海芋过氧化氢酶的分离纯化及其性质

Isolation,purification and characterization of Alocasia macrorrhiza catalase

为了探索海芋（Alocasia macrorrhiza）过氧化氢酶的分子结构和酶学性质,提取海芋新鲜叶片、叶柄和块茎的粗酶进行活力比较和同工酶分析。用Sephadex G 200凝胶过滤层析法将粗酶分离纯化,以PAGE电泳对酶纯度鉴定。结果表明：海芋过氧化氢酶较单一,没有同工酶。经层析法进行分离获得电泳纯的过氧化氢酶,其活力回收率为40.83%,纯化倍数为8.88倍。十二烷基硫酸钠-聚丙烯酰氨凝胶电泳（SDS-PAGE）分析结果发现：该酶由5条肽链组成,全酶相对分子质量为2.621 1×105。酶学性质研究结果表明：该酶最适反应温度40℃,最适p H为7.5。在25~45℃和p H 6.0~9.0下,该酶能保持较强的催化活力,稳定性较好。在最适条件下,该酶的Km值为16.43mmol/L,对底物的亲和力较好。甲醇、乙醇和异丙醇对此酶活力有显著的抑制作用。

In order to characterize catalase,crude enzyme was extracted from leaves,petioles,tuber of Alocasia macrorrhiza. The enzyme activities and isozymes were compared and analyzed among the organs.Sephadex G 200 gel chromatography was used to purify the enzyme. Polyacrylamide gel electrophoresis（ PAGE） was used to identify the enzyme purity. There was no isozyme of catalase in Alocasia macrorrhiza. The activity recovery was 40. 83% and the purification multiple was 8. 88 folds. It was indicated by SDS-PAGE that the catalase in Alocasia macrorrhiza was composed of 5 polypeptides. The molecular weight of this catalase was 2. 621 1 × 105. The optimal reaction temperature was 40 ℃ and the optimal p H was 7. 5. Under the optimal conditions,the Km of this catalase was 16. 43 mmol / L. The enzyme activity was inhibited by methanol,ethanol and isopropanol.