title: Purification and properties of three (1-->3)-beta-D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare).
abstract: Three (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1-->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1-->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1-->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1-->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1-->3;1-->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1-->3;1-->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1-->3;1-->4)-beta-D-glucans, (1-->6)-beta-D-glucan, CM-cellulose, insoluble (1-->3)-beta-D-glucans or aryl beta-D-glycosides.
authors.affiliation: Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Hrmova
authors.forename: G B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D043326:Glucan 1,3-beta-Glucosidase; D001467:Hordeum / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase