Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga ward, Shizuoka 422-8529, Japan.
Carbohydr Res. 2010 Dec 10;345(18):2623-9. doi: 10.1016/j.carres.2010.10.015. Epub 2010 Nov 8.
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.
通过使用β-乳酰基氨作为配体的亲和层析，实现了β-葡萄糖苷酶、内切-endo-β-（1→4）-葡聚糖酶 I（EG I）和外切-纤维素酶 I（CBH I）的选择性吸附和分离。该酶来源于粗糙脉孢菌（Hypocrea jecorina）的粗纤维素酶制剂。当粗纤维素酶被应用于基于β-乳酰基氨的亲和柱时，β-葡萄糖苷酶出现在未结合的部分。相比之下，EG I 和 CBH I 被保留在柱子上，然后通过适当调整洗脱条件将它们彼此分离。根据它们在柱上的洗脱条件，酶的相对亲和力强烈依赖于配体。通过亲和层析获得的高度纯化的 EG I 和 CBH I ，分别通过 Mono P 和 DEAE 层析进一步纯化。EG I 和 CBH I 仅在带有乳糖和 N-乙酰乳糖胺（LacNAc）的对硝基苯糖苷的酚键处切割。相比之下，尽管在动力学参数方面存在重要差异，但带有纤维二糖的对硝基苯糖苷的两个可切割键都受到水解的影响。
