Proteolytic degradation of sardine (Sardinella gibbosa) proteins by trypsin from skipjack tuna (Katsuwonus pelamis) spleen

Trypsin from the spleen of skipjack tuna (Katsuwonus pelamis) was purified by ammonium sulfate precipitation and a series of chromatographies, including Sephacryl S-100 and benzamidine-Sepharose 4 fast flow (high sub). The enzyme was purified 22.3-fold with a yield of 51.6%. The molecular weight of trypsin was estimated to be 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified trypsin was able to hydrolyse natural actomyosin (NAM) and myosin, but scarcely hydrolysed collagen. Myosin heavy chain was most susceptible to hydrolysis by trypsin as evidenced by the lowest band intensity remaining. The effect of NaCl on proteolytic activity was also studied. The band intensity of myosin heavy chain slightly increased as the NaCl concentration was increased, suggesting the inhibitory activity of NaCl. When hydrolytic activities of skipjack tuna spleen and bovine pancreas trypsins on sardine proteins, including NAM, myosin and collagen, were compared, it was found that trypsin from bovine pancreas showed a greater activity towards NAM and myosin than that from skipjack tuna spleen. However, neither enzymes could degrade collagen.