Purification and characterization of pepsin from stomach of Andrias davidianus

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.