Affinity purification and characterization of a yeast epoxide hydrolase |
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Authors: | AL Botes |
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Institution: | (1) Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, South Africa (Fax |
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Abstract: | Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min–1 mg protein–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts. |
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Keywords: | epoxide hydrolase purification Rhodosporidium toruloides yeast |
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