Characterization of proteases from stomachless marine organisms and their potential use in the food industry.

摘要

A trypsin (EC.3.4.21.4) and a trypsin-like enzyme were purified, respectively, from the pyloric ceca of mullet (Mugil cephalus) and Louisiana swamp crayfish (Procambarus clarkii) by chromatographic procedures.; Mullet trypsin catalyzed the hydrolysis of trypsin-specific synthetic substrates n-{dollar}{bsol}alpha{dollar}-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME). It was inhibited by phenyl methyl sulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), aprotinin, and benzamidine. Its molecular weight obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis was 24 kilodaltons (KD).; When compared to bovine trypsin, mullet trypsin was more stable at alkaline pH (7.5-9.0) than at acid pH. It was relatively unstable to temperature (30 min at 75{dollar}{bsol}sp{bsol}circ{dollar}C) and lost almost all of its activity, whereas bovine trypsin maintained all of its activity. Mullet trypsin was more efficient for amidase, esterase and protein hydrolase reactions and had a efficient for amidase, esterase and protein hydrolase reactions and had a higher substrate turnover number and a higher physiological efficiency (V{dollar}{bsol}sb{lcub}{bsol}rm max{rcub}{dollar}/K{dollar}{bsol}sb{lcub}{bsol}rm m{rcub}{bsol}sp{bsol}prime{dollar}) for the amidase reaction than bovine trypsin. Both trypsins were equally efficient in increasing resistance of milk to copper-induced lipid oxidation; however, mullet trypsin, but not the bovine form was totally inactivated by pasteurization.; Crayfish protease had tryptic-like activity in that it hydrolyzed ester bonds involving the carboxylic groups of arginine and lysine. It was also inhibited by PMSF, tosyl-L-lysine chloromethyl ketone (TLCK), SBTI, aprotinin and benzamidine. Two prototropic groups of apparent pKa's of 6.5 and 8.5 were found to be involved in the active site. These were similar to those of the amino acids histidine and serine. When TAME was used as substrate, the protease was stable between pH 7.5 and 9.0, but lost 50% of its activity at pH 5.0. The enzyme lost 50% of its activity upon heating for 30 min at 55{dollar}{bsol}sp{bsol}circ{dollar}C. The enzyme had a molecular weight of 33.6 KD and an isoelectric point of 3.0.; Crayfish protease (0.17 {dollar}{bsol}mu{dollar}g) inactivated commercial pectinesterase (1 PE unit) by 90% after 1 hour incubation at 4{dollar}{bsol}sp{bsol}circ{dollar}C, pH 7.0. It also inactivated 70% of PE in fresh squeezed orange juice, pH 4.0 at 4{dollar}{bsol}sp{bsol}circ{dollar}C after 1 day incubation when used in a ratio of 0.68 {dollar}{bsol}mu{dollar}g crayfish protease to 1 pectinesterase unit. Similarly, cloud stability was maintained for three more days in the orange juice as compared to untreated control.