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

Five isoforms of beta -galactosidase (EC 3.2.1.23), designated as e-galactosidases IV, were isolated from five-day-old mung bean (Vigna radiata) seedlings. beta -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. beta -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 beta -galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta -D-palactoside. and to a lesser extent, p-nitrophenyl-alpha -L-arabinoside and p-nitrophenyl-beta -D-fucoside. The enzymes were inhibited by D-galactono-l,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). P-Galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-l, 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 P-galactosidases I. II. III and V are immunologically similar to each other, while beta -galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified S-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds. (C) 2001 Elsevier Science Ltd. All rights reserved.