Laccases have many potential areas of commercial use including e.g. pulp bleaching, textile dye decolorisation, bioglueing, detoxification, biofuel cells and biosensors. Deeper understanding of the structure-function interactions would be needed to make better use of laccases in various applications. Our aim is to study and improve the performance, especially the enzyme-substrate interactions, of fungal laccases by means of protein engineering, including also directed evolution methods. Phlebia radiata is a basidiomycetous fungi expressing a typical, non-thermotolerant laccase (PrL) with a pH-optimum in acidic region. The crystal structure of the protein is not known, but a structural model could be built based on known fungal laccase structures. In order to be able to do directed evolution studies, a heterologous expression of PrL in Saccharomyces cerevisiae under an inducible GAL1 promoter was set-up. The cultivation of the recombinant yeast strain was optimised in microtiter plate format, and a robotic screen was also set-up. Targeted random mutagenesis was applied using error-prone PCR. Two random laccase mutant libraries have now been generated and screened for mutants having altered substrate specificity. 15 mutant clones showing changed substrate specificity for the non-phenolic ABTS as compared to the phenolic DMP compound were picked for a replica robotic assay. The characterization of the 5 best mutants is now being carried out.
|Published - 2004
|MoE publication type
|2nd European Meeting Oxizymes in Naples - Naples, Italy
Duration: 3 Jun 2004 → 5 Jun 2004
|2nd European Meeting Oxizymes in Naples
|3/06/04 → 5/06/04