甘薯叶过氧化物酶的分离纯化及其部分性质研究

Isolation,Purification and Enzymological Properties of Peroxidase from Sweet Potato(Ipomoea batatas Lam.) Leaves

经硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Sephacryl S-200凝胶过滤层析,从甘薯叶中得到过氧化物酶(POD)电泳纯制品。该酶比活力为91923.14U/mg,纯化倍数为255.69,回收率为1.59%。该酶分子量约为35kD,最适pH值为5.6,最适温度为60℃。该酶在20～50℃、pH4～8内稳定。以不同浓度H2O2为底物在pH7.2和25℃下测得该酶Km值为0.291mol/L。低浓度草酸、尿素、Li+、Na+、K+、Mg2+等对该酶有激活作用;SDS、KSCN、抗坏血酸(AsA)、Mn2+等对该酶有抑制作用;甲醇、乙醇、乙二醇、异丙醇对POD均有一定抑制作用,其抑制作用强弱顺序为异丙醇>乙醇>甲醇>乙二醇。实验表明,该酶稳定性较强。

In order to obtain purified peroxidase from sweet potato（Ipomoea batatas Lam.） leaves for the study of its enzymatic properties,ammonium sulfate precipitation,ion exchange chromatography on DEAE-Sepharose column and Sephacryl S-200 gel filtration were used to purify peroxidase（POD） from sweet potato leaves.The specific activity of purified POD was 91923.14 U/mg,which exhibited a purification fold of 255.69 and a recovery rate of 1.59%.The molecular weight of this enzyme was 35 kD,and the optimal pH and temperature were 5.6 and 60 ℃,respectively.The peroxidase was stable at 20-50 ℃ and pH 4-8.The apparent Km of this enzyme using H2O2 as substrate at different conditions was 0.291 mol/L at 25 ℃ and pH 7.2.Its activity was enhanced by urea,Li^＋,Na^＋,K^＋,Mg^2＋ and oxalic acid,yet inhibited by SDS,KSCN,AsA（ascorbic acid） and Mn2＋.Organic solvents such as methanol,ethanol,glycol and isopropanol could inhibit the activity of this enzyme and the order according to the inhibitory effect from strong to weak was isopropanol,ethanol,methanol and glycol.These results reveal that POD is very stable and therefore has promising application value.