Glycosidases of apple fruit: A multi‐functional β‐galactosidase

Extraction of Spartan apple (Malus domestica) fruit acetone powder and fractionation of the extract on DEAE‐agarose allowed detection and quantification of 10 glycosidases active toward 4‐methylumbelliferyl glycosides. Hydrolysis was measured fluorimetrically. The predominant activity, a β‐d‐galactosidase (EC 3.2.1.23), labile upon purification, was stabilized by soluble PVP. Molecular weights, measured by gel permeation HPLC, pH optima and Km values were obtained for most glycosidase activities. Multiple forms of several activities were found. The major α‐d‐ and β‐d‐galactosidases were resolved on phosphocellulose. The β‐d‐galactosidase so obtained had associated α‐l‐arabinopyranosidase and β‐d‐fucosidase activities which were retained upon GP‐HPLC. Mixed substrate kinetic analysis and inhibition analysis of this fraction indicated that the enzyme has 3 catalytic sites, 1 for each substrate, whose substrates mutually influence each other's activity positively.