ABSTRACT
I. INTRODUCTION There is an ever expanding number of techniques used to identify and sequence oligosaccharides. Many of the sequencing techniques require exoglycosidases to sequentially remove sugar residues from an oligosaccha ride. Because of this requirement, the more exoglycosidases available that are highly specific for either a particular sugar residue or glycosidic linkage, the more powerful these techniques become. Screening for novel specific exoglycosidases from bacterial species was initiated to try to augment the number of exoglycosidases available to the research community. Until this project was started, screening for exoglycosidases was primarily performed using chromogenic substrates such as /?-nitrophenyl-glycosides. Though this technique is simple and rapid, requiring only a color change upon incuba tion of the substrate with the extract to detect exoglycosidase activity, the results give no information about the enzyme’s linkage specificity or its ability to cleave an oligosaccharide substrate. To overcome these limitations and to detect exoglycosidases that do not cleave these synthetic substrates, a screening technique was developed that would use oligosaccharides as substrates for exoglycosidase digestion. This chapter describes the technique used at New England Biolabs for screening, purification, and characterization of novel exoglycosidases using fluorescently labeled oligo saccharides as the substrate for glycosidase digestion and thin-layer chro matography (TLC) for analysis of the cleavage products. The exoglycosi dases isolated and characterized as a result of this screening project will be described as well as a demonstration of the uses of these enzymes to confirm the sequence of an oligosaccharide.
