of freeness and/or ceramic fibers. The photodecomposition efficiency was affected by the void structure of the photocatalyst paper, and the initial degradation rate of acetaldehyde increased with an increase in the total pore volume of TiO2 paper. The paper voids presumably provided suitable conditions for TiO2 catalysis, resulting in higher photocatalytic performance by TiO2 paper than by TiO2 powder and a pulp/TiO2 mixture not in paper form.

The fungal metabolism of 4-nitrophenol (4-NP) was investigated using the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Despite its phenolic feature, 4-NP was not oxidized by extracellular ligninolytic peroxidases. However, 4-NP was converted to 1,2-dimethoxy-4-nitrobenzene via intermediate formation of 4-nitroanisole by the fungus only under ligninolytic conditions. The metabolism proceeded via hydroxylation of the aromatic ring and methylation of phenolic hydroxyl groups. Although the involvement of nitroreductase in the metabolism of 2,4-dinitrotoluene by many aerobic and anaerobic microorganisms including P. chrysosporium has been reported, no formation of 4-aminophenol was observed during 4-NP metabolism. The formation of 1,2-dimethoxy-4-nitrobenzene was effectively inhibited by exogenously added piperonyl butoxide, a cytochrome P450 inhibitor, suggesting that cytochrome P450 is involved in the hydroxylation reaction. Thus, P. chrysosporium seems to utilize hydroxylation and methylation reactions to produce a more susceptible structure for an oxidative metabolic system.

When the cultural atmosphere of the white-rot basidiomycete, Phanerochaete chrysosporium, was changed from air to 100% oxygen, the lyophilized mycelial weight increased and thickening of extracellular glucan layer was observed in 2-3 days. To better understand the oxygen-stress responsive mechanism of P. chrysosporium, the metabolomic differential display analysis was performed using metabolites isolated from fungal cells grown under either air or 100% O(2) atmosphere. In the GC-MS total ion chromatogram of methanol-extracts from fungal cells, at least 183 peaks were detected and 53 compounds were identified. Among them, veratryl alcohol (VA), threonate, and erythronate were identified as oxygen-stress responsive metabolites. The intracellular concentration of VA increased dramatically within 1 h after an oxygen purge, indicating that VA production is sensitive to the oxygen stress in P. chrysosporium.

Using the differential display reverse-transcriptional polymerase chain reaction (DDRT-PCR) technique, several cDNA fragments were isolated as chemical stress responsive genes from the white-rot basidiomycete, Coriolus versicolor, exposed to either 4-methyldibenzothiophene-5-oxide (4MDBTO) or dibenzothiophene-5-oxide (DBTO). A database search on deduced amino acid sequences of cDNAs revealed that they showed a high similarity with various proteins from other organisms. These results strongly suggested that cell responding systems might be involved in the fungal metabolism of exogenous chemicals by C. versicolor. One of the significantly up-regulated cDNA fragments by MDBTO, DD16gc, showed a high similarity to arylalcohol dehydrogenases (AADs) from several microorganisms. The full-length cDNA sequence of the DD16gc determined by 5' rapid amplification of cDNA ends method revealed that the gene consisted 1,295 nucleotide and poly(A) tail, encoding 394 amino acids in an open reading frame. The deduced protein showed a remarkable homology to AAD from Phanerochaete chrysosporium (66% identity) and to that from Saccharomyces cerevisiae (54% identity). The AAD gene was specifically transcripted under chemically-stressed conditions by 4MDBTO, suggesting that the enzyme encoded by the stress responsive gene may play an important role in the fungal conversion of 4MDBTO or its metabolic product(s).

