Laccases belong to the family of multicopper oxidases and they oxidise phenolic compounds with broad substrate specificity. A large variety of filamentous fungi have been shown to produce laccases, and these enzymes have been implicated in e.g. degradation of lignin, pigment formation, sporulation and plant pathogenesis. The best characterised laccases are those of the bacidiomycetous white-rot fungi. We have characterised a laccase enzyme from the ascomycete Melanocarpus albomyces and shown that it has very interesting technical properties, good thermostability and a neutral pH optimum. The three-dimensional structure of this laccase was solved and the gene encoding it was sequenced. These studies revealed some interesting points including N- and C-terminal processing of the protein and protrusion of the four C-terminal amino acids inside the protein near the active site. The M. albomyces laccase was expressed in Saccharomyces cerevisiae and Trichoderma reesei. Production in yeast yielded up to 3 mg/l of the enzyme, an expression level well adequate for protein engineering purposes. Both the use of a yeast prepro-sequence for expression and the truncation of the expression construct at the natural C-terminal cleavage site markedly improved the production level. When the laccase cDNA was expressed in Trichoderma reesei under the cbhl (cellobiohydrolase I) promoter, laccase production of more than 200 mg/l was obtained in shake flask cultivations. Fermentor cultivations have resulted in still higher laccase levels. The recombinant enzyme was purified and shown to have very similar biochemical and technical properties as the one produced by the native host. Northern analysis of the T. reesei transformants producing laccase suggested that expression of this laccase does not cause secretion stress to the host cells.
|Publication status||Published - 2004|
|MoE publication type||Not Eligible|
|Event||7th European Conference on Fungal Genetics - Copenhagen, Denmark|
Duration: 17 Apr 2004 → 20 Apr 2004
|Conference||7th European Conference on Fungal Genetics|
|Period||17/04/04 → 20/04/04|