title: 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.
authors.affiliation: Department of Food Engineering, Cumhuriyet Yerleşkesi, Ordu University, 52200, Ordu, Turkey. bgmazi@odu.edu.tr.|Department of Food Engineering, Middle East Technical University, 06531, Ankara, Turkey. bgmazi@odu.edu.tr.|Department of Food Science and Technology, University of California, Davis, CA, 95616, USA. bgmazi@odu.edu.tr.
authors.forename: Bekir G
authors.initials: BG
authors.lastname: Mazı
authors.affiliation: Department of Food Engineering, Middle East Technical University, 06531, Ankara, Turkey.
authors.forename: Haluk
authors.initials: H
authors.lastname: Hamamcı
authors.affiliation: Department of Food Science and Technology, University of California, Davis, CA, 95616, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Ogrydziak
authors.affiliation: Department of Food Science and Technology, University of California, Davis, CA, 95616, USA.|Department of Chemical Engineering, University of California, Davis, CA, 95616, USA.
authors.forename: Stephanie R
authors.initials: SR
authors.lastname: Dungan
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002850:Chromatography, Gel; D045424:Complex Mixtures; D004591:Electrophoresis, Polyacrylamide Gel; D004655:Emulsions / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000201:enzymology*; D007716:Kluyveromyces / Q000201:enzymology*; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis; D012965:Sodium Chloride / Q000494:pharmacology; D001616:beta-Galactosidase / Q000302:isolation & purification*
chemical_list: D045424:Complex Mixtures; D004655:Emulsions; D011506:Proteins; D012965:Sodium Chloride; D001616:beta-Galactosidase
keywords: Enzymes; Extraction; Microemulsion; Purification; Reversed micelles; β-Galactosidase