柿果实乙醇脱氢酶酶学特性研究

Enzymatic characterizations of alcohol dehydrogenase of persimmon fruit

以磨盘柿（Diospyroskaki 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、SephadexG-25脱盐柱脱盐、DEAESepharose 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pH5．0、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^＋0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Ca^2＋对酶活性有一定的抑制作用，Cu^2＋和Fe^3＋则完全抑制了酶的活性。在25℃，pH10．0的条件下ADH氧化乙醇的米氏动力学参数Km值为8．33mmol／L，最大反应速度Vmax为0．021μmol／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。

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.