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-Cu2+ and by the adsorption of the catalase on Sepabeads-IDA-Cu2+ support, a pure enzyme fraction was obtained and its N-terminal end was sequenced.
Laboratorio de Tecnología Enzimática, Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Cantoblanco, 28049 Madrid, Spain.
Aurelio
A
Hidalgo
Lorena
L
Betancor
Cesar
C
Mateo
Fernando
F
Lopez-Gallego
Renata
R
Moreno
Jose
J
Berenguer
José M
JM
Guisán
Roberto
R
Fernández-Lafuente
D000327:Adsorption; D002374:Catalase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004789:Enzyme Activation; D007475:Ion Exchange Resins / Q000737:chemistry*; D016963:Thermus thermophilus / Q000201:enzymology*
D007475:Ion Exchange Resins; D002374:Catalase