- Titel
- Catalytic features and synthetic potential of the hydrolase C13DAc, a tool for paclitaxel provision
- Zitierfähige Url:
- https://nbn-resolving.org/urn:nbn:de:bsz:14-qucosa2-1057841
- Übersetzter Titel (CHI)
- 水解酶 C13DAc 的催化特性与合成潜力——一种用于制备紫杉醇的工具
- Erstveröffentlichung
- 2026
- Datum der Einreichung
- 02.07.2026
- Datum der Verteidigung
- 30.06.2026
- Abstract (EN)
- Paclitaxel, an important anticancer agents, has long presented a major challenge to its efficient and scalable chemical synthesis due to the intrinsic structural complexity, thereby motivating the exploration of alternative synthetic strategies that are shorter, more efficient, and environmentally sustainable. Enzymatic approaches, with their inherent selectivity and mild reaction conditions, offer a promising solution; however, the lack of suitable enzymes capable of constructing the key ester linkage of paclitaxel remains a limitation. This dissertation addresses this challenge by establishing an enzymatic strategy for the semi-synthesis of paclitaxel through enzyme identification, mechanistic elucidation, and rational protein engineering. Building upon previous observations that a Nocardioides albus-derived enzyme exhibits selective hydrolytic activity toward the C13 ester bond of paclitaxel, this work systematically identifies and characterizes the corresponding enzyme, designated C13DAc. Comprehensive biochemical analysis revealed that C13DAc is a highly regioselective paclitaxel C13-deacetylase with a specific substrate scope and catalytic profile. Structural analysis, supported by three-dimensional structure prediction using AlphaFold and complementary bioinformatic approaches, enabled the identification of key active-site features governing its hydrolytic activity and specificity. These insights provided a mechanistic basis for understanding the enzyme’s natural function and its potential for catalytic reprogramming. A core accomplishment of the thesis was the reprogramming of C13DAc’s catalytic role. Although the unmodified enzyme inherently lacked the ability to catalyze paclitaxel formation under conditions tested, a structure-guided engineering strategy enabled the generation of tailored variants with potential of producing paclitaxel. Through molecular docking and Funclib design the modified C13DAc successfully mediated transesterification reactions using baccatin III as the acyl acceptor and N-benzoyl-(2R,3S)-3-phenylisoserine methyl ester as acyl donor, ultimately achieve in the enzymatic esterified construction of paclitaxel. The tailored variants of C13DAc displayed lower thermostability compared with unmodified protein. Collectively, this study demonstrates the successful transformation of a naturally hydrolytic enzyme into a catalyst capable of promoting the transesterification-based synthesis of paclitaxel. However, large scale synthesis of paclitaxel still impossible using the modified protein, which means the enzyme might need to be further tailored by enzyme engineering for future development. Beyond offering an environmentally sustainable enzymatic route for the semi-synthesis of paclitaxel, the strategies and mechanistic insights outlined in this study provide a framework for the rational design of biocatalysts, enabling enzyme catalysis to address transformations previously inaccessible to pharmaceutical manufacturing.
- Freie Schlagwörter (EN)
- Paclitaxel, Biocatalysis, Enzyme engineering, Rational design, Esterification
- Klassifikation (DDC)
- 570
- Klassifikation (RVK)
- VK 5577
- GutachterIn
- Prof. Dr. Marion B. Ansorge-Schumacher
- Prof. Dr. Wolfgang Kroutil
- Den akademischen Grad verleihende / prüfende Institution
- Technische Universität Dresden, Dresden
- Förder- / Projektangaben
- China Scholarship Council Enzymatic path to cancer therapy drugs Paclitaxel,Docetaxel and novel taxane derivatives
ID: 202106150020 - Version / Begutachtungsstatus
- publizierte Version / Verlagsversion
- URN Qucosa
- urn:nbn:de:bsz:14-qucosa2-1057841
- Veröffentlichungsdatum Qucosa
- 06.07.2026
- Dokumenttyp
- Dissertation
- Sprache des Dokumentes
- Englisch
- Lizenz / Rechtehinweis
CC BY 4.0- Inhaltsverzeichnis
Abbreviations IV Summary VII Abstract IX Contents XI 1 Introduction 1 1.1 Paclitaxel 1 1.2 Enzymes and biocatalysis 7 1.3 Enzyme engineering 12 1.4 Aim 15 2 Experiment method 19 2.1 Reagent and equipment 19 2.3 Microorganisms and cultivation 26 2.4 Nocardioides albus SC13911 cultivation and protein purification 28 2.5 Genes 32 2.6 Protein expression and purification 41 2.7 Activity measurement and analytic method 45 2.8 Mutagenesis49 2.9 Protein structure prediction and analysis 53 2.10 Enzymatic transesterification and hydrolysis 56 3 Isolation, identification and characterization of the enzyme C13DAc 58 3.1 Result and discussion 58 3.2 Gene identification of wild type c13dac 63 3.3 Gene codon optimization and heterologous gene expression 63 3.4 Biochemical features 66 3.5 Discussion 75 3.6 Conclusion 77 4. Structural insights and catalytic mechanism of C13DAc 79 4.1 Result and discussion 79 4.2 Discussion 96 4.3 Conclusion 98 5 Unlocking the synthetic potential of C13DAc: Transesterification ability of C13DAc 100 5.1 Result and discussion 100 5.2 Discussion 112 5.3 Conclusion 114 6 Rational design of C13DAc for paclitaxel synthesis 115 6.1 Funclib_C13DAc1 design 115 6.2 Funclib_C13DAc2 design 117 6.3 Transesterification synthesis of paclitaxel using Funclib_C13DAc2 118 6.4 Catalytic hydrolysis of paclitaxel using C13DAc and its variants 121 6.5 Thermostability of Funclib_C13DAc2 123 6.6 Discussion 124 6.7 Conclusion 127 7 Conclusion and Outlook 129 Appendix 1 131 Appendix 2 133 Appendix 3 136 Appendix 4 140 References 147 Acknowledgements 157 Publications 160 Previous publications 160