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KINETIC AND THERMODYNAMIC CHARACTERISTICS OF A DIGESTIVE PROTEASE FROM ATLANTIC COD, GADUS MORHUA
Author(s) -
SIMPSON B. K.,
SMITH J. P.,
YAYLAYAN V.,
HAARD N. F.
Publication year - 1989
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1989.tb00394.x
Subject(s) - gadus , chemistry , trypsin , protease , chromatography , hydrolysis , ammonium sulfate precipitation , substrate (aquarium) , kunitz sti protease inhibitor , atlantic cod , enzyme , biochemistry , size exclusion chromatography , biology , ecology , fishery , fish <actinopterygii>
A 23,500 dalton protease was isolated from the pyloric ceca of Atlantic cod by the successive steps of ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The protein fraction recovered after affinity chromatography migrated as one band in both Davis and Laemmli gels. The protease was classified as trypsin (EC 3.4.21.4) on the basis of its substrate specificity, molecular weight and response to known trypsin inhibitors. For trypsin hydrolysis of benzoyl‐DL‐arginine p‐nitroanilide, the substrate turnover number was 250 BAPA units per micromole trypsin (25°C), Km 1 was 1.48 mM, the Arrhenius energy of activation (Ea) was 8.9 kcal/mol, and the free energy of activation (ΔG*) was 12.8 kcal/mol. The Vmax for the hydrolysis of tosylarginine methyl ester was 18,210 TAME units per micromole trypsin and the Km 1 for the same reaction was 0.22 mM at 25°C.