ISOLATION, PURIFICATION AND CHARACTERIZATION OF A TRYPSIN FROM THE PYLORIC CECA OF MULLET (MUGIL-CEPHALUS)

1. A protease classified as trypsin on the basis of its mol. wt of 24,000, its ability to hydrolyze synthetic substrates N-alpha-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and the inhibitory effects of methyl sulfonyl fluoride (PMSF), SBTI, aprotinin and benzamidine on its activity was isolated from the pyloric ceca of mullet. 2. The enzyme exhibited optimal activity at pH 8 and a temperature of 55-degrees-C and was stable in the pH range of 7.5-9.0. The Arrhenius energy of activation (E(a)) for BAPA hydrolysis by this enzyme was 7.4 kcal/mol. 3. The enzyme showed greater affinity for TAME (K'(m) = 0.19 mM) as a substrate than for BAPA (K'(m) = 0.49 mM), and preferential hydrolysis of ester bonds (V(max) = 2640 TAME units/mu-mmol enzyme) than amide bonds (V(max) = 400 BAPA units/mu-mol enzyme).