The Pleurotus ostreatus laccase multi-gene family: isolation and heterologous expression of new family members
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This work was aimed at identifying and at characterizing new Pleurotus ostreatus laccases, in order to individuate the most suitable biocatalysts for specific applications. The existence of a laccase gene clustering was demonstrated in this basidiomycete fungus, and three new laccase genes were cloned, taking advantage of their closely related spatial organization on the fungus genome. cDNAs coding for two of the new laccases were isolated and expressed in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, in order to optimize their production and to characterize the recombinant proteins. Analysis of the P. ostreatus laccase gene family allowed the identification of a “laccase subfamily” consisting of three genes. A peculiar intron–exon structure was revealed for the gene of one of the new laccases, along with a high instability of the recombinant enzyme due to lability of its copper ligand. This study allowed enlarging the assortment of P. ostreatus laccases and increasing knowledge to improve laccase production.
KeywordsLaccase gene Fungus Recombinant heterologous expression Copper lability Promoter Phylogenetic analysis
This work was supported by the European Commission, Sixth Framework Program (SOPHIED contract NMP2-CT2004-505899), by grants from the Ministero dell’Università e della Ricerca Scientifica (Progetti di Rilevante Interesse Nazionale, PRIN), and from the Ministero Degli Affari Esteri di Intesa con il Ministero dell’Università e della Ricerca (Progetti di ricerca di base e tecnologica approvati nei protocolli di cooperazione scientifica e tecnologica bilaterale come previsto dal protocollo bilaterale tra Italia e Turchia). Stage of Vincenza Faraco at Department of Agrarian Production, Public University of Navarre was funded by University of Naples Federico II (Programma di scambi internazionali tra l’Università degli Studi di Napoli Federico II ed Istituti di ricerca stranieri per la mobilità di breve durata di docenti, studiosi e ricercatori). The authors thank Prof Ramírez of Department of Agrarian Production, Public University of Navarre for kindly making available the BAC (bacterial artificial chromosome) library of the Pleurotus ostreatus genome.
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