Purification and biochemical characterization of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caeruleus)

Chymotrypsin was isolated from the viscera of Monterey sardine by ammonium sulphate fractionation, gel filtration, and ionic exchange chromatography. The approximate molecular weight was 26,000 and its isoelectric point was about 5. Identity as chymotrypsin was established by its catalytic specificity for amide or ester bonds on the synthetic substrates succinyl-L-ala-ala-pro-L-pheitalanine-p-nitroanilide and benzoyl-L-tyrosine-ethyl-ester, showing esterase activity 3.2-fold higher than amidase. It was inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, partly inhibited by the specific chymotrypsin inhibitor N-toluenesulfonyl-L-phenylalanine chloromethyl-ketone, but not inhibited by EDTA or Benzamidine. Chymotrypsin showed its maximum activity at pH 8.0 and 50 degrees C for the hydrolysis of SAAPNA. The Michaelis-Menten constant was 0.074 mM with a catalysis constant of 18.6 seg(-1), and catalytic efficiency of 252 seg(-1) mM(-1). Results indicated that Monterey sardine chymotrypsin is a good catalyst and could be used as a biotechnological tool in food processing and using sardine industry wastes as a material for production of fine reagents. (c) 2005 Elsevier Ltd. All rights reserved.