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.
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.
A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.
The unplanned inclusion of antinutrients in fish food affects many biological processes, such as digestibility of amino acids and diet conversion, resulting in undesirable effects on body growth. Thus, the objective of this research was to propose the use of immobilized fish proteases in the detection of protease inhibitors, one of the most important antinutrients. In order to evaluate the detection of antinutritional factors through the immobilized trypsin, the enzyme was incubated with eight diets developed for commercial fish, and residual activity was measured. Comparatively, the tilapia trypsin showed an inhibition of antinutrients (protease inhibitors), present in the eight studied diets, up to 48% greater than the porcine trypsin immobilized in magnetic chitosan. Thus, it is possible to suggest the use of immobilized derivatives containing specific proteases of the target organism in the detection of antinutritional factors that reduce animal's digestive capacity and negatively influence their growth during husbandry.
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