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Engineering fungal laccases by directed molecular evolution and semi-rational approaches: application in bioremediation of polycyclic aromatic hydrocarbons (pahs) (DELAC)
Start date: 24 Sep 2007, End date: 23 Sep 2009 PROJECT  FINISHED 

The current research proposal claims to carry out the technology of the enzymatic bioremediation of polycyclic aromatic hydrocarbons (PAHs) by using biocatalysts (both native and tailored by directed molecular evolution). PAHs are a class of highly dangerous xenobiotics widely distributed in terrestrial and aquatic environments.Last trends in PAHs removal address to combine chemical and biological approaches for remediation of these persistent contaminants. However, high molecular weight PAHs cannot be successfully metabolized by neither autochthonous populations nor genetically manipulated bacteria, both under in situ and ex situ applications. In this context, the only organisms known to oxidize efficiently high molecular weight PAHs are the white rot fungi.Many shortcomings have hindered the application of such organisms in large-scale bioremediation processes. Laccase is the one of the most promising and versatile biocatalyst in PAHs oxidation. This proposal deals with the use of laccases in PAHs detoxification from sea spills and wastewaters. The dependence of redox mediators and the instability of laccases are the main hurdles for a practical application.The study of laccases in PAHs detoxification will be tackled by:- Biocatalyst engineering by directed molecular evolution to improve the enzyme stability against organic solvents.- The enhancing the redox potencial of lacccases will mean the increasing in the PAHs oxidation. The construction of combinatorial libraries by saturation mutagenesis will be performed to enhance the laccase ionization potential.- The biocatalyst will be immobilized on acrylic supports and used in a fixed-bed bioreactor to be tested with waste-waters contaminated with PAHs.The objectives of this Project are relevant to two of the activity areas of the Sixth Framework Program:- Life sciences, genomics and biotechnology for health;- Sustainable development, global change and ecosystems.
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