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 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.

1Progress, applications, challenges and prospects of protein purification technology

“…To sum up, the rough fractionation methods are relatively simple and have a large processing flux. They can not only remove a large number of impurities, but also concentrate the protein solution to achieve partial purification, while maintaining a high recovery rate ( Mazi et al, 2016 ). Among the above rough fractionation techniques, PEG precipitation, ultrafiltration, differential centrifugation and other methods can achieve the concentration of viruses in wastewater ( Dumke et al, 2021 ).…”

Mikrodalgada Kızartmanın Tavuk Göğüs Eti Miyofibriler ve Sarkoplazmik Proteinleri Üzerine Etkileri

“…After mixing with loading buffer at a ratio of 1:5 (loading buffer: sample solution), 5μl of each protein solution and 5μl of marker were loaded on a polyacrylamide gel (5% stacking and 12.5% separating gel) (Mazi et al 2016). The apparatus used was a Mini Protean II system (BIO-RAD Laboratories, Richmond, CA).…”