Purification of a β‐galactosidase from rice shoots and its involvement in hydrolysis of the natural substrate in cell walls

Abstract

Several glycosidase and glycanase activities have been detected in homogenates of rice ( Oryza sativa L. cv. Nipponbare) shoots after successive extraction with K‐phosphate (pH 7. 0) and buffer containing 3 M LiCl. The major β‐D‐galactosidase (EC 3. 2. 1. 23) present in the buffer‐soluble protein fraction was purified to electrophoretic homogeneity by a combination of chromatographic techniques including DEAE‐Sepharose CL‐6B, Sephacryl S‐200HR and p ‐aminophcnyl‐β‐D‐thiogalactopyranoside–Sepharose 4B. Analysis by denaturing gel electrophoresis revealed a single polypeptide chain with an apparent molecular mass of 42 kDa. Similar to the value of 40 kDa estimated for the native protein by gel‐permeation. The isoelectric point was pH 6. 0. The K m and V max values for p ‐nitrophenyl (PNP)‐β‐D‐galactopyra‐noside were 0. 63 m M and 0. 32 mmol (mg protein) −1 h −1 , respectively. Maximum activity in McIlvaine buffer occurred at pH 3. 4, and the activity was inhibited by Ag 2+ , Cu 2+ . Hg 2+ , p ‐chloromercuribenzoate (PCMB) and D‐galactono‐l,4‐lactone. The enzyme hydrolyzed larchwood arabinogalactan in an exo‐fashion, and acted weakly on arabinosyl and galactosyl residue‐rich polymer of pectic polysaccharides and cell walls from rice shoots.