Protein and gene structure of a blue laccase from Pleurotus ostreatus1

Abstract

A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified.

Optimum conditions for inducing laccase production in Lentinus crinitus

“…Peniophora sp was the best laccase producer under this condition (4140 U/L), but G. applanatum was particularly sensitive to the addition of copper, increasing the production (1850 U/L) in the presence of copper. Our results agree with those of Giardina et al (1999), who observed that P. ostreatus produced a significantly greater amount of laccase (30,000 U/L) in nitrogen-rich medium and in the presence of 150 mM CuSO 4 compared to that produced in the absence of copper (4000 U/L). Shutova et al (2008) used higher CuSO 4 concentrations than those used in our study (1500-2000 mM) and observed positive effects on laccase activity in Lentinus tigrinus (47,000 U/L).…”

Influence of Culture Conditions on Laccase Production, Growth, and Isoenzymes Patterns in Native White Rot Fungi from the Misiones Rainforest (Argentina)

“…Varied Lac isoenzyme patterns of fungal origin are obtainable according to the culture conditions used, and this enzymatic potential becomes relevant for the efficient colonization of the substrate and growth (Das et al 1997;Giardina et al 1999). Both ND-PAGE and PAGE methods applied in this work revealed the existence of isoenzymes of different MWs appearing at different conditions of pH and temperature.…”

Metal-responsive elements in Pleurotus ostreatus laccase gene promoters

“…The existence of multiple genes encoding different laccase isoenzymes has been demonstrated in several fungi (Mansur et al, 1997;Smith et al, 1998;Giardina et al, 1999). Moreover, laccase gene expression depends on cultural conditions, and differentially regulated systems to control laccase production have been reported (Collins & Dobson, 1997;Mansur et al 1998;Muñoz et al;.…”

“…Pleurotus ostreatus is a basidiomycete that secretes several laccase isoenzymes, four of which, named POXC (Giardina et al, 1996), POXA1w (Palmieri et al, 1997), POXA3 and POXA1b (Giardina et al, 1999), have been purified and characterized. Four different P. ostreatus laccase genes and their corresponding cDNAs have been cloned and sequenced: poxc (Giardina et al, 1996) (previously named pox2), poxa1b (Giardina et al, 1999), poxa3 (Gene Bank accession no.…”

“…Four different P. ostreatus laccase genes and their corresponding cDNAs have been cloned and sequenced: poxc (Giardina et al, 1996) (previously named pox2), poxa1b (Giardina et al, 1999), poxa3 (Gene Bank accession no. AJ344434) and pox1 (Giardina et al, 1995) (encoding an as yet unidentified laccase isoenzyme).…”