An enzyme engineering programme, combined with newly developed activity assays, will be run to generate insight into structure-function relationships and to, eventually, create better industrial biocatalysts.
Generated mutant enzymes will have been tested as improved alternatives in quorum sensing.
By X-ray diffraction, high resolution structures will be determined.
Identification and functional characterisation of new cofactor-independent oxygenases within the tautomerase superfamily, with special focus on hypothetical oxygenases for which the genomic context gives clues about their physiological function.
Crystal structures of mutant oxygenases will be obtained with the aim to better understand mechanistic properties of such oxygenases.
Biotechnological exploration of flavin-dependent monooxygenases for the production of indigoid dyes and application testing for developing a novel process for (in-situ) textile dyeing.
Production of the generated heme-dependent monoxygeneases for formulation as lyophilisates in 96 well plates to develop novel metabolite test kits for the hydroxylation of heterocyclic compounds which are key intermediates for active pharmaceutical intermediates (API) synthesis
Using the generated monooxygenases, biocatalytic conversions of heterocycles will be performed, and the formed products will be isolated and characterized.
The work will focus on eukaryotic flavin-containing monooxygenases that are essential for the detoxification of xenobiotics. The target enzymes will be produced and purified using protein chromatography.
Novel heterocycle hydroxylating heme-dependent monooxygenases: novel enzymes will be identified by in silico and functional screening of plant and fungal transcriptomes.
Investigation of three heme-dependent monooxygenases for the conversion of heterocyclic compounds. Through modelling and random mutagenesis positions will be identified and mutated to elucidate on the molecular level structure-function relationships.
Laboratory data will be used to optimise processes in real (pilot-scale) industrial bioreactors, where process conditions such as how oxygen availability affects operational LPMO performance and the overall outcome of biomass conversion processes will be assessed.
The studies monooxygenases will be subjected to multi-site mutagenesis. Three libraries will be prepared.
Elucidation of crystal structures of eukaryotic flavin-containing monooxygenases and analysis of the structural featues in order to understand enzyme-based catalysis.
In-depth mechanistic and structural studies of a cofactor-independent monooxygenase from the tautomerase superfamily that catalyses the oxidative cleavage of HPP.
Generate knowledge-based focussed enzyme mutant libraries
Insight into the mechanism of oxygen activation by the copper active site of LPMOs will be gained and a wholly new assay for the activity of new LPMOs will be developed based on Surface-Enhanced Raman spectroscopy (SERS) of polysaccharide-coated nanoparticles.
Screening of the generated libraries will result in discovery of improved variants. This directed evolution approach will focus on generating and studying engineered enzymes optimized for conversion of indole-related and pharmaceutical compounds.
Knowledge-based design of copper complexes will be tested as mimics of LPMOs.
The developed enzymes will be tested under industrially relevant conditions. The performance will be compared with existing traditional methods.
Isolation of new cofactor-independent dioxygenases from the alpha/beta hydrolase fold superfamily active toward the Pseudomonas quinolone signal (PQS), exploiting natural genetic diversity; and functional characterisation of PQS-cleaving enzymes, with a focus on substrate specificity and affinity, catalytic efficiency, and applicability to interfere with quorum sensing.
Using technology of SSB, mutant libraries will be prepared and transfered to the relevant partners.
Mutants of LPMOs will be prepared and checked for activity.
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Author(s): Andrea N. Fabara, Marco W. Fraaije
Published in: Applied Microbiology and Biotechnology, 104/3, 2020, Page(s) 925-933, ISSN 0175-7598
Publisher: Springer Verlag
Author(s): Sandra C. Wullich, Alba Arranz San Martín, Susanne Fetzner
Published in: Applied and Environmental Microbiology, 86/9, 2020, Page(s) 1-12, ISSN 0099-2240
Publisher: American Society for Microbiology
Author(s): Gustavo de Almeida Santos, Gaurao V. Dhoke, Mehdi D. Davari, Anna Joëlle Ruff, Ulrich Schwaneberg
Published in: International Journal of Molecular Sciences, 20/13, 2019, Page(s) 3353, ISSN 1422-0067
Publisher: Multidisciplinary Digital Publishing Institute (MDPI)
Author(s): Gaston Courtade, Luisa Ciano, Alessandro Paradisi, Peter J. Lindley, Zarah Forsberg, Morten Sørlie, Reinhard Wimmer, Gideon J. Davies, Vincent G. H. Eijsink, Paul H. Walton, Finn L. Aachmann
Published in: Proceedings of the National Academy of Sciences, 117/32, 2020, Page(s) 19178-19189, ISSN 0027-8424
Publisher: National Academy of Sciences
Author(s): Gautier Bailleul, Callum R. Nicoll, María Laura Mascotti, Andrea Mattevi, Marco W. Fraaije
Published in: Journal of Biological Chemistry, 296, 2021, Page(s) 100221, ISSN 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology Inc.
Author(s): F. Calderaro, M. Keser, M. Akeroyd, L. E. Bevers, V. G. H. Eijsink, A. Várnai, M. A. van den Berg
Published in: Biotechnology for Biofuels, 13/1, 2020, ISSN 1754-6834
Publisher: BMC (part of Springer Nature)
Author(s): Nico D. Fessner
Published in: ChemCatChem, 2019, ISSN 1867-3880
Publisher: Wiley - VCH Verlag GmbH & CO. KGaA
Author(s): Nikola Lončar, Filippo Fiorentini, Gautier Bailleul, Simone Savino, Elvira Romero, Andrea Mattevi, Marco W. Fraaije
Published in: Applied Microbiology and Biotechnology, 103/4, 2019, Page(s) 1755-1764, ISSN 0175-7598
Publisher: Springer Verlag
Author(s): Callum R. Nicoll, Gautier Bailleul, Filippo Fiorentini, María Laura Mascotti, Marco W. Fraaije, Andrea Mattevi
Published in: Nature Structural & Molecular Biology, 27/1, 2020, Page(s) 14-24, ISSN 1545-9993
Publisher: Nature Publishing Group
Author(s): Marie-Cathérine Sigmund, Gerrit J. Poelarends
Published in: Nature Catalysis, 3/9, 2020, Page(s) 690-702, ISSN 2520-1158
Publisher: Springer Nature