湖北钉螺酚氧化酶的部分纯化及酶学特征

目的制备并纯化钉螺酚氧化酶,探讨该酶最适pH、最适温度及其底物专一性等特征。方法利用匀浆、离心等方法制备粗制酚氧化酶酶液,用盐析、凝胶层析进行酶液纯化;在不同pH、不同温度下测定该酶比活力,寻求最适pH、最适温度;根据测定不同底物(邻苯二酚、L-多巴、焦没食子酸)对该酶的酶促反应米氏常数(Km),判定其底物专一性。结果经纯化后酶活力提高了13.62倍。该酶对邻苯二酚、L-多巴和焦性没食子酸的Km值分别为5.66±0.84mmol/L、4.72±0.45mmol/L和20.64±2.36mmol/L(前两者比较P>0.05,分别与后者比较P均小于0.05)。酚氧化酶在pH6.0、40℃时活性最高。结论通过盐析、凝胶过滤层析等方法纯化可使酶活性明显提高;钉螺酚氧化酶对邻苯二酚的亲和力近似于L-多巴,两者均高于对焦性没食子酸的亲和力。

Partial purification and characterization of phenoloxidase from Oncomelania hupensis

To prepare and purify phenoloxidase(PO) from Oncomelania hupensis, and to study its ensymatie properties, the crude enzyme was prepared by homogenation and centrifugation, and was purified by salt precipitation and gel filtration. Its ensymatie activity was detected at different pH and temperature conditions to obtain the optimal pH and optimal temperature. The Km of the enzyme reacted with catechol, pyrogallol and L-dopamine (L-DOPA) as substrates was examined. It was found that a 13.62-fold increase was from enzyme purification, and Km of PO with catechol, L-dopa and pyrogallol as substrates were 5.66-0.84 mmol/L, 4.72-0.45 mmol/L and 20.64-2.36 mmol/L, respectively(P>0.05 between the two formers and P<0.05 between others).The optimum pH was 6.0 and the optimum temperature was 40 ℃. It is clear that the activity of PO is distinctly improved by the methods of centrifugation, salt precipitation, dialysis and gel filtration. The affinity of PO with catechol is near to that with L-DOPA, both are higher than pyrogallol.