title: Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides.
abstract: Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24 kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8-10, respectively, using Nα-benzoyl-L: -arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6-8. Trypsin activity decreased with an increasing NaCl concentration (0-0.6 M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-L: -lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(L: -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.
authors: Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC. chliu@mail.npust.edu.tw
authors: Chun-Hung
authors: CH
authors: Liu
authors: Ya-Li
authors: YL
authors: Shiu
authors: Jue-Liang
authors: JL
authors: Hsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001835:Body Weight; D002432:Cecum / Q000201:enzymology; D004795:Enzyme Stability; D029941:Fish Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010473:Perciformes / Q000033:anatomy & histology / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014018:Tissue Distribution; D014357:Trypsin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D029941:Fish Proteins; D014357:Trypsin