Partial Purification and Characterization of .beta.-D-Galactosidase from Sweet Cherry, a Nonclimacteric Fruit

• Published in: Journal of agricultural and food chemistry 1994-10, Vol.42 (10), p.2177-2182
• Main Authors: Andrews, Preston K, Li, Shulin
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; ANTICORPS; ANTICUERPOS; BETA GALACTOSIDASA; BETA GALACTOSIDASE; Biological and medical sciences; CEREZA; CERISE; Enzymes and enzyme inhibitors; Food industries; Fruit and vegetable industries; Fundamental and applied biological sciences. Psychology; Hydrolases; MADURAMIENTO; MURISSAGE; PARED CELULAR; PAROI CELLULAIRE; PRUNUS AVIUM; PURIFICACION; PURIFICATION
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf00046a019

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 GA3, IAA, and Mg2+. The Km and Vmax of beta-Gal with p-nitrophenyl beta-D-galactopyranoside as substrate were 1.25 mM and 5 micromole.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