Purification and characterization of pepsin from stomach of Andrias davidianus
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摘要: 通过研究大鲵胃蛋白酶,可以了解大鲵消化机能,同时为食品加工提供新的材料。经80%硫酸铵沉淀、DEAE-52离子交换层析、SephadexG-100凝胶过滤层析以及HPLC,得到大鲵胃蛋白酶电泳纯制品。纯化倍数为239.87,回收率为4.4%,经SDS-PAGE测得该胃蛋白酶分子量为31ku,大鲵胃蛋白酶的最适温度是40℃,低于40℃稳定,当温度上升,活性迅速下降;最适pH为2,在pH2～6有很好的稳定性。EDTA、BrAc、NBS使该酶活性下降。以酪蛋白为底物,在pH2.0、40℃时测得米氏常数Km值为7.3×103mg/L,Vmax是2.674μg/min。Abstract: With A.davidianus pepsin as raw materials, extraction and purification of pepsin from stomach of A.davidianus were studied.Pepsin was isolated by extraction with PBS buffer, precipitation with 80% saturation ammonium sulfate, ion-exchange on DEAE-52 cellulose, gel filtration on Sephadex G-100 and HPLC.Finally the gotten pepsin was purified 239.87 times.The recovery rate was 4.4%.The pepsin was estimated to be 31ku with SDS-PAGE.The optimal temperature for pepsin activity was 40℃, lower than 40℃ the pepsin was stable, when the temperature raised, the activity decreased rapidly.The optimum pH was 2, the activity was stable in the pH range of 2 ~ 6.Further studies showed that the activity was rapidly decreased by EDTA, BrAc, NBS.Using casein as substrate, under the pH2.0, 40℃, Km was 7.3×103mg/L, Vmax was 2.674μg/min.
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