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 isoelectric point of 4.0 has been purified 2000-fold from extracts of rice bran, using fractional precipitation with ammonium sulfate, anion exchange chromatography, size-exclusion chromatography, chromatofocussing, and hydrophobic interaction chromatography. The enzyme can be classified 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 endohydrolases. 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-glucans of the kind commonly found in fungal cell walls are less susceptible to hydrolysis than essentially linear (1-->3)-beta-D-glucans. Kinetic analyses indicate an apparent Km of 42 microM, a kcat 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-glucanase contain blocks of amino acids that are conserved in other cereal (1-->3)-beta-glucanases. Although the precise tissue location and function of the enzyme in rice bran are not known, it is likely that it is concentrated in the aleurone layer and that it plays a preemptive role in the protection of ungerminated grain against pathogen attack.
Akiyama
T
T
National Hokkaido Agricultural Experimentation Station, Sapporo, Japan.
Shibuya
N
N
Hrmova
M
M
Fincher
G B
GB
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D017124:Conserved Sequence; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013696:Temperature
D004187:Disaccharides; D005936:Glucans; D011134:Polysaccharides; C014637:laminaribiose; C008247:laminaran; D004693:Glucan Endo-1,3-beta-D-Glucosidase