Ercolano, Carmen (2024) Discovery, characterization and engineering of enzymes involved in polymer modification for sustainable biocatalysis and biotransformation. [Tesi di dottorato]

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Abstract

Humans dominate the Earth, changing it in ways that menace planet's resilience. For the plausibility of a “tipping point”, this situation enhances the demand for biological solving from the local to the global scale, in particular, concerning the discovery of biocatalysts. In the frame of a circular bioeconomy, the discovery of enzymes able to breakdown natural and artificial polymers is gaining much attention. For the first objective of this PhD thesis, the scientific background concerns the dependence on fuels from fossil as energy source, contributing to global warming. Biofuels represent a valid alternative, and lignocellulose is a promising reservoir. As biocatalysts, great attention is given to (hyper)thermophilic glycoside hydrolases (GHs), which can operate under conditions in which the mesophilic ones quickly denature. To this aim, functional metagenomics can help find new GHs. The project aimed to create new protocols for analysing (hyper)thermophilic metagenomic data, using advanced bioinformatics for enzyme annotation and sample selection for fosmid library construction. Despite not identifying hyperthermophilic GH clones initially, the project developed and refined three novel functional metagenomic protocols, enhancing the search for enzymes suitable for high-temperature industrial applications. For the second objective, given the environmental accumulation of poly(ethylene terephthalate), the aim is to discover new PET Hydrolysing Enzymes (PHEs). Since the hydrolysis of PET increases with the rising temperature, screening for thermophilic enzymes may be helpful. In-silico metagenomics and protein engineering represent two approaches. For the enzyme discovery, two new PHEs, PP_EG and Sis, were identified, showing activity on ester and plastic substrates. Among them, Sis was biochemically characterized more in detail. For the engineering, PHEs already reported in the literature were exposed on the outer membrane of Escherichia coli exploiting Anchoring-and-Self-Labelling-protein-tags, showing activity on suitable substrates.

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