PURIFICATION OF A BETA-GALACTOSIDASE FROM RICE SHOOTS AND ITS INVOLVEMENT IN HYDROLYSIS OF THE NATURAL SUBSTRATE IN CELL-WALLS

Several glycosidase and glycanase activities have been detected in hom ogenates of rice (Oryza sativa L. cv. Nipponbare) shoots after success ive extraction with K-phosphate (pH 7.0) and buffer containing 3 M LiC l. The major beta-D-galactosidase (EC 3.2.1.23) present in the buffer- soluble protein fraction was purified to electrophoretic homogeneity b y a combination of chromatographic techniques including DEAE-Sepharose CL-6B, Sephacryl S-200HR and aminophenyl-beta-D-thiogalactopyranoside -Sepharose 4B. Analysis by denaturing gel electrophoresis revealed a s ingle polypeptide chain with an apparent molecular mass of 42 kDa, sim ilar to the value of 40 kDa estimated for the native protein by gel-pe rmeation. The isoelectric point was pH 6.0. The K(m) and V(max) values for p-nitrophenyl (PNP)-beta-D-galactopyranoside were 0.63 mM and 0.3 2 mmol (mg protein)-1 h-1, respectively. Maximum activity in McIlvaine buffer occurred at pH 3.4, and the activity was inhibited by Ag2+, Cu 2+, Hg2+, p-chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme hydrolyzed larchwood arabinogalactan in an exo-fashion, and acted weakly on arabinosyl and galactosyl residue-rich polymer of pec tic polysaccharides and cell walls from rice shoots.