Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40-60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-rho-tosyl-L-lysine-chloromethyl ketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 degrees C for the hydrolysis of alpha-N-benzoyl-DL-arginine-rho-nitroanilide (BAPNA). It was stable within the temperature range of 25-55 degrees C and pH range of 7.0-10.0. Purified trypsin had a Michaelis-Menten constant (K-m) and catalytic constant (k(cat)) of 0.507 mM and 4.71 s(-1), respectively, when BAPNA was used as the substrate. For the hydrolysis of alpha-N-rho-tosyl-L-arginine methyl ester (TAME), K-m and k(cat) were 0.328 mM and 112 s(-1), respectively. (C) 2009 Elsevier Ltd. All rights reserved.