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  • br Conclusions Extract of non germinated

    2023-01-31


    Conclusions Extract of non-germinated winter rape seeds cv. Bellevue was screened for the aminopeptidase activity. It revealed broad substrate specificity, with the highest activity toward Phe-pNA. Also in the partially purified sample, phenylalanine was preferentially hydrolysed. PMSF, known serine protease inhibitor and EDTA (metalloprotease inhibitor) were most efficient, suggesting that serine plays a crucial role in the catalytic process and that metal ions are required for full enzymatic activity. The enzyme was slightly activated by Ba2+, Na+ ions and no influence of Ca2+, Mg2+ or Al3+ ions was observed. The addition of Cu2+ Zn2+ and Ni2+ had significant inhibitory effect. The pI values as well as triapine and temperature optima were quite similar to other plant aminopeptidases and iminopeptidases. The obtained results suggest that there are few aminopeptidases (including prolyl aminopeptidase) present in the partially purified sample, having similar molecular weight and slightly different pI values.
    Acknowledgments
    Introduction Salting of food, particularly the salting of fish, is an ancient food preservation technique still in use around the world [1]. Layú, the traditional name for salted fish produced in southern China, is popular in the southern and eastern parts of China because of its special chewiness, flavor satisfying characteristics, and much longer shelf life [2]. Black carp (Mylopharyngodon piceus) and grass carp (Ctenopharyngodon idellus) are two species of freshwater fish that are usually used for Layú in China, due to their huge stocks, high quality proteins (15–26%, w/w) and relatively low market price. Generally, cleaned fish will be mixed with salt in a 10:1 or 20:1 ratio (fish:salt, w/w), depending on the desired final product taste. Then, salt-mixed fish are subjected to fermentation. After 15–20 days of maturing, it is suitable for consumption and can be stored for 2–3 months [2]. Five major factors are believed to influence the quality of the final product of salted fish: fish species, type of salt, the ratio of fish and salt, minor ingredients, and salting method [3]. The type of fish species also affects the type of proteins that serve as nutrients for microorganisms and substrates for enzymes, both of which hydrolyze proteins into small peptides and amino acids. Proteins are highly complex polymers made of up to 20 amino acids [4]. Muscle proteins in salted fish can be hydrolyzed into small peptides and amino acids, which contribute to the specific aroma and flavor in the fermentation of fish [1]. Proteolysis and lipolysis constitute two of the most important biochemical mechanisms occurring during the processing of salted fish with respect to the final sensory quality. Proteolysis contributes to the texture by breakdown of the muscle structure, to the taste through the generation of small peptides and free amino acids and to the aroma by further degradation of some free amino acids [5–7]. Although the amino acid composition of fresh fish does not vary greatly; however, the free amino acid composition changes considerably after processing and storage. There are several consecutive stages in proteolysis: (a) breakdown of major myofibrillar proteins by the action of endopeptidases, such as calpains and cathepsins, (b) generation of polypeptides that act as substrates for peptidases to generate small peptides and (c) intense generation of free amino acids by the action of exopeptidases like aminopeptidases and carboxypeptidases [5,8]. The generation of free amino acids is important because they directly contribute to taste. The contributions of cathepsins and calpains have been widely studied, but there exists a limited knowledge about the role of aminopeptidases in the generation of free amino acids and their contribution to flavor especially in Chinese traditional salted fish such as Layú [4,9]. Aminopeptidases (EC 3.4.11.1–15) are a group of exopeptidases with the ability to hydrolyze amino acid residues from the amino terminus of proteins or peptides and are classified according to the preference for amino-terminal amino acid of substrates, their location, sensitivity to inhibitors, and requirement of divalent metal ions for their enzymatic activities [9,10]. These enzymes are considered to have their original substrate specificities dependent on their physiological roles and are widely distributed throughout animals, plants, and microorganisms. They are vital for metabolic pathway regulation, cell maturation and turnover of proteins [11].