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White-rot basidiomycetes, the most common wood-rotting organisms, are characterized by their ability to produce extracellular oxidative enzymes, among which laccases are regarded as promising catalysts for many biotechnological applications. A significant obstacle to the exploitation of laccase-based bioprocesses is the large amounts of enzyme required. In this study the issue has been addressed by applying a classical breeding approach to increase laccase production yields in the white-rot fungus Pleurotus ostreatus. Starting from two different P. ostreatus varieties, three higher laccase-producing hybrids have been obtained by crossing selected compatible monokaryons. The three selected strains increased the titre of parental strains up to four fold, reaching an expression level of up to 100 000 U/L. One hybrid exhibited a more complex isoenzyme pattern, illustrating the potential of classical breeding to differentiate protein expression. [ABSTRACT FROM AUTHOR]

Abstract: Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. Most of the known laccases have fungal or plant origins, although few laccases have been also identified in bacteria and insects. Most of the fungal laccases reported thus far are extra-cellular enzymes, whereas only few enzymes from fruiting bodies have been described so far. Multiple isoforms of laccases are usually secreted by each fungus depending on species and environmental conditions. As a fact, a laccase gene family has been demonstrated in the white-rot fungus Pleurotus ostreatus. This work allowed identification and characterization of the first laccase isoenzyme from the fruiting body of P. ostreatus. Discovery through mass spectrometry of LACC12 proves the expression of a functional protein by the related deduced encoding transcript. The topology of phylogenetic tree of fungal laccases proves that LACC12 falls in cluster with the members of P. ostreatus LACC10 (=POXC) subfamily, although lacc12 deduced intron–exon structure differs from that of the subfamily members and the related locus is located in a different chromosome. Results show that the evolutionary pattern of lacc12 and that of the other laccase isozyme genes may have evolved independently, possibly through duplication–divergence events. The reported data add a new piece to the knowledge about P. ostreatus laccase multigene family and shed light on the role(s) played by individual laccase isoforms in P. ostreatus. [Copyright &y& Elsevier]

Modern biocatalysis requires fast, sensitive, and efficient high-throughput screening methods to screen enzyme libraries in order to seek out novel biocatalysts or enhanced variants for the production of chemicals. For instance, the synthesis of bio-based furan compounds like 2,5-diformylfuran (DFF) from 5-hydroxymethylfurfural (HMF) via aerobic oxidation is a crucial process in industrial chemistry. Laccases, known for their mild operating conditions, independence from cofactors, and versatility with various substrates, thanks to the use of chemical mediators, are appealing candidates for catalyzing HMF oxidation. Herein, Schiff-based polymers based on the coupling of DFF and 1,4-phenylenediamine (PPD) have been used in the set-up of a novel colorimetric assay for detecting the presence of DFF in different reaction mixtures. This method may be employed for the fast screening of enzymes (Z' values ranging from 0.68 to 0.72). The sensitivity of the method has been proved, and detection (8.4 μM) and quantification (25.5 μM) limits have been calculated. Notably, the assay displayed selectivity for DFF and enabled the measurement of kinetics in DFF production from HMF using three distinct laccase–mediator systems. [ABSTRACT FROM AUTHOR]