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(-1)s(-1), respectively. Enzyme activity increased in the presence of the ions (1mM) K(+), Li(+) and Ca(2+), but was inhibited by Fe(2+), Cd(2+), Cu(2+), Al(3+), Hg(2+), Zn(2+) and Pb(2+) as well as by the trypsin inhibitors TLCK and benzamidine.

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An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.

Intestine from Cyprinus carpio L., an important fish in the Brazilian Aquaculture, is proposed in this work as a source of alkaline protease. The intestine crude extract was submitted to a partial purification by ethanol precipitation. The fraction 30–70% (v/v) of ethanol concentration showed higher recovery and specific activity when compared with the crude extract and was submitted to further studies. The optimal temperature and pH were found to be 50C and 11.0, respectively. The enzymatic activity was activated by nonionic surfactants and retained more than 60% of their initial proteolytic activity in the presence of the ionic surfactants. Almost 50% of enzymatic activity was retained in the presence of 5% (v/v) of hydrogen peroxide. The high proteolytic activity at 50C and alkaline pH, together with its stability in the presence of the surfactants and oxidants tested, indicate that this alkaline protease can well be used in detergent formulations. PRACTICAL APPLICATIONS Hydrolytic enzymes have been employed in a range of applications including processes in the food, textile and pharmaceutics industries. Among theses enzymes, proteases correspond for the highest sales on the enzyme market. In the present time, most proteolytic enzymes are extracted from bacteria (genus Bacillus). These biomolecules can also be extracted from the viscera of several fish species. This study relates the partial purification and physical chemical characteristics of alkaline protease found in the Cyprinus carpio intestine, one of the most important species for the world aquaculture. Surfactants and oxidant agents were also used to assay the stability of this enzyme. Therefore, this communication contributes to the optimization of the use of fish by‐products as well as the discovery of enzymes displaying desirable properties.