Purification and Characterization of Trypsin From the Intestine of Genetically Improved Nile Tilapia (Oreochromis niloticus)

Abstract

Trypsin, with molecular weight of 28 kDa from the intestine of genetically improved Nile tilapia (Oreochromis niloticus), was purified by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. Purified trypsin had maximal activity at pH 8.0 and 60°C for hydrolysis of N α-p-tosyl-L-arginine methyl ester. The enzyme was stable at temperatures up to 50°C and pH range of 6.0–11.0. Its activity was strongly inhibited by metal ions such as Pb2+ and Fe3+ and protease inhibitors including soybean trypsin inhibitor and phenylmethylsulfonyl fluoride. Also, the ion Ca2+ slightly inhibited this activity. The Michaelis-Menten constant (K m) and catalytic constant (K cat) of purified trypsin were 0.036 mM and 152 s−1, respectively. Furthermore, trypsin contained low amounts of hydrophobic and aromatic amino acids as well as β-sheet (20.2%) and β-turn (25.0%).