Identification and characterization of glycoside hydrolases for biotechnological applications

The importance of carbohydrates in several biological processes is directly mirrored in a wide number of biotechnological applications, based mainly on glycoside hydrolases (GHs), including conversion of agricultural byproducts in fermentable sugars for the bioethanol production, the use of these biocatalysts in the formulation of laundry detergents and in food industry (e.g. hydrolysis of lactose and preparation of HFCS). Moreover, GHs are used also in several biomedical approaches, as the production of universal blood , the enzyme replacement therapy for the treatment of lysosomal storage diseases and as alterative to the chemical synthesis of therapeutic biomolecules as heparin and galactooligosaccharides. The development of a new class of enzyme, the glycosynthases (GS), obtained by mutating glycoside hydrolases, represent reliable alternative for the chemo-enzymatic synthesis of oligosaccharides. Here, the key of this approach is to cancel the hydrolytic activity of the enzyme by site-directed mutagenesis, but maintaining intact the structure of the active site. Therefore, by using substrate donor and certain reaction conditions, the engineered enzymes are able to synthesize products in quantitative yield. This thesis is directed to the identification and characterization of glycosyl hydrolase for biotechnological applications and was divided in two different parts. The first part (Chapters I and II) is aimed to the application of glycosidases in the oligosaccharides synthesis. In particular, I adressed my work to the study and characterization of the catalytic mechanism of a beta-galactosidase from the moderate thermophile Alicyclobacillus acidocaldarius (Aa?gal) for the development of a new ?-galactosynthase (Chapter I). Moreover I have characterized a new alpha-galactosynthase from the hyperthermophile Thermotoga maritima (TmGalA D327G) (Chapter II) to validate the approach based on the use of beta-glycosyl azide donors recently proposed. The second part of this thesis (Chapters III and IV) is dedicated to the characterization in detail of two new glycoside hydrolases: an alpha-glycosidase SSO1353 and an alpha-mannosidase (Ss-alpha-Man) both from the hyperthermophilic crenarchaeon Sulfolobus solfataricus. The characterization of SSO1353 was directed to the study of the catalytic mechanism and in particular to the identification of nucleophile and acid/base residues. Differently, the study of Ss-alpha-Man activity was directed to the analysis of its substrate specificity toward glycoconjugates and glycoproteins.