PURIFICATION OF alfa-LACTALBUMIN USING PEPTIDES AS AFFINITY CHROMATOGRAPHY LIGANDS

AUTOR(ES)

Patrick Vasconcelos Gurgel

FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

2000

RESUMO

A method for the purification of alfa-lactalbumin from whey protein isolate solutions using a hexapeptide ligand has been developed. The peptide WHWRKR was isolated from a peptide library generated by combinatorial chemistry, and displays affinity to alfa-lactalbumin. Other proteins from whey protein isolate bound nonspecifically to the resin used. Different strategies for production of alfa-lactalbumin from whey protein isolate using bioselective adsorption are reported. The use of a peptide ligand (WHWRKR) attached to a resin allowed production of an alfa-lactalbumin-rich fraction with a purity of 90.6% and a recovery of 47.9%, while also producing other fractions of commercial interest. The combined use of an amino resin followed by the WHWRKR resin produced a highly purified alfa-lactalbumin (100%) with a yield of 35.2%. An investigation on the mechanism of binding of alfa-lactalbumin to the peptide ligand WHWRKR and its variants HWRKR and acetylated WHWRKR immobilized on a polymethacrylate chromatographic resin was carried out. The presence of two temperature-dependent binding mechanisms and one temperature-independent mechanism was demonstrated. Injections of different forms of alfa-lactalbumin (apo-alfa-Iactalbumin, D87A mutant alfa-lactalbumin, and peptides 1-40/104-123 and 53-103) displayed similar behaviors when compared to native alfa-lactalbumin, while lysozyme showed little or no binding to the WHWRKR and AcWHWRKR resins. An alternative process for isolation of alfa-lactalbumin from WPI was developed, using consecutive injections of WPI with limited elution. Finally, the use of the peptide resin AcWHWRKR in batch mode with sweet whey was tested, demonstrating that although the resin docs bind alfa-lactalbumin properly, an efficient separation does not occur, due to the presence of glycomacropeptide in the elution fraction.

ASSUNTO(S)

ciencia e tecnologia de alimentos purification of alfa-lactalbumin

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