Diglycoside-Specific Glycosidases

The chapter focuses on β-primeverosidase, which is is one of the disaccharide-specific glycosidases, or “diglycosidases.” A β-primeverosidase responsible for the formation of aroma from the glycosidic aroma precursors was purified from the crude enzyme extract prepared from fresh juvenile tea leaves (Camellia sinensis). Many types of β-primeverosides having various kinds of aglycons have been isolated from many varieties of plants not only as aroma precursors in flowers and fruits, but also as cyanogenic glycosides and as polar constituents. β-Primeverosidase activity is examined by an assay using p-nitrophenyl (pNP) _-primeveroside or 2-phenylethyl β-primeveroside as a substrate. The activity is determined by measuring the liberation of p-nitrophenol or 2-phenylethanol from each glycoside. The enzymatic properties of β-primeverosidases from three different cultivars for green tea, oolong tea, and black tea arediscussed. Toclarify the substrate specificity of the β-primeverosidase with respect to the glycon moiety, nine kinds of diglycosides and a glucoside of 2-phenylethanol are synthesized. These glycosides are designed by considering the structures of the natural aroma precursors isolated from plants along with the ease of synthesis. The chapter also discusses the cloning of tea leaf β-primeverosidase, expression in Escherichia coli, mode of hydrolysis by β-primeverosidase, and diglycosidases from other plants and microorganisms.