Recovery and purification of a Kluyvermyces lactis β -galactosidase by Mixed Mode Chromatography
| Autor: | Micael de Andrade Lima, Luciana Rocha Barros Gonçalves, Ivanildo José da Silva Júnior, Maria de Fátima Matos de Freitas |
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| Rok vydání: | 2016 |
| Předmět: |
Clinical Biochemistry
02 engineering and technology 01 natural sciences Biochemistry Analytical Chemistry Kluyveromyces Adsorption Bacterial Proteins Desorption Protein precipitation Kluyveromyces lactis Chromatography biology Chemistry 010401 analytical chemistry Substrate (chemistry) Cell Biology General Medicine beta-Galactosidase 021001 nanoscience & nanotechnology biology.organism_classification 0104 chemical sciences Electrophoresis Mixed-mode chromatography Nonlinear Dynamics Fermentation 0210 nano-technology Chromatography Liquid |
| Zdroj: | Journal of Chromatography B. :181-191 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2016.01.053 |
| Popis: | Mixed Mode Chromatography (MMC) is a potential separation technique that allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. The aim of this work was to assess the recovery and purification of a Kluyveromyces lactis β-galactosidase employing MMC. Protein precipitation and dialysis were performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium. The best conditions for both adsorption and desorption were attained by a non-factorial Central Composite Experimental Design and employed in the chromatographic runs with resin CAPTO MMC. Fermentation yielded mean values of total enzyme concentration of 0.44 mg/mL, enzymatic activity (employing lactose as a substrate) of 74 U/mL and specific activity of 168 U/mg. The Purification Factor (PF) obtained was of 1.17. After precipitation and dialysis, the subsequent chromatographic run resulted in recovery values of 41.0 and 48.2% of total protein concentration and enzymatic activity, respectively. SDS-PAGE electrophoresis confirmed the purification evolution throughout the unit operations employed, attesting the feasibility of the technique to obtain enzymes with not only considerable degree of purity but also possessing high-added value. |
| Databáze: | OpenAIRE |
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