Final Activity Report Summary - DELAC (Engineering fungal laccases by directed molecular evolution and semi-rational approaches: application in bioremediation of polycyclic aromatic hydrocarbons)
The main aim of this project is to tailor biocatalysts for bioremediation of polycyclic aromatic hydrocarbons (PAHs) 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 either autochthonous populations or 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. However, most of these transformations must be carried out at high concentrations of organic co-solvents in which laccases undergo unfolding, therefore losing their activity.
We have tailored a thermostable laccase that tolerates high concentrations of co-solvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase -R2 variant- was capable of resisting a wide array of co-solvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of the catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the proteins surface stabilized the laccase by allowing additional electrostatic and hydrogen-bonding to occur. The evolved mutant MtL R2 was tested with mediator (1mM ABTS) for the oxidation of the PAHs such as anthracene in the presence of 40% acetonitrile.
The mutant showed the 90% conversion of anthracene into anthraquinone within the first 24 h, and it was recovered and reused with similar efficiency in the same oxidation process, demonstrating its stability under these reaction conditions. Furthurmore, the enzyme was covalently immobilised on polymethacrylate-based polymers activated with epoxy groups. The enzyme immobilised on Sepabeads EC-EP3 exhibited notable activity (203 U/g biocatalyst) along with remarkably improved stability towards pH, temperature and storage time, but no increased resistance to organic solvents. In addition, the immobilized laccase also exhibited good operational stability, maintaining 84% of its initial activity after 17 cycles.
The immobilised laccase was applied to the decolourisation of six synthetic dyes. It was found that some of the dyes needed the addition of 1-hydroxybenzotriazole for their decolourisation. Immobilized laccase retained 41% activity after five cycles for the decolourisation of methyl green in fixed-bed reactor experiments. The features of these immobilised biocatalysts are very attractive for their application for the decolourisation of dyes in the textile industry in batch and continuous fixed-bed bioreactors.
We have tailored a thermostable laccase that tolerates high concentrations of co-solvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase -R2 variant- was capable of resisting a wide array of co-solvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of the catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the proteins surface stabilized the laccase by allowing additional electrostatic and hydrogen-bonding to occur. The evolved mutant MtL R2 was tested with mediator (1mM ABTS) for the oxidation of the PAHs such as anthracene in the presence of 40% acetonitrile.
The mutant showed the 90% conversion of anthracene into anthraquinone within the first 24 h, and it was recovered and reused with similar efficiency in the same oxidation process, demonstrating its stability under these reaction conditions. Furthurmore, the enzyme was covalently immobilised on polymethacrylate-based polymers activated with epoxy groups. The enzyme immobilised on Sepabeads EC-EP3 exhibited notable activity (203 U/g biocatalyst) along with remarkably improved stability towards pH, temperature and storage time, but no increased resistance to organic solvents. In addition, the immobilized laccase also exhibited good operational stability, maintaining 84% of its initial activity after 17 cycles.
The immobilised laccase was applied to the decolourisation of six synthetic dyes. It was found that some of the dyes needed the addition of 1-hydroxybenzotriazole for their decolourisation. Immobilized laccase retained 41% activity after five cycles for the decolourisation of methyl green in fixed-bed reactor experiments. The features of these immobilised biocatalysts are very attractive for their application for the decolourisation of dyes in the textile industry in batch and continuous fixed-bed bioreactors.