Microperoxidase-11 (MP-11), a heme-containing undecapeptide, derived from horse heart cytochrome c was utilized as a peroxidative catalyst. Catalytic characteristics of MP-11 in hydrophilic organic media were studied using 2,6-dimethoxyphenol as a reducing substrate in a series of organic solvents at various concentrations, indicating that MP-11 was active in water-miscible organic solvents but at least 5% water was compulsory for the catalytic action. Thus, MP-11 was not active in hydrophobic solvents. The pH of the water portion in the media affected the reaction rate. The optimal pH was found to be 9, where a release of protons from either an oxidizing or reducing substrate to the media was facilitated. The decolorization of water-insoluble synthetic dyes by MP-11 in 90% methanol was attempted. MP-11 showed effective decolorization activities against either azo or anthraquinone dyes. The degradation pathway for Solvent Orange 7 was investigated in detail, showing that MP-11 catalyzed the oxidative cleavage of the azo linkage to generate 1,2-naphthoquinone and 2,4-dimethylphenol as key intermediates.

Under ligninolytic conditions, the white-rot basidiomycete Coriolus versicolor metabolized chloronitrofen (2, 4, 6-trichloro-4'-nitrodiphenyl ether; CNP) and nitrofen (2, 4-dichloro-4'-nitrodiphenyl ether; NIP), which constitute the largest class of commercially produced diphenyl ether herbicides. The pathway of CNP degradation was elucidated by the identification of fungal metabolites upon addition of CNP and its metabolic intermediates. The metabolic pathway was initially branched to form four metabolites – 2, 4, 6-trichloro-3-hydroxy-4'-nitrodiphenyl ether, 2, 4-dichloro-6-hydroxy-4'-nitrodiphenyl ether, NIP, and 2, 4, 6-trichloro-4'-aminodiphenyl ether – indicating the involvement of hydroxylation, oxidative dechlorination, reductive dechlorination, and nitro-reduction. Of these reactions, hydroxylation was relatively major compared to the others. Extracellular ligninolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase did not catalyze the oxidation of either CNP or NIP. Piperonyl butoxide, an inhibitor of cytochrome P450, suppressed fungal oxidation of CNP and NIP to their hydroxylated products. The inhibition resulted in increasing the amount of reductively dechlorinated and nitro-reduced products. These observations strongly suggest that basidiomycetes may possess a mechanism for a strict substrate recognition system and a corresponding metabolic response system to effectively degrade environmentally persistent aromatic compounds.

The fungal conversions of sulfur-containing heterocyclic compounds were investigated using the lignin-degrading basidiomycete Coriolus versicolor. The fungus metabolized a series of sulfur compounds – 25 structurally related thiophene derivatives – via several different pathways. Under primary metabolic conditions, C. versicolor utilized thiophenes, such as 2-hydroxymethyl-, 2-formyl-, and 2-carboxyl-thiophenes, as a nutrient sulfur source for growth; thus, the fungus degraded these compounds more effectively in a non-sulfur-containing medium than in conventional medium. The product analysis revealed that several redox reactions, decarboxylation reactions, and C-S cleavage reactions were involved in the fungal conversion of non-aromatic thiophenes. On the other hand, benzothiophene (BT) and dibenzothiophene (DBT) skeletons were converted to water-soluble products. All the products and metabolic intermediates were more hydrophilic than the starting substrates. These metabolic actions seemed to be a chemical stress response against exogenously added xenobiotics. These metabolic reactions were optimized under ligninolytic conditions, also suggesting the occurrence of a fungal xenobiotic response. Furthermore, the fungus converted a series of BTs and DBTs via several different pathways, which seemed to be controlled by the chemical structure of the substrates. DBT, 4-methylDBT, 4, 6-dimethylDBT, 2-methylBT, and 7-methylBT were immediately oxidized to their S-oxides. BTs and DBTs with the hydroxymethyl substituent were converted to their xylosides without S-oxidation. Those with carboxyl and formyl substituents were reduced to form a hydroxymethyl group, then xylosidated. These observations strongly suggested the involvement of a fungal substrate-recognition and metabolic response mechanism in the metabolism of sulfur-containing heterocyclic compounds by C. versicolor.

