Affinity purification and characterization of a yeast epoxide hydrolase

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 presen...

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Veröffentlicht in:Biotechnology letters Jg. 21; H. 6; S. 511 - 517
1. Verfasser: Botes, A.L
Format: Journal Article
Sprache:Englisch
Veröffentlicht: Dordrecht Springer 01.06.1999
Springer Nature B.V
Schlagworte:
adsorbents
amino acid composition
Amino acids
Biological and medical sciences
Biology of microorganisms of confirmed or potential industrial interest
Biotechnology
carbohydrate content
chromatography
electrophoresis
epoxide hydrolase
Fundamental and applied biological sciences. Psychology
glycosylation
Homogeneity
membrane proteins
Miscellaneous
Mission oriented research
Molecular weight
Proteins
Rhodosporidium toruloides
solubilization
Sporidiales
Yeast
Yeasts
Purification
Enzyme
Basidiomycetes
Epoxide hydrolase
Fungi
Enzymatic activity
Affinity chromatography
Ether hydrolases
Hydrolases
Chemical composition
Kinetic parameter
Physicochemical properties
Thallophyta
Rhodosporidium toruloides
ISSN:0141-5492, 1573-6776
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Zusammenfassung: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 approximately 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30-40 degrees 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 micromol 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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ISSN:0141-5492
1573-6776
DOI:10.1023/a:1005500407152