| 摘要: |
| 以磨盘柿(Diospyros kaki Thunb.)果皮为试材,提取乙醇脱氢酶(Alcohol Dehydrogenase)并进行纯化。经过30%~70%硫酸铵沉淀分离、Sephadex G-25脱盐柱脱盐、DEAE Sepharose Fast Flow离子交换层析纯化后,研究温度、pH、金属离子、底物对乙醇脱氢酶活性的影响。结果表明,纯化后乙醇脱氢酶纯化倍数为41.80倍,活性回收率为44.73%。将乙醇脱氢酶酶液分别置于20、30、40、45、50、55和60 ℃温育后测定,其最适反应温度为45 ℃;在pH 5.0、7.0、8.0、9.0、10.0和11.0下乙醇脱氢酶的最适pH为10.0;在甲醇、乙醇、丙醇、丁醇和戊醇为反应底物时,最适底物为丁醇;金属离子中,K+和Na+对活性略有增强的作用,二价金属离子除Mg2+几乎没有影响之外,Zn2+、Mn2+和Ca2+对酶活性有一定的抑制作用,Cu2+和Fe3+则完全抑制了酶的活性。在25 ℃,pH 10.0的条件下ADH氧化乙醇的米氏动力学参数Km值为8.33 mmol/L ,最大反应速度Vmax为0.021 μmol/min。乙醇脱氢酶的活性受温度、pH、金属离子、底物的影响较大,并且只有在合适的条件下,酶活性才能达到最大值。 |
| 关键词: 柿 乙醇脱氢酶 纯化 最适pH 金属离子 |
| DOI：10.11841/j.issn.1007-4333.2011.05.012 |
| 投稿时间：2011-03-09 |
| 基金项目:国家自然科学基金资助项目(30671713,30972395) |
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| Enzymatic characterizations of alcohol dehydrogenaseof persimmon fruit |
|
LIANG Yin-na, LI Bao
|
| (College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China) |
| Abstract: |
| Alcohol dehydrogenase(ADH, EC.1.1.1.1) was extracted from the pericarp tissues of 'Mopanshi’ persimmon (Diospyros kaki Thunb.). The ADH crude extracts was precipitated and isolated with 30%-70% (NH4)2SO4 solition,then desalted by Sephadex G-25 Desalting columns, and finally purified by DEAE Sepharose Fast flow ion-exchange columns. The enzyme was subjected to different temperatures (20, 30, 40, 45, 50, 55 and 60 ℃), different pHs (pH 5.0、7.0、8.0、9.0、10.0 and 11.0),metals (K+,Na+,Zn2+,Mg2+,Mn2+,Ca2+,Cu2+ and Fe3+) and substrates (methanol, ethanol,propyl alcohol, butyl alcohol and amyl alcohol).The results showed that the purification rate was up to 41.80 times.The active recovery rate was about 44.73%.The enzyme had highest activity for butyl alcohol oxidation at 45 ℃ and pH10.0,Km 8.33 mmol/L and Vmax 0.021 μmol/min. The enzyme activity was increased at the presence of K+ and Na+,but partially inhibited by Zn2+,Mn2+ and Ca2+,fully inhibited by Cu2+ and Fe3+.Mg2+ has no effect on the enzyme.It is evedent that ADH is largely influenced by temperature,acidity,metals and substrates.And high activity can only be obtained under proper conditions. |
| Key words: Mopanshi persimmon alcohol dehydrogenase(ADH) purification characterization |
