Single-Step Partial Purification of Intracellular β-Galactosidase from Kluyveromyces lactis Using Microemulsion Droplets.

Abstract

Partial purification of β-galactosidase from the crude extract of Kluyveromyces lactis was carried out using water-in-isooctane microemulsions formed by the anionic surfactant, sodium di-ethylhexyl sulfosuccinate (Aerosol OT). In order to obtain the crude extract, yeast cells of K. lactis were disrupted by a cell disrupter and separated. The purification of β-galactosidase from the extract by a recently developed one-step reversed micellar (i.e., microemulsion-based) extraction method was then tested, by measuring total protein mass and enzyme activity in the product stream and by analyzing its composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Effects of salt concentration, protein concentration, and pH on the extraction were investigated. Using this approach, a 5.4-fold purification of β-galactosidase was achieved with 96% total activity recovery, using a feed containing crude extract and 50mM K-phosphate buffer (pH 7.5) and 50mM KCl. Gel filtration chromatography showed that the single extraction was successful at removing low molecular weight impurity proteins (molecular weight (MW) < 42kDa) from the crude extract.