Revista de Microbiologia
Print version ISSN 0001-3714
SILVA, Maria Estela da and FRANCO, Telma Teixeira. Purification of microbial b-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning. Rev. Microbiol. [online]. 1999, vol.30, n.4, pp.324-331. ISSN 0001-3714. http://dx.doi.org/10.1590/S0001-37141999000400006.
This work investigated the partitioning of b-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-b-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme b-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 b-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 b-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.
Keywords : b-galactosidase; aqueous two-phase systems; protein purification; downstream-processing; affinity.