Low molecular weight serine protease from the viscera of sardinelle (Sardinella aurita) with collagenolytic activity: Purification and characterisation

A new low molecular weight (LMW) serine-protease from sardinelle (Sardinella aurita) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 3.82-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 14.2 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60 °C, respectively. The purified protease was strongly inhibited by phenylmethylsulphonyl fluoride, a serine-protease inhibitor, and soybean trypsin inhibitor. The N-terminal amino acid sequence of the first 10 amino acids of the purified protease was APVQPCVVVI. This sequence showed low homology with several peptidases, suggesting that the enzyme is a new protease. Interestingly, the protease was found to cleave collagen type I and hydrolyze succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin. Our findings indicate that the S. aurita protease is a new LMW enzyme with collagenolytic activity.