Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.
Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters.
Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga ward, Shizuoka 422-8529, Japan.
Makoto
M
Ogata
Yumiko
Y
Kameshima
Takeshi
T
Hattori
Kousuke
K
Michishita
Tomohiro
T
Suzuki
Hirokazu
H
Kawagishi
Kazuhide
K
Totani
Jun
J
Hiratake
Taichi
T
Usui
D002480:Cellulase / Q000302:isolation & purification*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D035901:Hypocrea / Q000201:enzymology*; D007525:Isoelectric Focusing; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D001617:beta-Glucosidase / Q000302:isolation & purification*
D001617:beta-Glucosidase; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase