Affinity purification and characterization of a yeast epoxide hydrolase
Affinity purification and characterization of a yeast epoxide hydrolase
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DOI：
10.1023/a:1005500407152
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发表时间：
1999-06-01
影响因子：
2.7
通讯作者：
Botes, AL
中科院分区：
文献类型：
--
作者：
Botes, AL
采用亲和配体吸附剂Mimetic绿色，在一个单一的色谱步骤中实现了从酵母Rhodosporidium toruloides CBS 0349的膜相关环氧化物水解酶的纯化至电泳均一性。超过68%的总环氧化物水解酶活性存在于整个细胞中回收的膜部分。该酶相对于溶解的膜蛋白纯化26倍，并以90%的产率获得。纯化的环氧化物水解酶具有类似于54 kDa的表观单体分子量和7.3的pI。该酶在30-40 ℃和pH 7.3-8.5下具有最佳活性。该酶是高度糖基化的，碳水化合物含量> 42%。纯化的酶对（+/-）-1，2-环氧辛烷的比活性为172 μ mol min（-1）mg蛋白（-1）。测定了蛋白质的氨基酸组成。这是第一个报告的酵母环氧化物水解酶纯化到均匀的毫克量。
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 similar to 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 mu 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.