Hiroyuki Wariishi

In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

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-benzo-quinone, 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.

Ultrahighly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors

A sensitive and simultaneous analytical technique for visualizing multiple endogenous molecules is now strongly required in biological science. Here, we show the applicability of a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) system for getting chemically diverse metabolite profiles on a single-mammalian cell. This ultrahighly sensitive MALDI-MS technique enabled a spatially resolved detection of a broad range of metabolites including nucleotides, cofactors, phosphorylated sugars, amino acids, lipids, and carboxylic acids in normal mouse brain tissue with their unique distributions. Furthermore, a combination of MS imaging and metabolic pathway analysis of a rat transient middle cerebral artery occlusion model visualized a spatiotemporal behavior of metabolites in the central metabolic pathway regulated by an ischemia reperfusion. These findings highlight potential applications of an in situ metabolomic imaging technique to visualize spatiotemporal dynamics of the tissue metabolome, which will facilitate biological discovery in both preclinical and clinical settings.

In situ synthesis of silver nanoparticles on zinc oxide whiskers incorporated in a paper matrix for antibacterial applications

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.

In situ metabolomic mass spectrometry imaging: Recent advances and difficulties ☆

MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a systems genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.

Proteomic and Metabolomic Analyses of the White-Rot Fungus Phanerochaete chrysosporium Exposed to Exogenous Benzoic Acid

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.

Highly sensitive matrix-assisted laser desorption ionization-mass spectrometry for high-throughput metabolic profiling.

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.

Oxidation of bisphenol A catalyzed by laccase hosted in reversed micelles in organic media

Abstract The oxidation of bisphenol A [BPA: 2,2-bis(4-hydroxyphenyl)propane] catalyzed in organic solvents by laccase (polyphenoloxidase, EC 1.10.3.2) entrapped in a reversed micellar (RM) system was investigated. The laccase/RM system effectively catalyzed the oxidation reaction in isooctane, 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 laccase/RM system, we examined the effects of pH in the water pools of RM, the concentration of laccase, and the degree of surfactant hydration ( W o ) on the laccase activity in organic media. The laccase/RM system showed a strong pH-dependency in organic media. Its optimum activity was obtained when the laccase/RM was prepared at pH=5. Interestingly, native laccase in an aqueous buffer solution exhibited an optimal activity at pH=3. The effect of a cosolubilized mediator (1-hydroxybenzotriazole, HBT) on the reaction was also investigated. BPA was effectively oxidized by the laccase/RM system in the absence of a mediator. On the other hand, the addition of 1-hydroxybenzotriazole strongly enhanced the catalytic performance of the laccase/RM system against the oxidation of various chlorophenols in organic media.

Bioconversion of Recalcitrant 4-Methyldibenzothiophene to Water-Extractable Products Using Lignin-Degrading Basidiomycete Coriolus versicolor

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‐(β‐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.

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.

Metabolomics-Driven Nutraceutical Evaluation of Diverse Green Tea Cultivars

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.