Wang Lu, Zhang Rongsheng, Eisenthal Robert, Hubble John
Department of Chemical Engineering, University of Bath, UK.
Biotechnol Appl Biochem. 2006 Jul;45(Pt 1):37-42. doi: 10.1042/BA20060011.
The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle.
本文探讨了使用基于CM-葡聚糖（羧甲基葡聚糖）的水凝胶从蛋清中选择性回收溶菌酶的方法。该水凝胶含有作为亲和配体的汽巴克隆蓝，并共固定化了牛血清白蛋白（BSA），旨在作为屏蔽剂以减少非特异性吸附。最初使用纯溶菌酶进行的研究表明，吸附容量随配体密度的增加而增加，并且吸附过程可用朗缪尔型等温线很好地描述。在单吸附质实验中，加入BSA作为假定的屏蔽剂并没有降低对溶菌酶的吸附容量。为了评估屏蔽策略的有效性，随后对确定的溶菌酶/卵清蛋白混合物和蛋清进行了实验。通过这些研究，确定了溶菌酶回收的最佳操作条件。这些条件包括：最佳初始蛋清浓度[在所选缓冲液中10%（v/v）的天然蛋清溶液]、亲和配体密度（1.86 mM）和配体与屏蔽剂的比例（4:1）。从蛋清中获得的溶菌酶纯度从使用非屏蔽水凝胶时的69%提高到使用固有屏蔽水凝胶时的94%。最后，研究了使用基于蛋白质而非葡聚糖主链的水凝胶的可能性。结果发现，在简化的合成过程中，BSA可以替代CM-葡聚糖作为凝胶主链，产生的凝胶在单次吸附-解吸循环中也被证明对溶菌酶回收有效，将蛋清溶液中30%的溶菌酶纯化至约92%。
