Study of digestive proteinases and proteinase inhibitors of Daphnia carinata

Quantification of proteases activities and their class structure have been studied in a cladoceran, Daphnia carinata. Protease activity ranged from 0.28 to 0.55 Unit mg−1 protein min−1 with an average value of 0.42±0.06 Unit mg−1 protein min−1. Chymotrypsin activity was more than twofold higher (0.49±0.09 Unit mg−1 protein min−1) than the trypsin activity (0.21±0.02 Unit mg−1 protein min−1). Protease activity and reduction of activity in bands of samples treated with specific inhibitors were documented in photometric assay and substrate SDS-PAGE. Proteinase activity against azocasein was inhibited (91.4±1.5%) with SBTI. PMSF reduced the enzyme activity by 53.1±6.5%, and the azocasein hydrolysis was reduced up to 64.6±3.8% by the specific inhibitor of trypsin, TLCK. In the present investigation, the molecular weight of various activity bands ranged from 16.3 to 51.1 kDa. The molecular weights of several protein bands are similar to protease activity zones. The knowledge of digestive enzyme profiles of fish food organisms generated in the present study may assist in the formulation of age-specific feed.