Isolation and characterisation of trypsin from sardinelle (<i>Sardinella aurita</i>) viscera

Abstract

Abstract 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 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N ‐α‐benzoyl‐ L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N ‐α‐benzoyl‐ DL ‐arginine‐ p ‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °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. Copyright © 2008 Society of Chemical Industry