In-silicA Protein Engineering: Tuning the Properties of Natural Enzymes

Giunta, Carolina Ilenia . In-silicA Protein Engineering: Tuning the Properties of Natural Enzymes. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/83071/

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Due to their excellent catalytic properties, enzymes are highly efficient tools for performing a wide variety of (bio)chemical transformation. However, the relative fragility of enzymes and their fast aging in non-physiological environments is a major limit for their further implementation in biocatalysis.
The bioconjugation of enzymes to a wide range of materials provides a facile, yet sophisticated strategy for tuning enzyme properties. Specifically, the immobilization of enzymes onto solid supports was proven to be effective in improving the stability of enzymes and in enhancing their catalytic properties. This strategy provides, also, the advantage of producing reusable catalysts, thus increasing the cost-effectiveness of their applications, e.g. in continuous flow processes. The local environment of enzymes greatly affects several of its properties including structure, turnover number, selectivity, specificity, promiscuity, and stability in harsh conditions. Therefore, the rational engineering of the local enzyme environment can be applied for tuning properties of enzymes. A brief state of the art of the methods used for the modification of properties of enzymes, including enzyme immobilization strategies and enzyme environment modification approaches, is provided in the introduction (Chapter 1).
The research carried out in the frame of this doctoral thesis combines the advantages of the enzyme bioconjugation to solid materials with the benefits of tuning the enzyme local environment, using a protein supramolecular engineering approach (Chapter 2). The design of the enzyme environment aimed at the improvement of the enzyme enantioselectivity and at the enhancement of the activity of enzymes at low temperatures and in organic solvent (i.e., acetonitrile).
Specifically, the synthesis of a nanobiocatalyst by shielding an immobilized ester hydrolase within an organosilica material of different compositions, is described (Chapter 3). The composition of the shielding material was modified to locally tune the enzyme nanoenvironment. Employing this method, the substrate promiscuous and not enantioselective ester hydrolase was endowed with enantioselectivity yet, conserving its high promiscuity. Furthermore, this nanobiocatalyst showed remarkably improved solvent stability when submitted to high solvent concentrations (i.e. acetonitrile). The versatility of this approach was proven with three structurally different ester hydrolases.
Furthermore, a novel synthetic method to produce nanobiocatalysts artificially endowed with cryophilic properties, is described (Chapter 4). The nanobiocatalysts, later called Aurozyme, consists of gold nanoparticles (AuNPs) and enzyme molecules co-immobilized onto a silica scaffold and shielded within a nanometer-thin organosilica protective layer. To produce such a hybrid structure, a method allowing the covalent immobilization of AuNPs on the surface of the silica nanoparticles (SPs) was developed and optimized. This method enables reaching a dense and homogeneous AuNPs surface coverage of SPs. After enzyme co-immobilization, a nanometer-thin protective organosilica layer was grown at the surface of the SPs. This layer was designed to fulfill the dual function of protecting the enzyme from the surrounding environment and allowing the confinement, at the nanometer scale, of the heat diffusing from the surface of AuNPs to the enzyme environment after surface plasmon resonance (SPR) activation. To establish a proof of concept, we used a lipase (i.e. lipase B from Candida Antarctica) and the β-galactosidase from Kluyveromyces lactis; enzymes extensively used in a wide range of industrial applications. We demonstrated a drastic biocatalytic activity improvement at temperatures ranging from 20°C to -10°C.
Advisors:Shagaldian, Patrick and Ward, Thomas R. R. and Walde, Peter
Faculties and Departments:05 Faculty of Science
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14127
Thesis status:Complete
Number of Pages:168
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss141277
edoc DOI:
Last Modified:08 Jul 2021 14:15
Deposited On:08 Jul 2021 14:15

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