Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus

Abstract

1. 1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH 4) 2SO 4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. 2. Both trypsins catalyzed the hydrolysis of N- benzoyl- dl-arginine p-nitroanilide (BAPNA), p- tosyl- l-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. 4. The enzymes had a pH optimum of 8–9 for the hydrolysis of BAPNA and 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. 5. Trypsin A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca 2+ for activity and stability.