Glycosidases and nanovesicles: novel biological tools for biotechnological applications.

Biocatalysis represents a versatile and valuable tool for industrial biotechnologies. The use of enzymes as biocatalysts has reached its present industrial level, due to their optimal reaction selectivity, high reaction rates, high product purity, and a significant decrease in the generation of chemical waste. The use of enzymes in industrial applications has been limited by several factors, mainly the high cost of enzymes, their instability, and their availability in small amounts. To overcome these problems, the quest for new catalytic activities and the development of new technical approaches, such as enzyme immobilization, to improve their stability and practical applications, remain a central focus of the current biotechnological research. In this PhD thesis, the biotechnological potential of a novel bacterial glycosidase (-RHA), and the characterization of two possible immobilization systems, bacterial OMVs and eukaryotic EVs is reported. More in detail, an optimized expression and purification procedure allowed to characterize a novel -RHA from the microorganism Novosphingobium sp. PP1Y, which resulted to be appealing from a biotechnological point of view for its interesting catalytic behaviour. Moreover, mutagenesis experiments, allowed a preliminary identification of the amminoacidic residues responsible for the catalytic activity of rRHA-P, which could be further mutagenized to fine-tune rRHA-P catalytic efficiency on selected substrates. In addition, two different potential scaffolds from bacteria (OMVs) and from an eukaryotic cell line (EVs) were isolated and characterized. Both systems resulted to be appealing either for enzyme immobilization or for drug-delivery strategies. In particular, OMVs isolated from N. sp. PP1Y were characterized by a peculiar biochemical composition, which showed some differences with the originating whole cells. EVs isolated from human macrophages resulted to have differential effects on inflammation activation, and their potential as a valid alternative to bacterial OMVs for the development of novel delivery Biosystems is discussed.