PARTIAL-PURIFICATION AND CHARACTERIZATION OF BETA-D-GALACTOSIDASE FROM SWEET CHERRY, A NONCLIMACTERIC FRUIT

beta-D-Galactosidase (beta-Gal, EC 3.2.1.23) was partially purified from sweet cherry (Prunus avium L. cv. Bing) fruit in four liquid chromatography steps, DEAE-Sephadex A-50, Sephadex G-75, and two Sephacryl S-200 columns. Partially purified beta-Gal produced two protein bands with pI values of 4.2 and 4.5, based on native IEF electrophoresis. Both proteins showed high enzyme activity. beta-Galase activity was severely inhibited by 1 mM Cu2+ in vitro but was stimulated by 1 mM GA(3), IAA, and Mg2+. Th, K-m and V-max of beta-Gal with p-nitrophenyl beta-D-galactopyranoside as substrate were 1.25 mM and 5 mu mol.mg(-1).min(-1), respectively. Maximum in situ beta-Gal activity expressed on a specific protein basis occurred about 2 weeks prior to fruit maturity but at maturity when expressed on a fresh weight basis. Polyclonal antibodies made against the 57 kDa beta-Gal reacted with four proteins from mature fruit. The estimated molecular masses of these proteins were 28, 43, 63, and 92 kDa. These results suggest that beta-Gal may contribute to cell wall hydrolysis during sweet cherry fruit softening.