芫荽酸性磷酸酶的提取、纯化及酶学性质研究  

Isolation, Purification and Characterization of Acid Phosphatase from Cilantro

作　　者：王丹[1] 万骥 傅婷[1] 唐云明[1] 

机构地区：[1]西南大学生命科学学院,淡水鱼类资源与生殖发育教育部重点实验室,三峡库区生态环境教育部重点实验室,重庆400715

出　　处：《食品科学》2015年第21期162-167,共6页Food Science

基　　金：重庆市科委重点攻关项目(CSTC;2011AB1027)

摘　　要：采用硫酸铵分级沉淀、CM-SepHarose离子交换层析、Superdex-200凝胶过滤层析法,从新鲜芫荽中分离纯化出电泳纯的酸性磷酸酶（acid pHospHatase,ACP）。该酶的酶活回收率为14.20%、纯化倍数为238.60、酶比活力为295.87 U/mg、亚基分子质量约为53.8 k D;芫荽ACP酶学性质研究结果表明：该酶的最适反应温度为55℃,在50℃以下时较稳定,因此该酶对温度较敏感;该酶的最适反应pH值为5.8,在pH 4.0~7.0之间较稳定,表明该酶耐受于酸性环境;芫荽ACP的对硝基苯酚磷酸二钠Km值为0.63 mmol/l,表明该酶与底物对硝基苯酚磷酸二钠具有较高的亲和力;甲醇、乙醇、异丙醇、抗坏血酸、草酸、Cu^2＋、Pb^2＋、Ag^＋对该酶具有强烈的抑制作用;Mg^2＋、Mn^2＋、Ba^2＋、K^＋对该酶具有一定的激活作用。Electrophoresis-purity acid phosphatase（ACP） from cilantro was obtained by homogenization, buffer solution extraction, ammonium sulfate fractional precipitation, CM-Sepharose ion exchange chromatography and Superdex-200 gel filtration. The results showed that after purification the recovery rate of ACP activity was 14.20% with a purification fold of 238.60, and the specific activity of ACP was 295.87 U/mg. The subunit molecular mass of the enzyme was approximately 53.8 kd. The characterization of ACP illustrated that the optimal reaction temperature was 55 ℃, and it was stable in the range of 20–50 ℃. Therefore, the enzyme was sensitive to temperature. The optimal reaction p H was 5.8, and it was relatively stable in the range of p H 4.0–7.0. The enzyme showed tolerance to acidic environment. Its Km was 0.63 mmol/l towards p-nitrophenyl phosphate disodium salt hexahydrate, indicating high affinity between the enzyme and the substrate. The enzyme activity of ACP could be strongly inhibited by methanol, ethanol, isopropanol, ascorbic acid, oxalic acid and Cu^2＋, Pb^2＋ and Ag^＋, while it could be enhanced to some extent by Mg^2＋, Mn^2＋, Ba^2＋ and K^＋.

关 键 词：芫荽 酸性磷酸酶 分离纯化 酶学性质 

分 类 号：Q946.5[生物学—植物学]