| Trypsin (EC3.4.21.4) belongs to the Ser-protease group and presents in fish pancreas and pyloric caeca. It is one of the main alkaline proteolytic enzymes in fish gastrointestinal, produced as inactive zymogen, which is activated in the gut to trypsin by enterokinase and Ca2+, and cutting the active peptide. The feeding habits of fish are generally divided into herbivorous, carnivorous and omnivorous. Selecting three kind of fishes as their representatives, grass carp(Ctenopharyngodon idellus), black carp (Mylopharyngodon piceu) and sharksucker (Carassius auratus), we extracted and purified their trypsins and compared their differences; measured in vitro their protein digestibility and enzyme dynamics; and so provided some theoretical basises for the research and development of different feeding fish feed and improving the utilization of feedprotein.1. Separation and purification of the trypsins from different feeding fishesThe 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 that: using40%-80%,30%-70%and20%-60%, respectively, ammonium sulfate saturation collected most of trypsin from grass carp, sharksuker and black carp; affinity chromatography in pH3collected the protein peak with trypsin activity; trypsin was the only band for SDS-PAGE with their molecular weight27.3KDa,26.7KDa and42.1KDa, respectively. So the final purifications were about122.25,130.46and128.39-fold, with the recovery rates14.64%,16.63%and17.65%, respectively.2. Comparative studies on the characterization of trypsins from different feeding fishesThe Michaelis Constant (Km) for trypsins from grass carp, sharksuker and black carp were determined to be0.030mol·L-1,0.036mol·L-1and0.027mol·L-1by using BAPNA as special substrate.For the hydrolysis of TAME hydrochloride, the optimum pH for trypsins from grass carp and sharksuker were8.5and black carp was9.0. Trypsins from grass carp and sharksuker were stable for pH6.0-10.0and black carp’s trypsin was stable for pH7.0-10.0. The optimum temperature for trypsins from thest fishes were40℃, and the thermal stability profile of the purified trypsins showed that they were stable for20-50℃. Trypsin activity was not affected by Na+and Ca2+, and inhibited by K+, but was lightly increased by Mg2+, and then gradually decreased with the increase of Mg2+. The enzyme was also strongly inhibited by EDTA.3. Comparative studies on the proteolytic activity of trypsines from different feeding fishThe experiments were conducted to study enzymolysis kinetics and digestive rate in vitro of trypsins from grass carp, sharksuker and black carp for hydrolyzing bovine albumin, casein, cottonseed cake, rapeseed cake, soybean cake, fish meal and wheat bran, respectively. The results showed that the amino acid generateing rate for grass, carp was:casein> rapeseed cake> cottonseed cake> fish meal> bovine albumin> soybean cake> wheat bran; for sharksuker was:casein> rapeseed cake> bovine albumin> fish meal> soybean cake> cottonseed cake> wheat bran; and for black carp was:casein> fish meal> cottonseed cake> rapeseed cake> soybean cake> bovine albumin> wheat bran. The results showed above all with significant difference (P<0.05). The proteolytic activity for fish meal by trypsin from black carp was highter than that for rapeseed cake and soybean cake, but the proteolytic activity for rapeseed cake and cottonseed cake by trypsin from grass carp was highter than that for fish meal. So it appears that, the trypsin from herbivorous fish has a more effective digestion for vegetable proteins, and trypsin from carnivorous fish has a more effective digestion for proteins from animals. |
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