Abstract
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.
Original language | English |
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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
Conference | 7th European Conference on Fungal Genetics |
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Country/Territory | Denmark |
City | Copenhagen |
Period | 17/04/04 → 20/04/04 |