A novel preparation method for surfactant-MnP-Mn(II) ternary complex utilizing water-in-oil emulsions has been developed. The surfactant-MnP complex was spectroscopically characterized, strongly suggesting that the heme environment of the surfactant-MnP complex in benzene is identical to that of native MnP in the aqueous buffer. o-Phenylenediamine oxidation catalyzed by the surfactant-MnP-Mn(II) ternary complex was performed in benzene. The ternary complex efficiently catalyzed the oxidation, and the complex was catalytically stable. Kinetic experiments revealed that the reaction mechanism was as follows: MnP is oxidized by H(2)O(2) and the oxidized intermediate catalyzes the oxidation of Mn(II) to Mn(III) and the latter, after complexed with malonate, readily oxidizes o-PDA inside the complex. Thus, the organic substrate o-PDA, but not Mn(III), shuttled between the surfactant-MnP-Mn(II) ternary complex and organic solvent.

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.

Under secondary metabolic conditions, the white-rot basidiomycete Coriolus versicolormetabolized 4-methyldibenzothiophene (MDBT), which is a recalcitrant organic sulfur contaminant found in petroleum. The pathway of the transformation of MDBT was elucidated by the identification of fungal metabolites upon the addition of MDBT and its metabolic intermediates. S-oxidation to form MDBT-5-oxide was the initial step of MDBT metabolism. Then, the metabolic pathway was branched to form MDBT-5-dioxide, which was a dead-end product, and hydroxymethylDBT (HMDBT)-5-oxide. Extracellular ligninolytic enzymes such as lignin and manganese peroxidases and laccase did not catalyze the oxidation of either MDBT or MDBT-5-oxide. HMDBT-5-oxide was then oxidized to HMDBT-5-dioxide. Piperonyl butoxide, an inhibitor of cytochrome P450, suppressed fungal oxidation of MDBT to its oxide, MDBT-5-oxide to dioxide and to HMDBT-5-oxide, and HMDBT-5-oxide to dioxide. The efficiency of the inhibition varied for each substrate, suggesting that each oxidation was catalyzed by different enzymes. The hydroxylation of methyl substituents to the hydroxymethyl group was suggested to be catalyzed by a novel monooxygenase. HMDBT-5-dioxide was finally xylosylated most likely by xylosyltrasferase to yield 10-(beta-D-xylopyranosyloxy)-4-methyldibenzothiophene-5-dioxide. The final xyloside product and metabolic intermediates are water-extractable compounds, which would give us a novel strategy for biodesulfurization technology.

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pK a of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s −1 , respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

The degradation of thiodiglycol (I) and benzyl sulfide (II) was attempted using Coriolus versicolor and Tyromyces palustris to investigate the potential ability of basidiomycetes to degrade Yperite (bis(2-chloroethyl) sulfide), a mass-produced and stored chemical warfare agent. I was very rapidly degraded by both fungi. The metabolic pathway of II was elucidated, showing that the initial step was the hydrolytic cleavage of the thioether bond to yield benzyl alcohol and benzyl mercaptan. Benzyl alcohol was further oxidized and finally mineralized. Benzyl mercaptan is reversibly converted to benzyl disulfide and also converted to benzyl alcohol. Finally, the effective degradation of bis(2-bromoethyl) sulfide strongly suggests that basidiomycete would be a potential tool for Yperite degradation.

The maturity of green coffee beans is the most influential determinant of the quality and flavor of the resultant coffee beverage. However, the chemical compounds that can be used to discriminate the maturity of the beans remain uncharacterized. We herein analyzed four distinct stages of maturity (immature, semi-mature, mature and overripe) of nine different varieties of green Coffea arabica beans hand-harvested from a single experimental field in Hawaii. After developing a high-throughput experimental system for sample preparation and liquid chromatography-mass spectrometry (LC-MS) measurement, we applied metabolic profiling, integrated with chemometric techniques, to explore the relationship between the metabolome and maturity of the sample in a non-biased way. For the multivariate statistical analyses, a partial least square (PLS) regression model was successfully created, which allowed us to accurately predict the maturity of the beans based on the metabolomic information. As a result, tryptophan was identified to be the best contributor to the regression model; the relative MS intensity of tryptophan was higher in immature beans than in those after the semi-mature stages in all arabica varieties investigated, demonstrating a universal discrimination factor for diverse arabica beans. Therefore, typtophan, either alone or together with other metabolites, may be utilized for traders as an assessment standard when purchasing qualified trading green arabica bean products. Furthermore, our results suggest that the tryptophan metabolism may be tightly linked to the development of coffee cherries and/or beans.

