Abstract
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.
Original language | English |
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Publication status | Published - 2004 |
MoE publication type | Not Eligible |
Event | 2nd European Meeting Oxizymes in Naples - Naples, Italy Duration: 3 Jun 2004 → 5 Jun 2004 |
Conference
Conference | 2nd European Meeting Oxizymes in Naples |
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Country/Territory | Italy |
City | Naples |
Period | 3/06/04 → 5/06/04 |