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

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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.

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Acknowledgments

Maxilact LX-5000 was a generous gift from DSM Food Specialties. Bekir G. Mazı is indebted to the Scientific and Technical Research Council of Turkey (Grant TUBITAK-BIDEB-2214) for the fellowship. This research was funded by grant METU-BAP-08-11-DPT.2002K120510 (Turkey) and TUBITAK-TOVAK-108O823 and seed grants from the UC Davis Robert Mondavi Institute and University Outreach and International Programs.

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Authors and Affiliations

  1. Department of Food Engineering, Cumhuriyet Yerleşkesi, Ordu University, 52200, Ordu, Turkey

    Bekir G. Mazı

  2. Department of Food Engineering, Middle East Technical University, 06531, Ankara, Turkey

    Bekir G. Mazı & Haluk Hamamcı

  3. Department of Food Science and Technology, University of California, Davis, CA, 95616, USA

    Bekir G. Mazı, David M. Ogrydziak & Stephanie R. Dungan

  4. Department of Chemical Engineering, University of California, Davis, CA, 95616, USA

    Stephanie R. Dungan

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  1. Bekir G. Mazı
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Correspondence to Bekir G. Mazı.

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Mazı, B.G., Hamamcı, H., Ogrydziak, D.M. et al. Single-Step Partial Purification of Intracellular β-Galactosidase from Kluyveromyces lactis Using Microemulsion Droplets. Appl Biochem Biotechnol 180, 1000–1015 (2016). https://doi.org/10.1007/s12010-016-2148-y

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