title: Enzymatic hydrolysis of 1,3-1,4-beta-glucosyl oligosaccharides by 1,3-1,4-beta-glucanase from Synechocystis PCC6803: a comparison with assays using polymer and chromophoric oligosaccharide substrates.
abstract: The specificity of 1,3-1,4-beta-glucanase from Synechocystis PCC6803 (SsGlc) was investigated using novel substrates 1,3-1,4-beta-glucosyl oligosaccharides, in which 1,3- and 1,4-linkages are located in various arrangements. After the enzymatic reaction, the reaction products were separated and determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As a result, SsGlc was found to hydrolyze the pentasaccharides, which possess three contiguous 1,4-beta-glycosidic linkages (cellotetraose sequence) adjacent to 1,3-beta-linkage, but none of the other oligosaccharides were hydrolyzed. To further analyze the specificity, kinetic measurements were performed using polymeric substrates and 4-methylumbelliferyl derivatives of laminaribiose and cellobiose (1,3-beta-(Glc)(2)-MU and 1,4-beta-(Glc)(2)-MU). The k(cat)/K(m) value obtained for barley beta-glucan was considerably larger than that for lichenan, indicating that SsGlc prefers 1,3-1,4-beta-glucan possessing a larger amount of cellotetraose sequence. This is consistent with the data obtained for 1,3-1,4-beta-glucosyl oligosaccharides. However, the k(cat)/K(m) value obtained for 1,4-beta-(Glc)(2)-MU was considerably lower than that for 1,3-beta-(Glc)(2)-MU, suggesting inconsistency with the data obtained from the other natural substrates. It is likely that the kinetic data obtained from such chromophoric substrates do not always reflect the true enzymatic properties.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. fukamizo@nara.kindai.ac.jp
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
authors.forename: Kanako
authors.initials: K
authors.lastname: Hayashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tamoi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Fujimura
authors.forename: Hideki
authors.initials: H
authors.lastname: Kurotaki
authors.forename: Anna
authors.initials: A
authors.lastname: Kulminskaya
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
mesh_terms: D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology*
chemical_list: D009844:Oligosaccharides; D011134:Polysaccharides; D006026:Glycoside Hydrolases; C028020:licheninase