Purification and characterization of gastric proteases from rainbow trout (Oncorhynchus mykiss)

The use of proteolytic enzymes from marine organisms as processing aids is becoming more widespread due to their unique properties, and their unique habitat conditions, compared to enzymes from mammalian sources. Furthermore, different fish species exhibit different properties depending on their environment, making them potential sources of enzymes for different applications. Fish guts are a good and cheap source of proteolytic enzymes, and they are produced in mass quantities, as a byproduct, during the processing of fish.; The gastric juice of vertebrates contains aspartic proteinases, which are secreted as zymogens and undergo conversion to active enzymes in the acidic conditions of the gastric environment. The major gastric proteases are pepsin, gastricsin, and chymosin. The classification of gastric proteases from aquatic organisms is not very well understood. Zymogens of proteases were extracted from stomachs, purified, and characterized in order to assess whether they are pepsin-like, gastricsin-like, chymosin-like or cathepsin D-like.; The pepsin activity of 6 month old rainbow trout increased with increasing water salinity, while no influence was seen on 12 month old rainbow trout and steelhead trout. Pepsin activity was highest in the steelhead trout at all the salinities, compared to that of rainbow trout.; Five zymogens (I–V) for gastric proteases were isolated. The order of elution from the DEAE cellulose column was I, II, IV, V, and III. From activity staining, III is the major zymogen, followed by II, I, V, and IV. Zymogen I was not retained by DEAE cellulose under the experimental conditions, and its activity was negligible unless the assay solutions contained NaCl. Activity staining in the presence of pepstatin and in the hemoglobin assay showed that zymogens V and IV were less sensitive to pepstatin than were the other isoforms and mammalian pepsin. Synthetic substrate APDT was scarcely hydrolyzed by any of the zymogens.; The structural characteristics such as molecular weight by SDS-PAGE, amino acid analysis, N-terminal sequence, and peptide mass mapping and some internal sequencing were performed on all the separated zymogens and activated enzymes. The structural homology of trout gastric zymogens with pepsin, gastricsin and chymosin was evaluated. Zymogens I, II and III appeared to be more pepsin-like, while zymogens V and IV appeared to be more gastricsin-like.