Dokument: Engineering of an organic solvent-tolerant esterase by modifying surface charges following simulation-based predictions
| Titel: | Engineering of an organic solvent-tolerant esterase by modifying surface charges following simulation-based predictions | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=57010 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20221207-132318-7 | |||||||
| Kollektion: | Publikationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Studienabschlussarbeit (z.B. Bachelor-, Master-, Examensarbeit) | |||||||
| Medientyp: | Text | |||||||
| Autor: | Scharbert, Lara [Autor] | |||||||
| Dateien: |
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| Beitragende: | Jun.-Prof. Strodel, Birgit [Gutachter] Prof. Dr. Jaeger, Karl-Erich [Gutachter] | |||||||
| Stichwörter: | organic solvent tolerance, molecular dynamics simulations, surface charge engineering, site-directed mutagenesis | |||||||
| Beschreibung: | The ability of enzymes to catalyze reactions in the presence of organic solvents has great industrial potential. However, organic solvent molecules can strip water molecules off the protein surface and enter the hydrophobic core, resulting in enzyme denaturation. No efficient, generally applicable strategy for enzyme-stability engineering in polar organic solvents has been reported to date. In this study, a mutagenesis strategy based on molecular dynamics (MD) simulations was developed, with the aim to strengthen the hydration shell around an enzyme to protect it from attack by organic solvents. This strategy is tested for the two esterases, PT35 and ED30. The former is considerably stable against denaturation and inactivation by organic solvents and is characterized by a high overall negative charge, whereas the structurally related ED30 is unstable in organic solvents. By removing negatively charged residues on the surface of PT35 and inserting negatively charged residues on the surface of ED30, PT35 becomes less stable and ED30 more stable. The selection of which of the surface residues to mutate was guided by various computational analyses, including MD simulations. The best in silico predictions, along with the wild-type enzymes, were biochemically characterized in the wet lab. A more acetonitrile resistant single mutant, K337E, was identified for ED30, while the D439K single mutant of PT35 became more sensitive towards acetonitrile. Interestingly, K337E was also found to be more thermally stable and D439K to be less thermally stable, which supports the assumption of a positive correlation between thermal and organic solvent tolerance. Non- additional mutation effects were observed for the triple-mutants K124D/R211E/K337E of ED30 and E199R/E328K/D439K of PT35. Analysis of the MD trajectories indicates that the formation and disruption of salt bridges contributes to the increased and decreased tolerance of the mutants, respectively. In summary, the MD-guided mutagenesis approach for engineering more organic solvent-tolerant enzymes looks promising and should therefore be continued in future studies. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Theoretische Chemie und Computerchemie | |||||||
| Dokument erstellt am: | 07.12.2022 | |||||||
| Dateien geändert am: | 07.12.2022 |
