PURIFICATION AND CHARACTERIZATION OF TRYPSIN FROM PYLORIC CAECA OF BIGEYE SNAPPER (PRICANTHUS MACRACANTHUS)

Trypsin from the pyloric caeca of bigeye snapper was purified and characterized. Trypsin had an apparent molecular weight of 23.8 kDa when analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and substrate‐gel electrophoresis. The trypsin fraction consisted of three isoforms as evidenced by the appearance of three different bands on native‐PAGE. Optimal activity was observed at 55C and pH range of 8–11. The activity of trypsin fraction was completely inhibited by soybean trypsin inhibitor and was partially inhibited by E‐64 and ethylenediaminetetraacetic acid. CaCl₂ partially protected the trypsin fraction from activity loss at 40C, while NaCl (0–20%) decreased the activity in a concentration‐dependent manner. The apparent Michaelis–Menten constant (K_m) and catalytic constant (k_cat) were 0.312 mM and 1.06 s, respectively when Nα‐Benzoyl‐dl ‐arginine ρ‐nitroanilide was used as a substrate. Trypsin from the pyloric caeca of bigeye snapper generally showed similar characteristics to other fish trypsins.