Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus)

An alkaline peptidase was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex® G-75), with final specific activity 86-fold higher than the enzyme extract and yield of 22.1%. The purified enzyme had an estimated molecular mass of 26.5 kDa and NH2-terminal amino acid sequence IVGGYECTMHSEAHE. Higher enzyme activity was observed at pH 8.5 and between 50 and 55 °C. The enzyme was completely inactivated after 30 min at 55 °C and it was significantly more stable at alkaline pH. Km, Kcat and K cat · K m - 1 values, using BApNA as substrate, were 0.266 mM, 0.93 s−1 and 3.48 mM−1 s−1, respectively. Enzyme activity increased in the presence of the ions (1 mM) K+, Li+ and Ca2+, but was inhibited by Fe2+, Cd2+, Cu2+, Al3+, Hg2+, Zn2+ and Pb2+ as well as by the trypsin inhibitors TLCK and benzamidine.