Preparation of a new thermo‐responsive adsorbent with maltose as a ligand and its application to affinity precipitation

A thermo‐responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation. Among the thermo‐responsive polymers synthesized as carriers for adsorbent, poly( N ‐acryloylpiperidine)–cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4°C, at which its solubility exhibits a sharp change. Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine–borane as a reducing reagent. This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4°C and was completely insoluble above 8°C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4°C, precipitation of the complex at 10°C, desorption by adding the desorption reagent at 4°C, and recovery of a target protein at 10°C. In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl‐α‐ D ‐mannopyranoside as a desorption reagent. In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance. Moreover, when pAPM was used for the purification of thermolabile α‐glucosidase from the cell‐free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206‐fold higher than that of the cell‐free extract without thermal deactivation of the enzyme. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 568–579, 1998.