Dokument: Computational Engineering of PET-degrading Enzymes
| Titel: | Computational Engineering of PET-degrading Enzymes | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=66267 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20240701-133806-1 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Jäckering, Anna [Autor] | |||||||
| Dateien: |
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| Beitragende: | Jun.-Prof. Strodel, Birgit [Gutachter] Prof. Dr. Gohlke, Holger [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | The escalating climate crisis necessitates innovative solutions, among which ecofriendly enzymatic degradation of plastic waste, particularly the widely used polymer Poly(Ethylene Terephthalate) (PET), stands out as a promising avenue. Despite
this, the intricate mechanisms of PET hydrolases remain incompletely understood, hindering the development of uniform enzyme engineering strategies to enhance degradation activity on an industrially viable scale. This study seeks to bridge this knowledge gap by exploring the PET binding and conversion mechanisms of PET hydrolases through advanced computational methodologies. We focused on two thermophilic PET hydrolases, PolyESter Hydrolase I (PES-H1) and Leaf-branch Compost Cutinase (LCC), along with their highly active variants PES-H1 L92F/Q94Y (PES-H1 FY) and LCC F243I/D238C/S283C/Y127G (LCC ICCG), with the LCC F243I/Y127G (LCC IG) variant serving as a representative for LCC ICCG in this study. We conducted a comprehensive computational analysis, ranging from substrate adsorption and final productive binding using extensive conventional Molecular Dynamics (MD) simulations and enhanced sampling Hamiltonian Replica Exchange Molecular Dynamics (HREMD) simulations, to the initial stage of catalysed PET degradation, known as acylation, explored via hybrid Quantum Mechanics / Molecular Mechanics (QM/MM) simulations. This effort resulted in the compilation of approximately 55 μs of simulation data, positioning our study as one of the most exhaustive computational examinations of PET hydrolases to date, further validated by experimental assessments. Our findings emphasised the critical roles of enzyme surface electrostatics and hydrophobicity in PET adsorption. Additionally, a few distal mutations significantly impaired in vitro functionality, highlighting the pivotal role of adsorption in PET degradation. We bridged the gap between adsorption and productive PET binding by employing HREMD simulations and Principle Component Analysis to sample and identify previously unexplored entry pathways of PET into the active site, a methodology with potential applications in studying active site entries of other polymers. Our simulations elucidated the crucial role of PET conformational flexibility in overcoming the energy barrier upon entry, and Free Energy Surface analyses facilitated the identification of hindering residues. These insights guided the enhancement of experimentally determined kinetic properties of LCC ICCG and PES-H 1FY variants. Furthermore, our QM/MM simulations revealed a concerted conformational shift in the PET benzene ring, changing its interaction with the ’wobbling’ tryptophan from T-stacking to face-to-face π-π interactions. Addressing methodological challenges, our research advocated for incorporating this previously overlooked conformational change and selecting appropriate QM methods to improve the consistency of future QM/MM investigations. In conclusion, this thesis has significantly advanced our understanding of the atomistic details underlying enzymatic PET degradation, offering novel approaches to investigate the elusive polymer entry pathways into enzyme binding sites. The methodologies and insights derived from this study lay a robust foundation for future holistic research, promoting consistency and facilitating the optimisation of PET hydrolases and potentially other polymer-degrading enzymes. This knowledge transfer holds transformative potential for large-scale industrial applications, paving the way for efficient and environmentally-responsible recycling of plastic waste. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Theoretische Chemie und Computerchemie | |||||||
| Dokument erstellt am: | 01.07.2024 | |||||||
| Dateien geändert am: | 01.07.2024 | |||||||
| Promotionsantrag am: | 02.05.2024 | |||||||
| Datum der Promotion: | 17.06.2024 |
