Peterson D R, Kirkwood S
Carbohydr Res. 1975 May;41:273-83. doi: 10.1016/s0008-6215(00)87025-1.
A method for the large-scale production of a (1 yields 3)-beta-D-glucan glucohydrolase (EC 3.2.1.58) from the culture filtrate of Basidiomycete QM806 is described. The final preparation is homogeneous by disc electrophoresis under non-dissociating and denaturing conditions, by ultracentrifugation, and by isoelectric focusing. Various physical and chemical characteristics of the enzyme have been determined, including terminal amino acid residues, extinction coefficient, and stability to pH extremes. The N-terminal amino acids are leucine and serine (Sanger's method) and the C-terminal amino acids are alanine, serine, and glycine (hydrazinolysis). pH profile studies show that no group titrating in the region 2.5-8 is directly involved with substrate binding and that a single group having a pKa of 6.5 is involved in the catalysis. Photooxidation of the enzyme caused rapid inactivation. The pH-dependence of this photooxidation, and amino acid analysis of the photooxidized enzyme, indicate that decomposition of histidine is probably responsible for the loss of activity. Other chemical modifications performed were: treatment with hydrogen peroxide under acidic conditions, esterification with diphenyldiazomethane, and oxidation with N-bromosuccinimide. Oxidation with N-bromosuccinimide indicated that a tryptophan side-chain is involved in, but not necessary for, the catalytic activity.
描述了一种从担子菌QM806的培养滤液中大规模生产(1→3)-β-D-葡聚糖葡萄糖水解酶（EC 3.2.1.58）的方法。最终制剂在非解离和变性条件下通过圆盘电泳、超速离心和等电聚焦均一。已经测定了该酶的各种物理和化学特性，包括末端氨基酸残基、消光系数以及对极端pH的稳定性。N末端氨基酸是亮氨酸和丝氨酸（桑格法），C末端氨基酸是丙氨酸、丝氨酸和甘氨酸（肼解）。pH曲线研究表明，在2.5-8区域中没有滴定的基团直接参与底物结合，并且具有6.5的pKa的单个基团参与催化。酶的光氧化导致快速失活。这种光氧化的pH依赖性以及光氧化酶的氨基酸分析表明，组氨酸的分解可能是活性丧失的原因。进行的其他化学修饰包括：在酸性条件下用过氧化氢处理、用二苯基重氮甲烷酯化以及用N-溴代琥珀酰亚胺氧化。用N-溴代琥珀酰亚胺氧化表明，色氨酸侧链参与催化活性，但不是必需的。
