Matches in SemOpenAlex for { <https://semopenalex.org/work/W859633244> ?p ?o ?g. }
Showing items 1 to 61 of
61
with 100 items per page.
- W859633244 abstract "This thesis describes the development and evaluation of a high-throughput screening method for the identification and isolation of lipolytic enzyme variants with altered enantioselectivity. It combines the cell surface display of lipolytic enzymes with a novel enrichment method and has the potential to considerably accelerate the identification of variants with desired properties. The E. coli cell surface display of lipolytic enzymes was achieved via recombinant production of the respective enzymes as fusion proteins with a truncated, esterolytic inactive variant of the P. aeruginosa esterase EstA. It was demonstrated that this system is able to mediate the surface display of several members of the family of lipolytic enzymes as passenger proteins. This kind of recombinant production not only creates an efficient genotype-phenotype coupling between an enzyme and a self-replicating particle (cell), it also allows immobilization of the respective enzyme to a solid matrix (cell surface) in the extracellular medium. Thus, surface exposed enzymes are directly accessible to the respective substrates and easily separable, which makes them useful tools as whole cell biocatalysts for organic synthesis. The presented high-throughput screening method is based on a specific labelling of E. coli cells, which display lipolytic enzyme variants on the cell surface. This labelling takes place as result of substrate hydrolysis, thus allowing the mapping of activity and substrate specificity in the form of a fluorescence signal on a single cell basis. The utilization of flow cytometry facilitates the simultaneous screening of a large number of variants (>107). The application of the different enantiomers of 2-methyldecanoic acid as esters of labelling substrates allowed the identification and isolation of P. aeruginosa EstA variants with inverted enantioselectivity from two enzyme libraries generated by random mutagenesis. Nucleotide sequence analysis of those variants revealed an amino a cid position with a decisive role for the enantioselectivity of EstA. Furthermore, it could be demonstrated that the developed strategy to utilize bacterial cell surface display for a direct functional analysis of proteins could be extended to the verification of the protein-protein interaction of the P. aeruginosa lipase-specific foldase LipH with the corresponding lipase LipA." @default.
- W859633244 created "2016-06-24" @default.
- W859633244 creator A5088712157 @default.
- W859633244 date "2022-02-16" @default.
- W859633244 modified "2023-09-25" @default.
- W859633244 title "Neue Zugänge zu enantioselektiven lipolytischen Enzymen durch fluoreszenzbasierte Durchmusterung kombinatorischer Bibliotheken" @default.
- W859633244 doi "https://doi.org/10.53846/goediss-251" @default.
- W859633244 hasPublicationYear "2022" @default.
- W859633244 type Work @default.
- W859633244 sameAs 859633244 @default.
- W859633244 citedByCount "0" @default.
- W859633244 crossrefType "dissertation" @default.
- W859633244 hasAuthorship W859633244A5088712157 @default.
- W859633244 hasBestOaLocation W8596332441 @default.
- W859633244 hasConcept C104317684 @default.
- W859633244 hasConcept C1491633281 @default.
- W859633244 hasConcept C153911025 @default.
- W859633244 hasConcept C181199279 @default.
- W859633244 hasConcept C185592680 @default.
- W859633244 hasConcept C186268636 @default.
- W859633244 hasConcept C18903297 @default.
- W859633244 hasConcept C2777289219 @default.
- W859633244 hasConcept C2779281246 @default.
- W859633244 hasConcept C28406088 @default.
- W859633244 hasConcept C40767141 @default.
- W859633244 hasConcept C51323132 @default.
- W859633244 hasConcept C553184892 @default.
- W859633244 hasConcept C55493867 @default.
- W859633244 hasConcept C86803240 @default.
- W859633244 hasConceptScore W859633244C104317684 @default.
- W859633244 hasConceptScore W859633244C1491633281 @default.
- W859633244 hasConceptScore W859633244C153911025 @default.
- W859633244 hasConceptScore W859633244C181199279 @default.
- W859633244 hasConceptScore W859633244C185592680 @default.
- W859633244 hasConceptScore W859633244C186268636 @default.
- W859633244 hasConceptScore W859633244C18903297 @default.
- W859633244 hasConceptScore W859633244C2777289219 @default.
- W859633244 hasConceptScore W859633244C2779281246 @default.
- W859633244 hasConceptScore W859633244C28406088 @default.
- W859633244 hasConceptScore W859633244C40767141 @default.
- W859633244 hasConceptScore W859633244C51323132 @default.
- W859633244 hasConceptScore W859633244C553184892 @default.
- W859633244 hasConceptScore W859633244C55493867 @default.
- W859633244 hasConceptScore W859633244C86803240 @default.
- W859633244 hasLocation W8596332441 @default.
- W859633244 hasOpenAccess W859633244 @default.
- W859633244 hasPrimaryLocation W8596332441 @default.
- W859633244 hasRelatedWork W1603694014 @default.
- W859633244 hasRelatedWork W1675864211 @default.
- W859633244 hasRelatedWork W1965618318 @default.
- W859633244 hasRelatedWork W1967732746 @default.
- W859633244 hasRelatedWork W1977765199 @default.
- W859633244 hasRelatedWork W1999195584 @default.
- W859633244 hasRelatedWork W2019689594 @default.
- W859633244 hasRelatedWork W2064042198 @default.
- W859633244 hasRelatedWork W2901055207 @default.
- W859633244 hasRelatedWork W60097589 @default.
- W859633244 isParatext "false" @default.
- W859633244 isRetracted "false" @default.
- W859633244 magId "859633244" @default.
- W859633244 workType "dissertation" @default.