caa a22 4500 605505551 CHVBK 20210128100622.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6686-y doi (NATIONALLICENCE)springer-10.1007/s00253-015-6686-y A selection assay for haloalkane dehalogenase activity based on toxic substrates [Elektronische Daten] [Michael Fibinger, Timo Davids, Dominique Böttcher, Uwe Bornscheuer] Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 106 cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases. Springer-Verlag Berlin Heidelberg, 2015 Toxicity nationallicence Continuous selection process nationallicence Haloalkane dehalogenase nationallicence Growth assay nationallicence Fibinger Michael Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany aut Davids Timo Davids Biotechnologie GmbH, Röntgenstraße 3, D-93055, Regensburg, Germany aut Böttcher Dominique Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany aut Bornscheuer Uwe Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8955-8962 0175-7598 99:21<8955 2015 99 253 https://doi.org/10.1007/s00253-015-6686-y text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6686-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fibinger Michael Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany aut NATIONALLICENCE 700 1- Davids Timo Davids Biotechnologie GmbH, Röntgenstraße 3, D-93055, Regensburg, Germany aut NATIONALLICENCE 700 1- Böttcher Dominique Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany aut NATIONALLICENCE 700 1- Bornscheuer Uwe Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8955-8962 0175-7598 99:21<8955 2015 99 253