PURIFICATION AND CHARACTERIZATION OF NOVEL GLYCOSIDASES FROM THE BACTERIAL GENUS XANTHOMONAS

Enzymatic analysis of oligosaccharides using exoglycosidases has become a powerful tool for determining the sequence and structure of sugar chains. The principal limitation to these methods has been the lack of highly purified and wellcharacterized enzymes. Using fluorescently labelled carbohydrate substrates and TLC, we have developed a method to identify glycosidases with novel specificities, This screening method led to the discovery that bacteria of the genus Xanthomonas are a rich source of exoglycosidases. From Xanthomonas manihotis, eight novel exoglycosidases have been isolated and characterized, A novel beta-N-acetylglucosaminidase has been purified that, unlike those previously described, will cleave N-acetylglucosamine without cleaving N-acetylgalactosamine residues. A novel beta-galactosidase has been isolated that preferentially hydrolyses beta(1-->3) galactosyl linkages, Three alpha-mannosidases have been isolated that serve as useful reagents in the analysis of high-mannose oligosaccharide structures: alpha 1-3,6 mannosidase, alpha 1-6 mannosidase and alpha 1-2,3 mannosidase. An alpha 1-3,6 galactosidase has been purified that does not hydrolyse terminal alpha 1-4 galactose residues, Two fucosidases, alpha 1-3,4 fucosidase and alpha 1-2 fucosidase, are similar to enzymes purified from other sources, Together, these glycosidases provide powerful reagents for determining the sequence of complex carbohydrates. Equally important is their usefulness in selectively removing specific sugar residues and thereby creating novel carbohydrates for analysing the biological roles of oligosaccharides.