Laccases (EC 1.10.3.2) catalyze oxidation of phenolic hydroxyls in lignins via removal of an electron and a proton to form phenoxy radicals during reduction of O2. Assaying of laccase activity is currently carried out on soluble low molecular weight molecules (e.g. ABTS and syringaldazine) which poorly resemble the lignin polymer. We employed electron paramagnetic resonance spectroscopy (EPR) for real time measurement of enzyme catalyzed radical formation on three types of lignin, ie two types of organosolv lignin, and a lignin rich residue from wheat straw hydrolysis, brought about by two different fungal laccases, derived from Trametes versicolor and Myceliophthora thermophila, respectively. In addition, the effect of selected mediators on the radical formation in the lignins facilitated by the laccases was measured. Laccase addition to suspensions of the individual lignin samples produced immediate time and enzyme dose dependent increases in intensity in the EPR signal allowing a direct quantitative monitoring of the radical formation and thus providing for laccase enzyme kinetics assessment on lignin. The experimental data verified that the laccases acted upon the insoluble lignin substrates in the suspensions and that a direct radical formation action of fungal laccases occurs on lignin without addition of mediators. EPR methodology provides a new type of enzyme assay of laccases on lignin and it will be discussed how such type of assay provides more insight into the interaction between laccases, lignins and mediators.

Real-time measurement of laccase catalyzed formation of radicals in lignin by electron paramagnetic resonance spectroscopy

255th National Meeting (2018)

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