Induction, Isolation, and Characterization of Two Laccases from the White Rot BasidiomyceteCoriolopsis rigida

Abstract

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH 2 ). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH 2 extended the reactions catalyzed by these enzymes to the production of H 2 O 2 , the oxidation of Mn 2؉ , the reduction of Fe 3؉ , and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.Lignin is an aromatic heteropolymer of phenyl-propanoid units which confers structural rigidity to woody plant tissues and protects them from microbial attack (25). To depolymerize and mineralize lignin, white rot fungi have developed a nonspecific oxidative system including several extracellular oxidoreductases, low-molecular-weight metabolites, and activated oxygen species (47). The ability of white rot fungi to degrade a wide number of organopollutants is in part due to the action of this nonspecific system (42). Extracellular enzymes involved in the degradation of lignin and xenobiotics by white rot fungi include several kinds of laccases (34, 49), peroxidases (8, 33), and oxidases producing H 2 O 2 (20,32,50).The enzymatic composition of the ligninolytic system depends on the fungal species, with laccase being the common component (24,43). For this reason, a wide number of studies have focused on demonstrating the participation of laccase in significant ligninolytic events which were first attributed to other enzymes of the ligninolytic system. These events include the oxidation of nonphenolic lignin units, which comprise ca. 80% of the polymer, the generation of the H 2 O 2 required for both peroxidase activities and hydroxyl radical ( ⅐ OH) formation, and the production of Mn 3ϩ from the Mn 2ϩ pr...

Influence of pH on the growth, laccase activity and RBBR decolorization by tropical basidiomycetes

“…castanella and L. crinitus laccases from other basidiomycetes presented optimum pH 3.0 using ABTS as substrate (Saparrat et al, 2002;Ryan et al, 2003;Zouari-Mechichi et al, 2006). The present results demonstrated that i) the pH of the culture medium influenced the growth and in vitro decolorization of RBBR by L. crinitus CCB274 and P. castanella CCB444; ii) the fungi were able to alter the initial pH of the medium so that it would reach the optimum pH for growth; iii) RBBR decolorization by these fungi seemed to be the result of the action of laccase-type oxidases.…”

Screening of inducers for laccase production by Lentinula edodes in liquid medium

“…1B). Copper is a laccase cofactor which presents four cupric ions each associated with one single polypeptide chain (23) but it also has been proved that this element may play an important role in laccase genes regulation at transcription level as showed in Trametes versicolor (7) Phanerochaete chrysosporium (9), Pleurotus ostreatus (18) and Coriolopsis rigida (20). The 250 µM Cu provided the highest laccase activity in LN cultures, which is in accordance to the findings for other fungi, like T. versicolor (400 µM) (7), P. ostreatus (1.0 mM) (3) and V. volvacea (200 µM) (6).…”

“…1.10.30.2) are multi-copper containing proteins that are widely distributed among fungi (23,25) but are also found in bacteria (1), insects (22) and plants (15). These enzymes are involved in lignin degradation (15) and their low substrate specificity allows them to oxidize a wide range of compounds, which makes them suitable for biotechnological and environmental applications (20). However, high amounts of the enzyme are needed for these applications, as well as to study and understand its properties and role in lignin biodegradation.…”

“…However, high amounts of the enzyme are needed for these applications, as well as to study and understand its properties and role in lignin biodegradation. In this way, researches have been developed in order to increase laccase production by searching for new sources of it (16,17) and by screening for inducers of laccase production by microorganisms, like aminoacids (8), aromatic compounds (10), plant extracts (2) and copper (9,20). The white rot fungus shiitake (Lentinula edodes (Berk.)…”

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes Involved in Lignin- Degradation by Lentinula edodes on Native-PAGE

“…This Lcc activity increased gradually after day 52 while MnP activity was completely suppressed. Lcc has been shown to be induced by aromatic compounds and metallic ions such as copper (Collins & Dobson, 1997;Saparrat et al, 2002;Scheel et al, 2000;Shutova et al, 2008;Soden & Dobson, 2001). Indeed, copper has been reported to be a strong laccase inducer in the white-rot fungi Pleurotus ostreatus (Palmier et al, 2000), Trametes pubescens (Galhaup & Haltrich, 2001;Galhaup et al, 2002), and T. versicolor (Collins & Dobson, 1997).…”