Abstract
Although the model agaricomycete Coprinopsis cinerea possess 17 different laccase genes, up to now only four C. cinerea laccases have been purified and characterized to some degree. By exchanging the nucleotide sequence of the deduced signal peptide of Lcc8 it was possible to homologously express lcc8 in C. cinerea under control of the Agaricus bisporus gdpII promoter and the C. cinerea lcc1 terminator. The purified Lcc8 showed two bands in the SDS-PAGE with a molecular weight of 64 kDa and 77 kDa, respectively. The IEF determined pI values of 3.3 and 3.4 for both bands. The optimal pH for oxidation of the substrates ABTS, 2,6-dimethoxyphenol, guaiacol and syringaldazine was pH 4.0, pH 5.0, pH 4.5 and pH 5.0, respectively. Best pH for enzyme storage was pH 8.0. The optimal temperature for oxidation of ABTS was 63 °C, while Lcc8 showed activity of at least 50% over 300 min at 50 °C. The comparable high stability of Lcc8 at alkaline pH and higher temperatures can be of interest for biotechnical applications.
Highlights
Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) are phenoloxidases capable of oxidizing phenolic and aromatic compounds (Leonowicz et al 2001)
To express laccase Lcc8 homologously in C. cinerea, the most suitable plasmid pYSK7 constructed by Kilaru et al (2006b) for the expression of Lcc1 was modified in this study
The resulting plasmid pYMS33 comprised of the lcc8 gene under control of the glyceraldehyde 3-phosphate dehydrogenase II promotor from Agaricus bisporus and the lcc1-terminator of C. cinerea laccase lcc1 both revealed to be functional in C. cinerea laccase expression (Kilaru et al 2006b)
Summary
Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) are phenoloxidases capable of oxidizing phenolic and aromatic compounds (Leonowicz et al 2001). In nature, they are widespread in the fungal kingdom in the phylum Basidiomycota, but they occur in plants, insects and bacteria (Claus 2004). A very high titer of recombinant enzyme has been achieved with the homologous expression of the lac gene in the basidiomycete Pycnoporus cinnabarinus (Alves et al 2004) with a laccase yield of 1.2 g L−1. Research on basidiomycetous expression systems for industrial relevant enzymes is spare and might be underestimated, resulting in limited studies on recombinant laccase production in fungi of the phylum Basidiomycota
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