title: Mode of action of a β-(1→6)-glucanase from Penicillium multicolor.
abstract: β-(1→6)-Glucanase from the culture filtrate of Penicillium multicolor LAM7153 was purified by ammonium sulfate precipitation, followed by cation-exchange and affinity chromatography using gentiotetraose (Gen₄) as ligand. The hydrolytic mode of action of the purified protein on β-(1→6)-glucan (pustulan) was elucidated in real time during the reaction by HPAEC-PAD analysis. Gentiooligosaccharides (DP 2-9, Gen₂₋₉), methyl β-gentiooligosides (DP 2-6, Gen₂₋₆ β-OMe), and p-nitrophenyl β-gentiooligosides (DP 2-6, Gen₂₋₆ β-pNP) were used as substrates to provide analytical insight into how the cleavage of pustulan (DP¯ 320) is actually achieved by the enzyme. The enzyme was shown to completely hydrolyze pustulan in three steps as follows. In the initial stage, the enzyme quickly cleaved the glucan with a pattern resembling an endo-hydrolase to produce a short-chain glucan (DP¯ 45) as an intermediate. In the midterm stage, the resulting short-chain glucan was further cleaved into two fractions corresponding to DP 15-7 and DP 2-4 with great regularity. In the final stage, the lower oligomers corresponding to DP 3 and DP 4 were very slowly hydrolyzed into glucose and gentiobiose (Gen₂). As a result, the hydrolytic cooperation of both an endo-type and saccharifying-type reaction by a single enzyme, which plays a bifunctional role, led to complete hydrolysis of the glucan. Thus, β-(1→6)-glucanase varies its mode of action depending on the chain length derived from the glucan.
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga Ward, Shizuoka 422-8529, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hattori
authors.forename: Yasuna
authors.initials: Y
authors.lastname: Kato
authors.forename: Shuji
authors.initials: S
authors.lastname: Uno
authors.forename: Taichi
authors.initials: T
authors.lastname: Usui
mesh_terms: D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000096:biosynthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology*; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002241:Carbohydrates; D001617:beta-Glucosidase