Abstract
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 26.5kDa 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 30min at 55°C and it was significantly more stable at alkaline pH. Km, Kcat and Kcat·Km-1 values, using BApNA as substrate, were 0.266mM, 0.93s−1 and 3.48mM−1s−1, respectively. Enzyme activity increased in the presence of the ions (1mM) 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.
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