Trypsins, fish

Attempts have also been made to exploit the relatively high molecular activity of cod trypsin at low temperatures by incorporating the enzyme into herring "fermentations that proceed at 10°C. The preparation of brine-fermented round herring (matjes) is limited to certain seasons because of the balance of digestive enzymes in the fish at this time. Other studies have indicated that proteinases are important components in matje fermentation 41),... 

The pH-stat method can be modified and applied to a particular enzyme or substrate to assay any enzyme/substrate combination. The substrate can come from any source of protein such as poultry, milk, soybean, or fish processing byproducts. The amount of protein in the reaction should not exceed 8% (as calculated in Basic Protocol 1). The enzyme can be any alkaline endopeptidase such as Alcalase, trypsin, or chymotrypsin, and should be used in the proportions indicated in Basic Protocol 1. The selection of the appropriate enzyme depends on its efficiency and cost. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Thermal Stability. Increased catalytic efficiency at low temperatures is associated often with a decrease in thermal stability (41). Proteolytic enzymes from the pyloric caeca of cod, herring, and mackerel are more heat labile than bovine trypsin (42) the thyroid protease of burbot from cold waters had lower thermal stability than that from burbot in a warmer habitat (43). The half-life of myofibrillar ATPase at 37°C averages 1 min for the enzyme from Antarctic Ocean fish species, 70 min for the enzyme from fish species in the Indian Ocean, and 600 min for the enzyme from East African hot spring fish (33). 

Substrate Affinity. Enzyme-substrate affinity, as estimated by the Michaelis constant (Km), is affected also by habitat temperature (37). In many cases, the temperature range at which the Michaelis constant is minimal coincides closely with the temperature of the organism s native environment. Wiggs (43) observed that the Km of thyroid protease from burbot is lower for winter fish than for summer fish. Hofer et al. (44) also showed that the Km of trypsin from various species, including fish, correlated with the temperature preferendum of each species. The response of Km to temperature appears to relate to the balance of electrostatic and hydrophobic interactions of the active site with substrate and intramolecular bonds within the enzyme (38,39,45,46,47). 

Dissolution and Separation by Special Applications Enzymes and Microextraction Several enzymes such as trypsin, protease type XIV, lipase and/or cellulase, are used for enzymatic hydrolysis. For the determination of Hg2+ and Me-Hg in fish... 

Such chemical changes may lead to compounds that are not hydrolyzable by intestinal enzymes or to modifications of the peptide side chains that render certain amino acids unavailable. Mild heat treatments in the presence of water can significantly improve the protein s nutritional value in some cases. Sulfur-containing amino acids may become more available and certain antinutritional factors such as the trypsin inhibitors of soybeans may be deactivated. Excessive heat in the absence of water can be detrimental to protein quality for example, in fish proteins, tryptophan, arginine, methionine, and lysine may be damaged. A number of chemical reactions may take place during heat treatment including decomposition, dehydration of serine and threonine, loss of sulfur from cysteine, oxidation of cysteine and methio-... 

Romarheim, O.H. M.A. Aslaksen T. Storebakken A. Krogdahl A. Skrede. Effect of extrusion on trypsin inhibitor activity and nutrient digestibility of diets based on fish meal, soybean meal and white flakes. jArch. Anim. Nutr. 2005, 59, 365—375. 

Digestive serine proteases are present in the pyloric ceca, the pancreatic tissues, and the intestines of animals and have been reported in numerous species of Archaea (Eichler, 2001). Serine proteases are inactive at acidic pH and have high activity under neutral to slightly alkaline conditions (Simpson, 2000). Although fish serine proteases are quite similar to their mammalian counterparts, they have been reported to be more active under alkaline rather than neutral conditions (Shahidi and Janak Kamil, 2001). Some of the most well-known serine proteases from marine sources include trypsins, chymotrypsins, collagenases, and elastases. 

Fish Hoki skin gelatin Hydrolysis with trypsin HGPLGPL Mendis et al. (2005)... 

Trypsin, chymotrypsin, and cathepsin C (all from bovine), and the synthetic substrates glycyl-L-phenylalanine p-naphtylamide (GPNA), 4-phenylazo benzyloxy-car-bonyl-pro-leu-gly-pro-D-arg (PZ-peptide), N-benzoyl-L-tyrosine ethyl ester (BTEE), and Na-benzoyl-DL-arginine p-nitroanilide (BAPNA), were purchased from Sigma Chemical Co (St. Louis, MO). Fresh bluefish (Pomatomus saltatrix) and sheephead samples were purchased from a local fish market (Waldman Plus, Montreal, PQ) and kept in iCe until ready for use. 

The crude fish enzyme extracts were prepared as per the method of Baranowski et al. (1984), and stored in ice for use in the pressure treatments and subsequent enzyme assays. The spectrophotometric methods of Hummel (1959) and Erlanger et al (1961) were used to assay for chymotrypsin-like and trypsin-like enzyme activities using BTEE and BAPNA as substrates, respectively. Cathepsin C activity was assayed using gly-phe-NA as substrate (Lee et al, 1971), and collagenase activity in the fish extracts were assayed as per the method of Wunsch and Heidrich (1963). Protein content of the crude enzyme extracts from fish was determined by the method of Hartree (1972). 

Figure 2. Hydrostatic pressure inactivation of proteases from fish and mammalian sources (a) => cathepsin C (b) => chymotrypsin and (c) => trypsin.

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