(Supra)molecular Enzyme Engineering - Design, Synthesis and Characterization of Fine-Tuned Biocatalysts

Briand, Manon L.. (Supra)molecular Enzyme Engineering - Design, Synthesis and Characterization of Fine-Tuned Biocatalysts. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/83299/

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Enzymes, owing to the rich diversity of reactions they catalyze, are of great importance in both industry and
academia. Efforts are being exerted continuously to promote their use in order to comply with the growing demand
for green and sustainable chemical manufacture. This is done in particular through the development of methods
allowing to modify and enhance the properties of these natural biocatalysts. This doctoral thesis focuses on the
design, synthesis and characterization of systems enabling the tuning of enzyme properties to simplify and expand
their use within the scientific community.
Herein, we report the first method of enzyme protection allowing the production of partially shielded enzymes.
We show that enzyme partial shielding provides nanobiocatalysts capable of processing large protein substrates.
The method is applied to two model systems, sortase A and trypsin. We demonstrate that the partially
shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on monoclonal antibodies.
Moreover, we show that the partially shielded trypsin is capable of performing proteolytic reactions with
better efficiency than its soluble counterpart. The enzyme temporal stability is increased drastically, demonstrating
the highly efficient protective effect of the partial shield on the immobilized enzymes. Since proteolysis often
requires protein unfolding prior to digestion, the partial shielding strategy is further explored to provide proteolytic
nanobiocatalysts with disulfide bond reducing properties. To do so, immobilized papain and subtilisin A are partially
shielded in a mercaptosilica layer. The nanobiocatalysts produced are shown to efficiently perform simultaneous
disulfide bond reduction and protein digestion. Although the mercaptosilica shield possesses strong reducing
properties against protein substrates, it does not induce protease unfolding and leads to a drastic increase of
enzyme stability.
The absence of control over enzyme orientation during immobilization results in undefined positioning of the
enzyme active site in the protective layer. Steric hindrance of the enzyme active site can therefore lead to the
production of heterogeneous reaction products. This is the main limitation when it comes to the production of
bioconjugate therapeutics. Herein, we report the first method enabling simultaneous site-selective immobilization
and protection of enzymes having large protein substrates. The developed system is based on a Twin-Strep tagged
sortase A and relies on a protective bilayer aiming at increasing the nanobiocatalyst homogeneity. We show that
this method allows the production of nanobiocatalysts capable of performing bioconjugation reactions on antibodies
with improved efficiency.
In addition, the tuning of sortase A properties by the introduction of various chemically tailored active sites
into the enzyme scaffold is explored. The catalytic properties of the produced artificial metalloenzymes are studied.
The copper-based hybrid conjugates are shown to perform regioselective Diels-Alder reactions between 2-
azachalcone derivatives and dienes. The iron-based metalloenzymes are demonstrated to mimic catecholase activity.
Sortase is demonstrated to be a promising protein scaffold for the design of hybrid catalysts.
The presented work provides new methods that can be implemented in the design of innovative processes
aiming at improving enzyme properties.
Advisors:Shahgaldian, Patrick and Meier, Wolfgang P. and Walde, Peter
Faculties and Departments:05 Faculty of Science
UniBasel Contributors:Meier, Wolfgang P.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14120
Thesis status:Complete
Number of Pages:145
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss141205
edoc DOI:
Last Modified:29 Jun 2021 04:30
Deposited On:28 Jun 2021 09:55

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