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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Vydané v:	Biotechnology letters Ročník 21; číslo 6; s. 511 - 517
Hlavný autor:	Botes, A.L
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	Dordrecht Springer 01.06.1999 Springer Nature B.V
Predmet:	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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Shrnutí:	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.
Bibliografia:	SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 14 ObjectType-Article-2 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0141-5492 1573-6776
DOI:	10.1023/a:1005500407152