Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.

Biocatalysis and Biosensing Laboratory Department of Food Science and Technology BOKU University of Natural Resources and Life Sciences Muthgasse 18 1190 Vienna Austria

BioCeV Institute of Microbiology Czech Academy of Sciences Prumyslova 595 Vestec 252 50 Czech Republic

Research Division Biochemical Technology Institute of Chemical Environmental and Bioscience Engineering TU Wien 1060 Vienna Austria

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$a Csarman, Florian $u Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. Electronic address: florian.csarman@boku.ac.at

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$a Functional expression and characterization of two laccases from the brown rot Fomitopsis pinicola / $c F. Csarman, T. Obermann, MC. Zanjko, P. Man, P. Halada, B. Seiboth, R. Ludwig

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$a Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.

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$a Obermann, Tobias $u Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria; BioCeV - Institute of Microbiology, Czech Academy of Sciences, Prumyslova 595, Vestec, 252 50, Czech Republic. Electronic address: obermann.tobias@gmail.com

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$a Zanjko, Mihael Colar $u Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. Electronic address: colar.mihael@gmail.com

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$a Man, Petr $u BioCeV - Institute of Microbiology, Czech Academy of Sciences, Prumyslova 595, Vestec, 252 50, Czech Republic. Electronic address: pman@biomed.cas.cz

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$a Halada, Petr $u BioCeV - Institute of Microbiology, Czech Academy of Sciences, Prumyslova 595, Vestec, 252 50, Czech Republic. Electronic address: halada@biomed.cas.cz

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$a Seiboth, Bernhard $u Research Division Biochemical Technology, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, 1060 Vienna, Austria. Electronic address: bernhard.seiboth@tuwien.ac.at

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$a Ludwig, Roland $u Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. Electronic address: roland.ludwig@boku.ac.at

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