Discovery of two novel laccase-like multicopper oxidases from Pleurotus citrinopileatus and their application in phenolic oligomer synthesis.

BACKGROUND: Laccases and laccase-like multicopper oxidases (LMCOs) oxidize a vast array of phenolic compounds and amines, releasing water as a byproduct. Their low substrate specificity is responsible for their tremendous biotechnological interest, since they have been used for numerous applications. However, the laccases characterized so far correspond to only a small fraction of the laccase genes identified in fungal genomes. Therefore, the knowledge regarding the biochemistry and physiological role of minor laccase-like isoforms is still limited. RESULTS: In the present work, we describe the isolation, purification and characterization of two novel LMCOs, PcLac1 and PcLac2, from Pleurotus citrinopileatus. Both LMCOs were purified with ion-exchange chromatographic methods. PcLac2 was found to oxidize a broader substrate range than PcLac1, but both LMCOs showed similar formal potentials, lower than those reported previously for laccases from white-rot fungi. Proteomic analysis of both proteins revealed their similarity with other well-characterized laccases from Pleurotus strains. Both LMCOs were applied to the oxidation of ferulic and sinapic acid, yielding oligomers with possible antioxidant activity. CONCLUSIONS: Overall, the findings of the present work can offer new insights regarding the biochemistry and variability of low-redox potential laccases of fungal origin. Low-redox potential biocatalysts could offer higher substrate selectivity than their high-redox counterparts, and thus, they could be of applied value in the field of biocatalysis.

FTacV study of electroactive immobilized enzyme/free substrate reactions: Enzymatic catalysis of epinephrine by a multicopper oxidase from Thermothelomyces thermophila.

In the present work, a kinetic analysis is made concerning the reaction of an electroactive immobilized enzyme with a free substrate, based on a Michaelis-Menten scheme. The proposed kinetic equations are investigated numerically for conditions describing large amplitude fast Fourier transform alternating current voltammetry (FTacV), under different reaction states (transient or steady state for the reaction intermediate as well as quasi or complete reversibility of the electrochemical step). The dependence of two chief observables that occur from the analysis of the results of the method, that is, the maximum of the harmonics and the potential shift of the corresponding dominant peaks, on substrate concentration is presented. The FTacV method is applied experimentally for an immobilized laccase-like multicopper oxidase from Thermothelomyces thermophila, TtLMCO1, and its reaction with epinephrine. From the experimental findings it is shown that the intrinsic characteristics of the system do not lead to the extraction of the desired kinetic data although indications on the relation between the kinetic constants is revealed. Finally, the response of the third harmonic for the first additions of epinephrine at subnanomolarity range can be exploited for the detection of epinephrine at rather low concentrations.

Direct electron transfer of lytic polysaccharide monooxygenases (LPMOs) and determination of their formal potentials by large amplitude Fourier transform alternating current cyclic voltammetry.

MtLPMO9 and FoLPMO9 are two lytic polysaccharide monooxygenases (LPMOs), from the filamentous fungi Thermothelomyces thermophila and Fusarium oxysporum, respectively. In the present study an attempt has been made to achieve direct electron transfer between these enzymes and a glassy carbon electrode by immobilization in Nafion polyelectrolyte. The method used to ascertain the feasibility of direct electron transfer was large amplitude Fourier transform alternating current voltammetry (FTacV) and the formal potentials of these enzymes were determined at different temperatures. The findings of this paper indicate that LPMOs can be studied by direct electron transfer, which could be exploited in the near future for their biochemical characterization.

Kinetic and amperometric study of the MtPerII peroxidase isolated from the ascomycete fungus Myceliophthora thermophila.

The enzyme MtPerII is a new peroxidase which has been isolated only recently from fungus Myceliophthora thermophila and has significant thermostability and stability at high H2O2 concentrations. In the present work, an electrochemical kinetic study, based on cyclic voltammetry, is performed for the first time for the catalytic decomposition of H2O2 by MtPerII, at 18°C. Leuco methylene blue (LMB) is used as a mediator and the catalytic and Michaelis constants are determined, assuming a Michaelis-Menten mechanism. Experimental evidence suggest the absence of inhibition by H2O2, for concentrations up to 16mM, and increasing catalytic activity for temperatures up to 50°C. Moreover, a modified electrode is constructed, by attempting the entrapment of MtPerII on a dodecanothiol self-assembled monolayer on gold. The modified electrode is studied chronoamperometrically in solutions containing methylene blue mediator and different concentrations of H2O2. It is shown that adsorbed MtPerII retains its activity and the modified electrode exhibits a considerably high linear region for the detection of H2O2. The experimental findings indicate that MtPerII is a new candidate for analytical and industrial applications.

Magnetic effect for electrochemically driven cellular convection.

Hydrodynamic instability analogous to Rayleigh-Bénard convection is observed in an electrolytic solution between two parallel copper wire electrodes. The laser interferometric technique can reveal the dissipation structure created by the motion of the fluid, which is controlled electrochemically. It is shown that under the presence of horizontal magnetic field the roll cells move horizontally along the electrodes. The electrochemically driven convection is simply controlled and monitored by setting and measuring the electrochemical parameters and forms many kinds of spatiotemporal patterns, especially under the magnetic field. The phenomenon is modeled by considering a Boussinesq fluid under a concentration gradient. The stability of the resulting equations is studied by linear stability analysis. The time dependent nonlinear system is investigated numerically and the main features of the experimental response are reproduced.