This work investigated the partitioning of beta -galactosidase from Kluyveromyces fragilis in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-beta -D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme beta -galactosidase from Kluyveromyces fragilis was developed. In the first step, a system composed of 6% PEG 4000-APGP and 8% dextran 505 was used, where beta -galactosidase was strongly partitioned to the top phase (K = 2,330). In the second step, a system formed of 13% PEG-APGP and 9% phosphate salt was used to revert the value of the partition coefficient of beta -galactosidase (K = 2 x 10(-5)) in order to provide the purification and recovery of 39% of the enzyme in the bottom salt-rich phase.304324331