Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation

Abstract

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42-and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65°C and pH 4, and that for laccase II was reached at 55°C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy-and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide anion was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions.

Purification and characterization of the laccase secreted by the white rot fungus Perenniporia tephropora and its role in the decolourization of synthetic dyes

“…The pI of P. tephropora laccase (pH 3AE3) was in the acidic range reported for laccases from other white rot fungi, such as Pleurotus eryngii and Pleurotus ostreatus (pI 2AE9-4AE7) (Palmieri et al 1993;Muñoz et al 1997), Coriolopsis rigida (Saparrat et al 2002) Pycnoporus cinnabarinus (pI 3AE7) (Eggert et al 1996).…”

“…Indeed, there was a factor of specificity of 17 between this substrate and 2,6-DMP. Fungal laccases are known to possess a very wide range of substrate affinities (Mayer 1987;Eggert et al 1997) and even the laccase isozymes of the same strain may also show differential substrate affinities (Fukushima and Kirk 1995;Muñoz et al 1997). The effects of several laccase inhibitors were determined with 2,6-DMP (5 mmol l )1 ) as a substrate in 100 mmol l )1 sodium acetate buffer (pH 4AE0).…”

Purification and characterization of a growth-regulating laccase from Pleurotus florida

“…The molecular mass of the native protein was estimated to be 73 kDa by gel permeation chromatography (data not shown) and 70 kDa based on SDS-PAGE ( Fig. 1), indicating its monomeric nature like the P. eryngii laccase (MUNOZ et al 1997). Isoelectric focusing of L 2 of P. florida showed homogeneous protein band with a pI value of 4.2 (data not shown).…”

“…A number of laccase isozymes have been isolated and purified from Pleurotus spp. (MUNOZ et al 1997, PALMIERI et al 1993, SANNIA et al 1986, RESCIGNO et al 1993, YOUN et al 1995 but role of these enzyme in producer organisms has not yet been reported. Pleurotus florida produces two extracellular laccase isozymes (L 1 and L 2 ) and involvement of L 2 in the regulation of mycelial growth and yield of the mushroom has been suggested (DAS et al 1997).…”

Optimizing Laccase Application on Creeping Bentgrass (Agrostis stolonifera L.) to Facilitate Biodethatching

“…This cleavage of different covalent bonds formed within lignin macromolecules and between lignin and structural sugars open up the biomass structure, leading to increased availability of easily degradable sugars by microbes. The laccase enzyme is stable over a wide range of pH and temperatures (Baldrian, 2006;Munoz et al, 1997;Stoilova et al, 2010;Thurston, 1994). By using laccase enzyme, the rate of thatch decomposition can be improved over a wide range of environmental conditions by enhancing microbial decomposition of easily decomposable cellulose and hemicellulose.…”