| Schizothorax prenanti, a kind of cold water fish, belongs to Cypriniformes, Schizothoracinae, Schizothorax. It widely distributes in the upper and middle reaches of the changjiang river, Jinsha river, Minjiang river, Tatu river, Qingyi river and the lower reaches of the Wujiang river. This paper studies the fish typsin on the Enzyme levels, which is important not only for the fish digestive physiolegy, but also provides theoretical support for the development of fish fodder.1. Study on the purification of trypsin from Schizothorax prenantiThree-step purification of trypsin involves Salt precipitation, ion exchange chromatography and affinity chromatography. Schizothorax prenanti was killed off on the ridge, removed the liver and pancreas, washed with double distilled water(4℃)and dried up with filter paper and weighed. Then it was homogenized with 0.02% NaN350mM Tris-HCl (4℃, pH8.0) buffer at 1:4 (W/V), activated overnight and then centrifuged, and the crude enzyme extract was the supernatant. Supernatant was carried out with 20-60% ammonium sulfate salt, and then centrifuged 30min (11000rpm,4℃) and dissolved precipitate with the same buffer. Liquid was dialysized and dissolved. The crude enzyme solution after dialysis purified by chromatography, first by ion exchange chromatography, with Amersham's DEAE-SepharoseTM Fast Flow (1.2×20 cm) as ion exchange packing. Then it was subjected to elution in buffer gradient containing 0-0.5 M NaCl in Tris-HCl. elution peak with trypsin activity will be collected. And then it was purified by affinity chromatography with HiTrap Benzamidine FF (high sub) pro-and pre-column. Then it was subjected to buffer elution of pH3.0 glycine, and elution peak with trypsin activity will be collected. protein content and trypsin activity of the collection of components were determined by Bradford and Worthington respectively, and calculate the yield and purification fold. The results showed that the final preparations from Schizothorax prenanti was 456-fold, with the recovery of 18.1%.2. Study on the characterization of trypsin from Schizothorax prenanti Through SDS-PAGE, electrophoresis is finally showing a single band, with the molecular weight of trypsin 25.2KDa measured by the Bio-Rad gel imaging system. With BAPNA as the substrate, The Michaelis constant (Km) of Schizothorax prenanti is 0.136mM measured by BAPNA.Through the method of Worthington,the optimum temperature of Schizothorax prenanti is 20℃, the optimum pH is 8.0. The thermal stability of the purified enzyme showed that the enzyme is stable below 20℃. Trypsin of Schizothorax prenanti is stable at pH7.0-9.0.30min of metal ions and inhibitors purified trypsin, after measured by the Worthington method, shows that:the enzyme activity was decreased by Ca2+, The trypsin activity was increased by Mg2+and Cu2+. The enzyme activity was lightly increased by K+and Na+, then gradually decreased with the increase of K+and Na+concentration. The trypsin was effectively repressed by protease inhibitors, such as PMSF.EDTA and benzamidine, but was increased by B-mercaptoethanol.3. Study on the proteolytic activity of trypsin from Schizothorax prenantiStudy involves the enzymolysis kinetics and digestive rate in vitro of trypsin from Schizothorax prenanti to bovine albumin, casein, soybean meal, cottonseed meal, rapeseed meal and fish meal. The results showed that the amino acid generate rate is: fish meal> rapeseed meal> casein> soybean> cottonseed> bovine serum albumin. the proteolytic activity of fish meal by trypsin from Schizothorax prenanti was the hightest, and the digestive ability of bovine albumin was the lowest.
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