One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads.
Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.
Pessela
Benevides C C
BC
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain.
Mateo
Cesar
C
Carrascosa
Alfonso V
AV
Vian
Alejandro
A
García
José L
JL
Rivas
German
G
Alfonso
Carlos
C
Guisan
José M
JM
Fernández-Lafuente
Roberto
R
D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D004852:Epoxy Compounds; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008670:Metals; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013824:Thermus / Q000201:enzymology*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D002614:Chelating Agents; D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D008670:Metals; D011994:Recombinant Proteins; D001616:beta-Galactosidase