Characterization and Potential Utilization of Proteases From the Hepatopancreas of Crawfish, Procambarus Clarkii.

Four electrophoretically homogenous anionic trypsins, tentatively named trypsin A, B, C, and D, were isolated from crawfish hepatopancreas. Purity was increased 56, 100, 87, and 64-fold with approximately 2.4, 8.1, 5.1, and 3.0% yield for trypsin A, B, C, and D, respectively. The molecular weights of trypsin A, B, C, and D were estimated to be 23,800, 27,900, 24,800, and 31,400, respectively, using Sephacryl S-200 gel filtration. Isoelectric points and amino acid profiles of the trypsins were similar. Caseinolytic activity was consistently highest between pH 5.5 and 10.0 for all trypsins. Amidolytic and esterolytic activities were maximum in a narrower range, between pH 7.5 and 8.5. Trypsins were unstable at acidic pH. Thermal stabilities of the enzymes increased in the presence of casein. Trypsin C and D had higher thermal stability than trypsin A and B. Maximum activities of trypsin A, B, C, and D were achieved at concentrations of 0.5, 0.05, 0.025 and 0.1 mM Ca$\sp{2+}$ ions, respectively. Sensitivity to inhibitors of these trypsins was similar to trypsin-like serine enzymes found in marine animals. Trypsin D had the highest substrate turnover number for caseinolytic reaction followed by trypsin A compared with other trypsins at physiological pH. This suggests that these two enzymes may play a primary role in the development of mushiness in tail meat. Crawfish trypsins had similar activation energies for the esterolytic reaction using either N$\sb\alpha$-p-tosyl-L-arginine methyl ester or benzoyl-L-arginine ethyl ester of approximately 6.4 to 9.0 Kcal/mole. The activation energies of the hydrolysis of benzoyl-D,L-arginine-p-nitroanilide ranged from 5.8 to 6.2 Kcal/mole and those for the hydrolysis of casein varied between 9.8 to 13.0 Kcal/mole. The optimum reaction conditions for the hydrolysis of crab waste protein by crawfish enzyme was established for enzyme concentration, pH and incubation time. One-hundred fifteen volatile flavor components were identified in enzyme treated crab waste and one-hundred sixteen in untreated crab waste. Volatile flavor compounds increased significantly in enzyme treated crab waste compared to untreated crab waste, however, high molecular weight aldehydes and aromatic hydrocarbons decreased in enzyme treated crab waste. 2-Ethyl-6-methylpyrazine and 2,3-dimethylpyrazine were among 12 identified pyrazine compounds with markedly higher concentrations in enzyme-treated sample