Purification of a Catalase from Thermus thermophilus via IMAC Chromatography: Effect of the Support

A hexameric Mn‐catalase was purified from crude extracts of Thermus thermophilus using ammonium sulfate precipitation and ion metal‐chelate affinity chromatography (IMAC). Eupergit 250 and Sepabeads FP‐EP3 epoxy supports derivatized with iminodiacetic acid (IDA) and copper were used, at similar micromole/packed milliliter of support. Although Eupergit 250‐IDA‐Cu support adsorbed 80% of the total proteins in the extract, it exhibited a minimum affinity for the catalase. On the other hand, Sepabeads FP‐EP3‐IDA‐Cu allowed the full adsorption of the catalase activity, which could be desorbed in fractions of different purity. This was attributed to a different geometrical congruence of the support surfaces with the enzyme surface, resulting in a different ability to form multipoint interactions with the proteins. Thus, by a cleanup step, followed by a negative chromatographic step using Eupergit 250‐IDA‐Cu 2+ and by the adsorption of the catalase on Sepabeads‐IDA‐Cu 2+ support, a pure enzyme fraction was obtained and its N‐terminal end was sequenced.