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

2016-11-01
MAZI, BEKİR GÖKÇEN
Hamamcı, Haluk
Ogrydziak, David M.
Dungan, Stephanie R.
Partial purification of beta-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 beta-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 beta-galactosidase was achieved with 96 % total activity recovery, using a feed containing crude extract and 50 mM K-phosphate buffer (pH 7.5) and 50 mM KCl. Gel filtration chromatography showed that the single extraction was successful at removing low molecular weight impurity proteins (molecular weight (MW) < 42 kDa) from the crude extract.
Citation Formats
B. G. MAZI, H. Hamamcı, D. M. Ogrydziak, and S. R. Dungan, “Single-Step Partial Purification of Intracellular beta-Galactosidase from Kluyveromyces lactis Using Microemulsion Droplets,” pp. 1000–1015, 2016, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/31661.