氧化葡萄糖酸杆菌DSM 2003膜结合乙醇脱氢酶的纯化鉴定和性质研究

Purification, Identification and Characterization of Membrane-bound Alcohol Dehydrogenase from Gluconobacter oxydans DSM 2003

通过超速离心收集膜组分、表面活性剂溶解膜蛋白、CM-纤维素柱层析纯化等步骤,从氧化葡萄糖酸杆菌DSM2003中获得电泳纯的具有1,2-丙二醇脱氢酶活性的脱氢酶,此酶由两个亚基组成,其表观相对分子质量分别为80000和50000。经MALDI-TOFMS-MS质谱分析与肽质量指纹图谱检索鉴定,证明纯化得到的酶是乙醇脱氢酶。酶学性质分析表明,该酶的最适反应温度为30℃,最适pH值为5.5～6.0;该酶能催化多种一元醇、二元醇,但对包含3个以上羟基的多元醇基本无氧化活性;其催化活性随着底物碳链长度的增加而减小;大多数金属离子及抑制剂(Cu2+、Fe3+、Ca2+、EDTA等)对此酶活性均有抑制作用。

The membrane fraction of Gluconobacter oxydans DSM 2003 was collected by ultra-centrifugation, and an enzyme with dehydrogenase activity to 1,2-propanediol was isolated from the membrane fraction and purified by CM-cellulose column chromatography. The purified enzyme was composed of two subunits with molecular weights of approximately 80 and 50 kD, respectively. Meanwhile, this enzyme was confirmed to be alcohol dehydrogenase （ADH） by mass spectrometry. The optimal reaction pH and temperature for this enzyme were 5.5 - 6.0 and 30 ℃, respectively. Although this enzyme exhibited a broad substrate spectrum including primary and secondary alcohols, alcohols containing 3 hydroxyl groups and more failed to be oxidized by it. The activity of this enzyme was inhibited by most of the selected metal ions, especially by Cu2＋ and Fe3＋.