Purification and characterization of trypsin from the digestive system of carp Catla catla (Hamilton)

Abstract

Trypsin was purified from the digestive system of carp Catla catla (Hamilton) by ammonium sulfate fractionation, diethylaminoethyl-cellulose column chromatography, and Benzamidine Sepharose 4 fast flow column affinity chromatography. Trypsin was purified 26.2-fold with an 11.1% yield. The purified enzyme was active between pH 7.0 and 9.8, and maximal activity of the enzyme was observed at pH 7.0. Highest activity was found at 40°C. The activity was reduced to 52.84% at 60°C and was completely lost at 70°C. An addition of 2 mM CaCl2 enhanced trypsin activity during the 8-h incubation. The Km, Kcat, and catalytic efficiency values of purified enzyme were 0.062 mM and 19.23/s, and 310.16/s/mM, respectively. The enzyme activity was inhibited by soybean trypsin inhibitor, phenylmethylsulfonylflouride, and N-α-p-tosyl-l-lysine chloromethyl ketone. The molecular mass of the purified enzyme was 20.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Mass spectrometry study of purified enzyme gave the peptide sequences LGEHNIAVNEGTEQFIDSVK (MW = 2,027.9568) and HPSYNSRNLDNDIM (MW = 1,692.6952) showing identical sequence with trypsin from various fishes.