猪肝乙醇脱氢酶的分离纯化及部分性质

新鲜猪肝经匀浆、缓冲液抽提、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析及Superdex-200凝胶过滤层析，获得电泳纯的乙醇脱氢酶（alcohol dehydrogenase，ADH）。纯化结果显示：该酶比活力为1 622.33 U/mg，回收率为29.05%，纯化倍数为34.58；该酶分子质量约为171.79 kD，亚基分子质量约为43.68 kD。ADH酶学性质研究显示：最适反应温度和pH值分别为45 ℃和10.0；在25～45 ℃及pH 7.5～9.0范围内稳定性较好；最适条件下测得该酶对乙醇的Km值为19 mmol/L；正丁醇、氯仿、异丙醇、十二烷基硫酸钠、草酸、Zn2＋、Cu2＋、Ag＋对该酶的抑制作用最强，Mg2＋对该酶有激活作用，EDTA对该酶有双重作用。

Isolation,Purification and Partial Characterization of Alcohol Dehydrogenase from Pig Liver

Electrophoresis-purity alcohol dehydrogenase (ADH) from pig liver was obtained through homogenization, buffer
extraction, ammonium sulfate fractionation, DEAE-Sepharose ion-exchange chromatography and Superdex-200 gel filtration
chromatography. Results showed that the specific activity of the purified ADH was 1 622.33 U/mg with an activity recovery
of 29.05% and a purification fold of 34.58. The relative molecular weight of the ADH was approximately 171.79 kD, in
which the subunit molecular mass was roughly 43.68 kD. The enzymatic properties showed that the optimum temperature and pH
for the ADH were 45 ℃ and 10.0, respectively. The enzyme was stable at 25–45 ℃ and pH 7.5–9.0, and its apparent Km towards
ethanol was 19 mmol/L. The enzyme activity of ADH could be strongly inhibited by n-butanol, chloroform, isopropanol, sodium
dodecyl sulfate, oxalic acid, Zn2+, Cu2+, and Ag+, and activated by Mg2+. EDTA had a dual effect on this enzyme.