This work investigated the effect of the molecular weight of polyethyleneglycol (PEG) upon the partition coefficient of beta-galactosidases from three different microorganisms: Escherichia coli, Klueveromyces lactis and Aspergillus orizae. It was found that PEG 6,000 and PEG 8,000/phosphate were the best systems for achieving the highest purification factors of E. coli beta-galactosidase. However, the other two yeast beta-galactosidases were not efficiently separated from their contaminants in any of the PEG/salt systems. In order to improve the separation of Klueveromyces lactis beta-galactosidase from the main protein contaminants, the biospecific ligand p-aminophenyl 1-thio-beta-D-galactopyranoside (APGP) was attached to activated PEG 4000. The affinity PEG having APGP bound to its backbone was synthesized in two steps. The partitioning of Klueveromyces lactis beta-galactosidase in aqueous two-phase systems prepared with 6% APGP-PEG4000 + 12% dextran T505,000 increased 1.6-fold the purification factor of the target enzyme, allowing the recovery of 83% of the enzyme in the top PEG-rich phase.
beta-galactosidase; protein recovery; aqueous two-phase systems; downstream processing
