Purification and characterization of trypsin from the poikilotherm Gadus morhua

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Abstract
A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularily with dogfish and some mammalian trypsins. The apparent Km values determined at 25.degree. C for the predominant form of Atlantic cod cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 .mu.M and 77 .mu.M respectively, which are notably lower values than those determined for bovine trypsin (46 .mu.M and 650 .mu.M respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.