Purification, characterization and NH2-terminal sequencing of an endo-(1→3,1→4)-β-glucanase from rice (Oryza sativa L.) bran

A (1 --> 3,1 --> 4)-beta-glucanase was purified from rice bran to electrophoretic homogeneity by fractionation with ammonium sulfate followed by ion-exchange chromatography's on DEAE- and CM-Sepharose, chromatofocusing on Polybuffer exchanger 94 and hydrophobic interaction chromatography on Phenyl-Sepharose. The purified enzyme had a molecular mass of 31 kDa as judged by SDS-PAGE and a basic isoelectric point of 7.9. The enzyme specifically hydrolyzed (1 --> 3,1 --> 4)-beta-glucans such as beta-glucans of barley and oat, and lichenins, but not CM-cellulose and laminarin. The purified enzyme was most active at pH 4.5 and stable for 10 min at 40 degrees C. When barley (1 --> 3,1 --> 4)-beta-glucan was used as the substrate, two oligosaccharides were formed as the major products of hydrolysis after prolonged incubation with the enzyme. No glucose was detected however, indicating that the enzyme may be classified as an endo-(1 --> 3,1 --> 4)-beta-glucanase (EC 3.2.1.73). NH2-terminal amino acid sequencing (40 residues) of rice endo-(1 --> 3,1 --> 4)-beta-glucanase revealed that it had a high similarity (83-85%) to that of endo-(1 --> 3,1 --> 4)-beta-glucanase from barley and wheat. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.