A series of hemoproteins were characterized using Raman spectroscopic technique under non-resonant (near-infrared excited) conditions. All the proteins used in this study contain an iron-protoporphyrin IX with a coordinated histidine as a proximal ligand. Hemoproteins exhibited a near-infrared Raman shift at 1372 cm-1, only when heme was in the ferric state, while the peak completely disappeared when heme iron was reduced. The intensity of this peak was weakened upon the coordination of electron-donating ligands to heme iron. Therefore, the characteristics of this peak are different from the oxidation marker band assigned by the resonance Raman spectroscopy, rather, the intensity is strongly related to the sixth ligand field strength. In addition, the peak intensity may also reflect the distance between heme iron and the sixth ligand.

The effect of oxalate, malonate, lactate, and succinate chelators on the reduction of Phanerochaete chrysosporium manganese peroxidase compound II by MnII was investigated using stopped-flow techniques. All rate data were collected from single-turnover experiments under pseudo-first-order conditions. With oxalate, the reduction of compound II by MnII exhibited saturation behavior when the observed pseudo-first-order rate constants were plotted against oxalate concentration. The plots passed through the origin, indicating that the reduction by MnII is irreversible at all concentrations of oxalate. Maximal stimulation of the rate of compound II reduction occurred at 2 mM oxalate, the concentration of oxalate found in the extracellular medium of agitated cultures of this fungus. In contrast, maximal stimulation of the reduction of compound II by MnII only was observed at high (> 20 mM) nonphysiological concentrations of malonate and lactate. Furthermore, at low concentrations of malonate and lactate, the reduction of compound II appeared to be reversible. These results suggest that at physiological concentrations oxalate chelates and stabilizes MnIII, enhancing its efficient removal from the enzyme. The rate constants for compound II reduction exhibited bell-shaped curves as a function of pH and had optima at pHs 5.0-5.4. In the presence of succinate, triphasic kinetics were observed for compound II reduction by MnII. In contrast to the reduction of compound II by MnII, various chelators had no observable effect on the formation of compound I. However, they did affect the steady-state oxidation of 2,6-dimethoxyphenol.

Background Green tea has various health promotion effects. Although there are numerous tea cultivars, little is known about the differences in their nutraceutical properties. Metabolic profiling techniques can provide information on the relationship between the metabolome and factors such as phenotype or quality. Here, we performed metabolomic analyses to explore the relationship between the metabolome and health-promoting attributes (bioactivity) of diverse Japanese green tea cultivars. Methodology/Principal Findings We investigated the ability of leaf extracts from 43 Japanese green tea cultivars to inhibit thrombin-induced phosphorylation of myosin regulatory light chain (MRLC) in human umbilical vein endothelial cells (HUVECs). This thrombin-induced phosphorylation is a potential hallmark of vascular endothelial dysfunction. Among the tested cultivars, Cha Chuukanbohon Nou-6 (Nou-6) and Sunrouge (SR) strongly inhibited MRLC phosphorylation. To evaluate the bioactivity of green tea cultivars using a metabolomics approach, the metabolite profiles of all tea extracts were determined by high-performance liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses, principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), revealed differences among green tea cultivars with respect to their ability to inhibit MRLC phosphorylation. In the SR cultivar, polyphenols were associated with its unique metabolic profile and its bioactivity. In addition, using partial least-squares (PLS) regression analysis, we succeeded in constructing a reliable bioactivity-prediction model to predict the inhibitory effect of tea cultivars based on their metabolome. This model was based on certain identified metabolites that were associated with bioactivity. When added to an extract from the non-bioactive cultivar Yabukita, several metabolites enriched in SR were able to transform the extract into a bioactive extract. Conclusions/Significance Our findings suggest that metabolic profiling is a useful approach for nutraceutical evaluation of the health promotion effects of diverse tea cultivars. This may propose a novel strategy for functional food design.

