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


MAZI B. G. , HAMAMCI H., Ogrydziak D. M. , Dungan S. R.

APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol.180, no.5, pp.1000-1015, 2016 (Peer-Reviewed Journal) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 180 Issue: 5
  • Publication Date: 2016
  • Doi Number: 10.1007/s12010-016-2148-y
  • Journal Name: APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
  • Journal Indexes: Science Citation Index Expanded, Scopus
  • Page Numbers: pp.1000-1015
  • Keywords: Microemulsion, Enzymes, Purification, beta-Galactosidase, Extraction, Reversed micelles, REVERSE MICELLAR EXTRACTION, BACK-EXTRACTION, PROTEINS, ENZYMES, WHEY, SEPARATION

Abstract

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.