Purification and characterization of a β-glucosidase from rye ( Secale cereale L.) seedlings

Abstract

Cyclic hydroxamic acids and a glucosidase that occur in rye seedlings were investigated. The concentration of the glucoside of 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) in shoots increased soon after germination and decreased to a lower, constant level as the plants started autotrophic growth. Cyclic hydroxamic acid glucoside β-glucosidase activity also occurred transiently, and the timing of the increase and decrease was concurrent with that of cyclic hydroxamic acid glucosides. The glucosidase was isolated from 48-h-old rye shoots and purified to apparent homogeneity by using isoelectric precipitation, anion exchange chromatography, and gel filtration. The isoelectric point and the optimum reaction temperature were 4.9–5.1 and 25–30°C, respectively. The N-terminal amino acid sequence was almost identical to that of the wheat glucosidases, but did not show any similarity to the sequences of other glucosidases of plant origin. SDS– and native–PAGE analyses showed that rye had several isozymes of glucosidase, and each isozyme was an oligomer of 60-kDa monomers with a molecular mass of ∼300 kDa. The enzyme was highly active not only for DIMBOA-Glc but also for its 7-demethoxy analogue, DIBOA-Glc, which was different from the specificities of maize and wheat glucosidases.