Description
Enzymes catalyzing redox reactions (oxidoreductases) represent an environmentally-friendly alternative to harsh chemical reagents in industrial processes that include oxidative transformations for the production of chemicals and other value-added products. Fungi and other microorganisms provide the wider and more easily exploitable source for this type of enzymes. However, the penetration of microbial oxidoreductases in the industrial markets is still low despite the recent discovery of very promising enzymes. In the above scenario, the HIPOP project focuses first on the search for new basidiomycete peroxidases, one of the most interesting types of oxidoreductases due to their high redox-potential and peroxygenase activity recently described in some of them. The huge amount of genomic resources available nowadays, and to be generated in the near future, will be exploited in the search for new peroxidase genes. This will include the different types of peroxidase genes to be identified in a large JGI (DOE, US) project on 30 new basidiomycete genomes, where CIB coordinates the analysis of all the oxidoreductase genes, as well as in other related genomes available. Genome screening and gene heterologous expression will provide us a collection of new peroxidases to be evaluated in different oxidation reactions. With this purpose, some of the main issues presently limiting the industrial application of this type of enzymes will be addressed (e.g. their suicide inactivation by peroxide). Moreover, the catalytic properties of the most interesting enzyme candidates (including self-sufficient mono-oxygenase activity and oxidation of bulky substrates at the protein surface by long-range electron transfer to heme) will be modulated for higher industrial applicability. In both cases, a rational design based on information on structure-function relationships will be used, and changes in the amino acid sequences will be introduced by site-directed mutagenesis. This rational design will require a previous functional (including substrate specificity, and steady and transient-state kinetics), stability (to peroxide excess and other factors) and structural (including crystal structures, identification of catalytic residues, and detection of eventual protein radicals) characterization of the selected enzymes. The above approach will be possible by the large experience of the CIB group in peroxidase structure-function and phylogenetic relationships, together with some selected collaborations with other institutions (including JGI-DOE, University of Sienna, and Barcelona Supercomputing Center) and CSIC groups. In this way, a toolbox of new and more stable peroxidases with different catalytic properties of interest will be obtained. The high redox-potential and wide substrate specificity of some peroxidases, which will be further improved within the project, make them suitable to substitute harsh reagents in the oxidation of recalcitrant compounds. On the other hand, peroxidases with natural or engineered mono-oxygenase activity have also a great biotechnological potential for selective oxyfunctionalization reactions required in organic synthesis, which are difficult to achieve by conventional chemical tools. The biotechnological potential of the new or improved peroxidases available at the end of the HIPOP project will be further evaluated by Novozymes, the project EPO and world leader in the production of industrial enzymes.
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