Purification and characterization of isoforms of β-galactosidases in mung bean seedlings

Five isoforms of β-galactosidase (EC 3.2.1.23), designated as β-galactosidases I–V, were isolated from five-day-old mung bean ( Vigna radiata) seedlings. β-Galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose, and chromatofocusing. β-Galactosidases I, II and III have the same molecular mass of 87 kDa, comprising two non-identical subunits with molecular masses of 38 and 48 kDa, while β-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-β- d-galactoside, and to a lesser extent, p-nitrophenyl-α- l-arabinoside and p-nitrophenyl-β- d-fucoside. The enzymes were inhibited by d-galactono-1,4-lactone, d-galactose, Hg 2+, Ag + and sodium dodecyl sulfate (SDS). β-Galactosidases I, II and III were shown to be competitively inhibited by either d-galactono-1, 4-lactone or d-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that β-galactosidases I, II, III and V are immunologically similar to each other, while β-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified β-galactosidases II and III were capable of releasing d-galactose residue from the hemicellulose fraction isolated from mung bean seeds.