PURIFICATION AND CHARACTERIZATION OF A (1-]3)-BETA-D-GLUCAN ENDOHYDROLASE FROM RICE (ORYZA-SATIVA) BRAN

A (1 --> 3)-beta-glucanase with an apparent M(r) of 29,000 and an isoe lectric point of 4.0 has been purified 2000-fold from extracts of rice bran, using fractional precipitation with ammonium sulfate, anion exc hange chromatography, size-exclusion chromatography, chromatofocussing , and hydrophobic interaction chromatography. The enzyme can be classi fied with the EC 3.2.1.39 group, because it releases laminarabiose and higher laminara-oligosaccharides from linear (1 --> 3)-beta-D-glucans with an action pattern that is typical of(1 --> 3)-beta-D-glucan endo hydrolases. However, the introduction of substituents or branching in the (1 --> 3)-beta-D-glucan substrates causes a marked decrease in the rate of hydrolysis. Thus, substituted or branched (1 --> 3)-beta-D-gl ucans of the kind commonly found in fungal cell walls are less suscept ible to hydrolysis than essentially Linear(1 --> 3)-beta-D-glucans. Ki netic analyses indicate an apparent K-m of 42 mu M, a k(cat) constant of 67 s(-1), and a pH optimum of 5.0 during hydrolysis of the (1 --> 3 )-beta-D-glucan, laminaran, from Laminaria digitata. The first 60 NH2- terminal amino acid residues of the purified rice (1 --> 3)-beta-gluca nase contain blocks of amino acids that are conserved in other cereal( 1 --> 3)-beta-glucanases. Although the precise tissue location and fun ction of the enzyme in rice bran are not known, it is likely that it i s concentrated in the aleurone layer and that it plays a pre-emptive r ole in the protection of ungerminated grain against pathogen attack. ( C) 1997 Elsevier Science Ltd.