Purification Of Trypsin From Cyprinus Carpio Var.Wuyuanensis And Immobilization Of Trypsin

With an important physiological role in the digestion of protein in fish, trypsin (EC 3.4.21.4) belongs to the Ser-protease group and has endopeptidase activity. Cyprinus carpio var. Wuyuanensis is a major strain for Jiangxi's aquaculture, being assigned to Cypriniformes, Cyprinidae, Cyprinus. So far there have been very limited researches about its digestive physiology and the purification and properties for its trysin, which plays an important role in feed development and feeding habits for this fish. This paper did some work on the above two aspects, and tried preliminarily for the immobilization of trypsin, in order to make use of trypsin further on a higher level.1. The purification of trypsin mainly concerned four steps:crude enzyme extraction, ammonium sulfate fractionation, affinity chromatography, and molecular weight determination with SDS-PAGE.The results showed:with 25mM calcium ion, trypsin extract worked well after 20 hours of activation; 30%-70% ammonium sulfate saturation collected most of the enzyme; affinity chromatography in pH3 collected the protein peak with trypsin activity; trypsin was the only band for SDS-PAGE with its molecular weight 26.5kD. So the final purification elevate to 151.4 times, with specific activity 84.81U/mg, and recovery rate 17.4%.2. Some characteristics of trypsin:about pH, temperature and KmRising between pH 6-7.5, the specific activity for trypsin was maximum at pH 7.5, and then decreased slowly for pH 7.5～10. Similarly, the specific activity rose between 4～40℃, with the maximum at 40℃, but reduced significantly at 60℃. And Km for trypsin was 0.056 mol/L-1, using BAPNA as its specific substrate.3. Immobilization for trypsinThe results showed:Best chitosan particles could be made with 2.5% chitosan gel dripping in alkaline solution and crosslinking with 0.5% glutaraldehyde.Fixed trypsin: Using 6mg free trypsin/gram of chitosan particles got the best immobilzation effect, with specific activity 101U/g and recovery rate 42.6%.Some characteristics of the chitosan-immobilized trypsin:Optimum pH for immobilized trypsin was 8, with its alkaline adaptability increased, and showing 87.7% the highest activity even at pH 10. Immobilized trypsin got its maximum activity between 40～60℃, and at 80℃still showing 60% of it.Km value of immobilized trypsin was about double for the free trypsin, and after five repeated reactions, still showing 30% enzymatic activity.