| 摘要: |
| 以30%～60%硫酸铵饱和度分级沉淀、DEAE Sepharose CL-6B、Sephadex G-200、Phenyl Sepharose CL-4B和羟基磷灰石层析纯化了小麦5-氨基酮戊酸脱水酶(ALAD),其在pH 8. 5时比活为31 U·mg-1蛋白;酶纯化了91倍,得率5%,酶亚基分子质量约52 ku,全酶分子质量约330 ku,表明酶由6个亚基组成;酶的等电点为4. 8,在400 nm有吸收峰,最适反应温度45℃,M g2+激活酶,Zn2+和磷酸吡哆醛抑制酶。纯化的ALAD浓缩后, -20℃下在0. 1mol·L-1, pH 8. 5的Tris-HCl,内含50%的甘油, 5mmol·L-1的巯基乙醇和MgCl2中黑暗贮藏30 d酶活不损失。 |
| 关键词: 5-氨基酮戊酸脱水酶,小麦,酶活性 |
| DOI： |
| 分类号:S512.101,Q555+.6 |
| 基金项目:国家自然科学基金（39570066） |
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| Purification and some properties of 5-aminolevulinate dehydratase from wheat leaf |
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| Abstract: |
| 5-aminolevulinate dehydratase (ALAD) is purified to homogeneity from
wheat leaves with 30%～60% ammonium sulphate fractionation, chromatography DEAE
Sepharose CL-6B,Sephadex G-200,Phenyl Sepharose CL-4B and hydroxylatite.The purified enzyme has a specific activity of 31U/mg protein at pH 8. 5 with a fold of 91 and
yield of 5%. It has a relative Mr of 330 ku and subunit Mr of 52 ku, thus showing six identical subunits. IEF confirms its pI is about 4. 8.The enzyme has an absorption maximum at 400 nm in Tris-HCl buffer, pH 8. 5. It is maximally active at a temperature of
45℃ and activated by Mg2+, but inhibited by Zn2+. The purified enzyme after concentrated is stored without a loss of activity for amonth at -20℃ in the dark in the 0. 1mol·L-1 Tris-HCl buffer, pH 8. 5, including 50% glycerol 5 mmol·L-1
2-mercaptoethanol and MgCl2. |
| Key words: 5-aminolevulinate dehydratase (ALAD),wheat,enzyme activity |
