In order to understand how ligninolytic basidiomycetes manage to degrade recalcitrant aromatic compounds and to take advantage of this to design new bio-processes the following studies were carried out in the present EU project (QLK3-99-590): i) To optimize an expression systems for fungal peroxidases and laccases (to be used for protein engineering and industrial fermentation) by comparing refolding of protein from bacterial inclusion bodies, improved expression in industrial ascomycetes, and development of a basidiomycete expression system; ii) to characterize in terms of structure-function relationships some of the most relevant metalloenzymes produced by basidiomycetes and, based on the above information, to improve the catalytic properties of some of them for industrial application by protein engineering techniques; iii) to develop efficient fermentation processes for the production of the most promising metalloenzymes using the expression tools developed, and iv) to evaluate their industrial applicability in food production, paper pulp manufacture (as alternative to contaminating processes) and/or other environmentally-sound applications.
     The work includes the following tasks: i) Cloning DNA encoding peroxidases and laccases (for optimization of expression, site-directed mutagenesis, and recombinant enzyme fermentation); ii) Expression in Escherichia coli and production of active enzyme (refolding of protein from inclusion bodies); iii) Fungal metalloenzyme expression in Ascomycetes (using Emericella nidulans as a reference and Aspergillus oryzae as a fungal host for industrial expression); iv) Metalloenzyme expression in Basidiomycetes (using Schizophyllum commune as a model to develop a new expression system in the industrial basidiomycete Pycnoporus cinnabarinus); v) Isolation and characterization of relevant enzymes (including new peroxidase); vi) NMR spectroscopy and crystallographic analysis (to obtain molecular models and structural information on new and recombinant enzymes); vii) Structure-function and protein modification studies (for elucidation of key aspects of enzyme catalysis and production of improved variants); viii) Fermentation technology for metalloenzyme production (using the most adequate host systems); and ix) Evaluation of the industrial application of the enzymes studied (including food and pulp-paper sectors).
     Because of high multi-disciplinarity required, the project consortium includes groups with expertise in: i) biotechnology (CIB-CSIC, ES; and UHEL, FI); ii) fungal genetics (RuG, NL; and INRA, FR); iii) peroxidase engineering (USussex, GB); and iv) protein NMR (UFIR, IT) and x-ray diffraction analyses (ETH, CH); together with v) two enzyme-producing companies with experience in recombinant protein production (Frimond, BE; and Novozymes, DK). The above consortium (with partners from eight EU members and Switzerland) gives the project a strong European dimension.