Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus

Abstract

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the "blue copper oxidase" family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the K m was 7.1 M. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-aminoacid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.Litter mineralization is an important component of biogeochemical cycles in terrestrial environments. Lignin is the most difficult litter polymer to degrade, and the only organisms known to completely mineralize lignin are white-rot fungi (6,17). In the last two decades, several such organisms have been studied. At present, three main enzymes (i.e., manganese and lignin peroxidases and laccases) (15, 30) are implicated in the biodegradation of lignin. In addition to fundamental studies on lignin mineralization, these enzymes also have potential uses in industrial processes such as the bleaching of paper pulp (1) or the remediation of xenobiotics in effluents (3). There is no clear relationship between the distribution of ligninolytic enzymes and lignin degradation, since white-rot fungi with only one, with a combination of two, or with all three enzymes are known and can degrade lignin (15,27,34).The role of laccases in lignin degradation has only recently become well established (38). Laccases (p-diphenol oxidase, EC 1.10.3.2) are polyphenol oxidases that catalyze the reduction of oxygen to water with a concomitant oxidation of phenolic compounds. They are typically glycoproteins containing 2 to 4 atoms of copper per molecule and are found in plants and fungi (25,33,35).The evergreen oak (Quercus ilex) forms a characteristic forest climax common in the Western Mediterranean area (22,28). The leaf of Q. ilex, highly lignous and covered by a thick and waxy upper cuticle, is typical of sclerous plants exposed to dryness, particularly during the summer. The white-rot fungus Marasmius quercophilus colonizes dead leaves of Quercus ilex (32). Under favorable temperature and humidity conditions, i.e., in autumn and sometimes in May and June, this fungus becomes predominant among litter fungi and produces ma...

Enhancement of minor laccases production in the basidiomycete Marasmius quercophilus C30

“…Anionic chromatography was performed on an 8-dayold METCU-pHB liquid culture (2 l) as previously described [6]. Fractions containing laccase activity were concentrated and analyzed by electrophoresis.…”

“…quercophilus C30 was isolated and propagated as previously described [5,6]. Three 250-ml Erlenmeyer £asks containing either 50 ml of MET (2% malt extract+Tween 80, 0.05%), 50 ml of METCu (MET+CuSO 4 5 mg l 31 ) or 50 ml of METCu-pHB (MET+CuSO 4 5 mg l 31 +p-hydroxybenzoic acid 250 mg l 31 ) were inoculated with 2 ml of mycelial suspension and incubated at 28³C on a reciprocal shaker (50 rpm).…”

“…The C30 isolate produces several laccases among which LAC1, the major one, has been puri¢ed and fully characterized [6]. Low expression levels of other laccases, which could have a speci¢c activity between one and two orders of magnitude higher than that of LAC1, have been mentioned in earlier studies [5,7].…”

The fungal ability for biobleaching/biopulping/bioremediation of lignin-like compounds of agro-industrial raw material

“…99,100 Laccase was the only ligninolytic enzyme in Marasmius quercophilus. 101 The selected ligninolytic fungi for degradation of wood chips or non woody plants such as kenaf, straw, and corn stalks, should have preferred action against hemicelluloses and lignin united with low activity on cellulose. 7 The chips are briefly steamed to reduce native microorganisms, cooled with forced air, and inoculated with the biopulping fungus before be piled and ventilated with filtered and humidified air for 1-4 weeks prior to processing.…”

Biochemical and Molecular Analysis of Laccase Enzyme in Saprobic Fungus; Sordaria fimicola

“…Fungal species belong to Ascomycota have different laccase encoding genes which are involved in the oxidization of syringaldazine dye (Dedeyan et al, 2000). In the current research S. fimicola fungus was evaluated for its laccase enzyme potential, further laccase gene analysis was carried out in order to exploit them to produce laccase enzyme at large scale.…”