Laccases are multicopper oxidases wide distributed in nature and involved in a range of different processes (delignification, lignification, morphogenesis, oxidative stress response in plants, etc). Laccases are considered as ideal “green catalysts” since are capable of oxidizing dozens of compounds, using O2 from air and releasing H2O as the only by-product. Above all, the high redox potential laccases (with redox potentials around +800 mV at the T1 copper site) produced by white-rot basidiomycetous fungi, are of huge biotechnological significance because of their higher catalytic capabilities for bioremediation porpoises, paper pulp bleaching, organic synthesis, design of biosensors and biofuel cells, and processing of lignocellulose materials for biofuels production or manufacture of new products derived from lignin or cellulose.
For a number of applications, the presence of redox mediator compounds is required. Thus, the range of reducing substrates oxidized by the enzyme can be further expanded. In this context, it has been described the capability of certain compounds naturally-occurring as plant extractives or forming lignin-hemicellulose linkages in herbaceous plants or released during oxidation of lignin polymer, to act as redox mediators of fungal laccases. The presence of these natural compounds notably promotes the oxidative transformation of recalcitrant pollutants or lignin polymer in reactions catalyzed by laccase. The possibility to obtain these mediators from
natural resources wastes at low cost, makes the difference with the expensive synthesis of artificial mediators, providing an eco-friendly alternative to the use of artificial mediators.
On the other hand, biotechnological exploitation of fungal laccases demands the production at industrial scale of stable and active enzymes under the operational conditions. Therefore, protein engineering is required to attain high expression levels of efficient and robust enzymes. Directed molecular evolution is a powerful protein engineering tool to improve known features or to generate novel enzymatic functions not required in natural environments. The design of high redox potential laccases by directed molecular evolution represents an attractive alternative of huge possibilities keeping in mind the high evolvability of these systems owing their catalytic promiscuity and thermostability. Based on our former experience, the functional expression of high-redox potential laccases in S. cerevisiae can be significantly enhanced by directed evolution. Accordingly, the expression systems previously engineered through directed evolution are the platform in which EVOFACEL is based.
Combination of different in vitro and in vivo techniques (the latter using the eukaryotic machinery of S. cerevisiae) will be used to create genetic diversity from our mutant libraries and other laccase genes of interest. Generation of new laccases will stress on creation of chimerical genes from laccases from different (natural or synthetic) sources. On the other hand, fine-tunning of new high-troughput screening methods for laccase activity will be addressed on the use of natural compounds of biotechnological interest. Engineering of laccases with improved catalytic efficiency towards this type of compounds might be of relevance for building up enzymatic consortiums directed towards the integral use of plant biomass.