Paragliola, Francesca Maria Pia (2024) Discovery and functional characterization of novel Glycoside Hydrolases: from in-silico identification to biotechnological applications. [Tesi di dottorato]

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Abstract

There is an urgent need to replace traditional chemical processes in industries which generate toxic by-products with more sustainable and economically efficient solutions. Enzyme-based catalysts offer a promising ecological alternative, particularly those derived from microorganisms in extreme environments. These biocatalysts, such as carbohydrate-active enzymes (CAZymes), play essential roles in breaking down and synthesising complex sugars. Glycosidehydrolases (GHs) are especially important for industrial applications, including biofuel production and biopolymer manufacturing. Enzyme discovery was the keyword of this PhD thesis. The goal is to identify and characterise new GHs through metagenomic and genomic approaches for biotechnological applications and to deepen our understanding of the GH family. Metagenomic analysis performed in this work allows the identification of approximately 8,000 new putative GH enzymes from three extreme environments. It develops a novel database, MetaGH, representing a solid reservoir of novel hypothetical glycosidases with potential utility in industrial biotransformations. Additionally, by combining in-silico metagenomic analysis of the Pisciarelli hot springs in Naples with detailed biochemical studies, a novel member of the GH122 family was identified. This study allowed for a deeper understanding of the functional biodiversity within this, to date, poorly characterised family and provided the background to identify the molecular determinants of the different activities identified so far. Beyond metagenomics, genome analysis, explicitly focusing on Polysaccharide Utilisation Loci (PUL), has emerged as a resource for identifying new GH families. PUL consists of physically linked and functionally related genes found in microorganisms such as Bacteroidetes, which enable the degradation of complex carbohydrates. Starting with identifying sequences of Proteins of Unknown Function (PUF) present in the PUL, the team of Professor Nicolas Terrapon discovered several new CAZymes through bioinformatics tools and high-throughput platforms. Using this approach, 6 novel GHs were identified and biochemical characterised. These novel enzymes will be the seed for determining novel GH families. This work has focused on advancing glycoside hydrolase research on multiple fronts, from in-silico identification to biochemical characterisation.

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