Purification and Partial Characterization of a Thermostable Laccase from Pycnoporus sanguineus CS-2 with Ability to Oxidize High Redox Potential Substrates and Recalcitrant Dyes

Abstract

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are enzymes that catalyze the oxidation of phenolic compounds and aromatic amines with the simultaneous reduction of molecular oxygen to water [1]. They are widely distributed in many plants and fungi, some insects and bacteria, being particularly abundant in white-rot basidiomycetes [2]. Typical fungal laccases are described as glycosylated multicopper proteins, which are produced as extracellular monomeric forms of around 60-80 kDa, containing four copper atoms and 15-20% carbohydrates. Operatively, they are moderately thermotolerant, showing optima activity at 50-55 °C, and under acidic conditions (pH 3-5); although their maxima stability occurs in the alkaline zone (pH 8-9) [3]. Their copper atoms are distributed in three different sites bringing unique spectroscopic properties: The type 1 copper (CuT1) atom, is responsible of the intense blue color of enzymes by light absorption around 610 nm; The type 2 copper (CuT2) atom exhibits a weak absorption in the visible region; and the two type 3 copper (CuT3) atoms are present as a binuclear center, which has an absorption maximum about 330 nm. Moreover, CuT2 and CuT3 copper atoms are structural and functionally arranged as a trinuclear cluster. The four copper atoms form part of the active site of enzyme contributing directly to reaction. CuT1 is involved in the initial electron subtraction from reducer substrates, while trinuclear