A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.