Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by-products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G-75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G-75 gel filtration, resulting in a 5.42-fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase-specific activity on N-alpha-benzoyl-L-arginine ethyl ester (BAEE) that was four times greater than its amidase-specific activity on N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 degrees C respectively using BAEE as a substrate. The trypsin kinetic constants K-m and k(cat) on BAPNA were 1.67 mmol L-1 and 3.87 s(-1) respectively, while the catalytic efficiency k(cat)/K-m was 2.31 s(-1) L mmol(-1). CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish-processing and food industries. (c) 2008 Society of Chemical Industry