Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea.
Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.
Castillo-Yáñez
Francisco Javier
FJ
Universidad de Sonora, Apartado Postal 1735. C.P. 83000, Hermosillo, Sonora 83000, Mexico.
Pacheco-Aguilar
Ramón
R
García-Carreño
Fernando Luis
FL
Navarrete-Del Toro
María de Los Angeles
Mde L
D000645:Ammonium Sulfate / Q000378:metabolism; D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D002140:California; D002432:Cecum / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005399:Fishes / Q000502:physiology*; D010140:Pacific Ocean; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011708:Pylorus / Q000201:enzymology*; D014106:Tosylarginine Methyl Ester / Q000378:metabolism; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology
D001550:Benzamidines; D004791:Enzyme Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D014106:Tosylarginine Methyl Ester; D014360:Trypsin Inhibitor, Kunitz Soybean; D014357:Trypsin; C032157:benzamidine; D000645:Ammonium Sulfate