Browsing by Author "Urzúa, U"

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    Kinetics of Mn3+-oxalate formation and decay in reactions catalyzed by manganese peroxidase of Ceriporiopsis subvermispora
    (1998) Urzúa, U; Kersten, PJ; Vicuña, R
    The kinetics of Mn3+-oxalate formation and decay were investigated in reactions catalyzed by manganese peroxidase (MnP) from the basiomycete Ceriporiopsis subvermispora in the absence of externally added hydrogen peroxide, A characteristic lag observed in the formation of this complex was shortened by glyoxylate or catalytic amounts of Mn3+ or hydrogen peroxide, MnP titers had a minor effect on this lag and did not influence the decay rate of the complex. In contrast, Mn2+ and oxalate drastically affected maximal concentrations of the Mn3+-oxalate complex formed, the decay of which was accelerated at high Mn2+ levels. The highest concentration of complex was obtained at pH 4.0, whereas an inverse relationship was found between the pH of the reaction and the decay rate of the complex with MnP present. In the absence of MnP, the best fit for the decay kinetics of the complex gave an order of 3/2 at concentrations in the range of 30-100 mu M, with a k(obs) = 0.012 min(-1) M-0.5 at pH 4.0. The rate constant increases at lower pH values and decreases at high oxalate concentrations. The physiological relevance of these findings is discussed. (C) 1998 Academic Press.
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    Manganese peroxidase dependent oxidation of glyoxylic and oxalic acids synthesized by Ceriporiopsis subvermispora produces extracellular hydrogen peroxide
    (1998) Urzúa, U; Kersten, PJ; Vicuña, R
    The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase, In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted, The in vivo oxidation of oxalate was measured; (CO2)-C-14 evolution was monitored after addition of exogenous [C-14]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO2 from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5, However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed, Both MnP activity and oxidation of [C-14]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [C-14]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures, IG addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction, Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.
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    Oxalate oxidase from Ceriporiopsis subvermispora
    (1999) Aguilar, C; Urzúa, U; Koenig, C; Vicuña, R
    The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pi of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has K-m and k(cat) values of 0.1 mM and 88 s(-1), respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed. (C) 1999 Academic Press.
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    Oxidation of kojic acid catalyzed by manganese peroxidase from Ceriporiopsis subvermispora in the absence of hydrogen peroxide
    (2002) Bastidas, F; Urzúa, U; Vicuña, R
    We have previously reported the oxidation of kojic acid catalyzed by manganese peroxidase (MnP) from Ceriporiopsis subvermispora. This reaction is strictly dependent on Mn(II), although it does not require the addition of hydrogen peroxide. We have extended these studies because this reaction can be considered as a model system for the in situ generation of hydrogen peroxide in natural environments. We show here that oxidation of kojic acid with horseradish peroxidase (HRP) plus hydrogen peroxide or with manganic acetate rendered a product with identical chromatographic and spectral properties as the one obtained in the reaction catalyzed by MnP. The initial lag observed in the latter reaction decreased significantly upon UV irradiation of the substrate. On the other hand, ascorbic acid increased the lag and did not affect the yield of the reaction. The superoxide anion trapping agents glutathione, nitr blue tetrazolium, and superoxide dismutase markedly affected the reaction. In contrast, addition of the hydroxyl radical scavengers mannitol and salicylic acid had no effect. Based on these results, a mechanism for the MnP-catalyzed reaction is proposed.