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Purification and characterization of trypsin from the pyloric ceca of the new zealand hoki fish (macruronus novaezealandiae)

, : Purification and characterization of trypsin from the pyloric ceca of the new zealand hoki fish (macruronus novaezealandiae). Journal of food biochemistry 31(6): 772-796

Trypsin was purified from the pyloric ceca of hoki fish by (NH)SO fractionation, acetone precipitation and affinity chromatography. The purified trypsin migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the isoelectric point was determined as 6.5. The molecular weight was determined as 26,000 Da by SDS-PAGE and as 23,791 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. The enzyme was stable within the pH range of 6.0-11.0, and exhibited optimum activity for the hydrolysis of N-l-benzoyl- dl-arginine-p-nitroanilide ( dl-BAPNA) at pH 9.0. The enzyme was stable up to 40C, and its optimum temperature for dl-BAPNA hydrolysis was 60C, and was inhibited by soybean trypsin inhibitor, aprotinin, phenyl methyl sulfonyl fluoride and benzamidine. The apparent Michaelis-Menten constant (Km') and substrate turnover number (kcat) for the dl-BAPNA hydrolase reaction was 0.06 mM and 0.33 sp#, respectively, while the corresponding values for tosyl arginine methyl ester hydrolysis were 2.08 mM and 19.0 sp#, respectively. The N-terminal 20 amino acid residues of hoki trypsin, IVGGQECVPNSQPFMASLNY, displayed considerable homology with other fish trypsins. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Thus, it is expected that like other trypsins, hoki trypsin would also be useful in biomedical, food and beverage industry applications. Examples of the practical applications of trypsins include their use in mammalian cell culture to disaggregate adherent cells for research and the production of recombinant proteins; in cancer therapy to break down tumors and cancer cells; and in wound care, for treatment of acne and other skin disorders. Other uses are as a digestive aid, for treating pain, inflammations, arthritis, tendonitis, ulcers, hemorrhoids and navel infections in newborns. Trypsins are also used in diabetes diagnosis and therapy; in detergents and bating of leather; and in the food industry for the production of protein hydrolysates, as meat tenderizer and as an aid to improve yields in oil extractions.


DOI: 10.1111/j.1745-4514.2007.00141.x

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