Piyachomkwan K, Penner M H
Department of Food Science and Technology, Oregon State University, Corvallis 97331-6602, USA.
Anal Biochem. 1998 Jan 15;255(2):223-35. doi: 10.1006/abio.1997.2390.
The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters.
研究了配体偶联化学和流动相组成对外切作用纤维素酶与固定化互补配体相互作用的影响。对氨基苯基1-硫代-β-D-纤维二糖苷（APTC）用作外切作用纤维素酶（纤维二糖水解酶，CBHs）优先结合的代表性亲和配体。来自里氏木霉的“粗制”纤维素酶制剂用作酶源。在几种情况下，该制剂中两种主要外切作用CBHs，即CBH I和CBH II的吸附特性显示出明显不同。基于柱洗脱行为和分配平衡实验，它们的相对亲和力显示出高度依赖于用于配体偶联的官能团、官能团水解程度、流动相组成以及酶的固有性质。使用含有氰酸酯、N-羟基琥珀酰亚胺和环氧官能团的琼脂糖载体说明了对支持基质化学性质的依赖性。在明显的最佳条件下进行比较时，CBH II对固定化APTC的亲和力约为CBH I的10倍。然而，通过调整实验参数可以实现CBH I或CBH II的选择性吸附。