The crystal structure of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium has been determined to 2.6 A resolution by usine multiple isomorphous replacement methods and simulated annealing refinement. Of the 343 residues, residues 3-335 have been accounted for in the electron density map, including four disulfide bonds. The overall three-dimensional structure is very similar to the only other peroxidase in this group for which a high-resolution crystal structure is available, cytochrome c peroxidase, despite the fact that the sequence identity is only approximately 20%, LiP has four disulfide bonds, while cytochrome c peroxidase has none, and LiP is larger (343 vs. 294 residues). The basic helical fold and connectivity defined by 11 helical segments with the heme sandwiched between the distal and proximal helices found in cytochrome c peroxidase is maintained in LiP. Both enzymes have a histidine as a proximal heme ligand, which is hydrogen bonded to a buried aspartic acid side chain. The distal or peroxide binding pocket also is similar, including the distal arginine and histidine. The most striking difference is that, whereas cytochrome c peroxidase has tryptophans contacting the distal and proximal heme surfaces, LiP has phenylalanines. This in part explains why, in the reaction with peroxides, cytochrome c peroxidase forms an amino acid-centered free radical, whereas LiP forms a porphyrin pi cation radical.

Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.

Au nanoparticles (AuNPs) were successfully synthesized in situ on a microstructured paper matrix composed of ceramic fibers as the main framework and ZnO whiskers as a preferential support for AuNPs. The paper-like ceramic fiber/ZnO whisker composites were prepared using a papermaking technique, then soaked in an aqueous solution of the Au(III) complex HAuCl4. AuNPs with size

Glyco-modification of a solid cellulose surface with lactosevia an enzymatic reaction was successfully achieved using a surfactant-enveloped enzyme (SEE) in a lithium chloride/dimethylacetamide solvent system. The unique biocatalyst, SEE, which is active in organic media, was prepared by protecting the surface of cellulase with the specific nonionic surfactant dioleyl-N-D-glucona-L-glutamate. Lactose, a second cellulase substrate, was introduced onto cellulose surfaces viaSEE-mediated enzymatic reaction. Fluorescent labeling and imaging revealed the presence of galactose residues from lactose on the modified cellulose surface. X-Ray diffractometry showed that the crystal structure of cellulose remained unchanged even after glyco-modification. Cell adhesion assays were carried out using rat liver cells on which galactose-specific receptors are present, and the initial cell attachment (within 12 h) on the lactose-modified cellulose filter was greater than that on an original (unmodified) cellulose matrix. The SEE-mediated biocatalysis enabled efficient glyco-modification of the solid cellulose surface in a one-step reaction, without complicated pre-treatment of the cellulose matrix and donor sugar. Consequently, this simple and effective approach to surface modification of a solid cellulose matrix with bio-functional sugars would be expected to have wide potential applications in glycomaterials engineering.

Silver nanoparticles (AgNPs) were successfully synthesized in situ on a paper matrix composed of ceramic fibers as the main framework and zinc oxide (ZnO) whiskers as a selective support for AgNPs. Paper-like ceramic fiber/ZnO whisker composites were prepared in advance using a high-speed, low-cost papermaking technique, then immersed in an aqueous solution of silver nitrate for 6 h. AgNPs with particle size 5–20 nm were spontaneously formed on the ZnO whiskers through selective ion-exchange between Ag and Zn species, and simultaneous ZnO-mediated photo-reduction under natural light irradiation. As-prepared material (AgNPs@ZnO paper) was subjected to antibacterial assay by the disk diffusion method using Escherichia coli. The AgNPs@ZnO paper exhibited excellent antibacterial activity and durability for repeated use, as compared with paper composites containing either ionic Ag components or commercial crystalline Ag microparticles. The facile and direct synthesis of AgNPs on a paper-like matrix is a unique approach for the immobilization of highly active metal NPs onto easy-to-handle support materials, and the AgNPs@ZnO paper is expected to be a promising bioactive material having both antibacterial function and paper-like utility.

Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H 2 O 2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [ 14 C]phenylhydrazine and H 2 O 2 results in 77% loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso -phenyl, iron-phenyl, or N -phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be δ- meso -azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active site of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.