Purification,Characterization And Application Of Two Aspartic Proteases From Golden Pompano Viscera

Golden pomfret is an important marine fish in southern China with tender meat,delicious taste and high nutritional value. In recent years, the market demand of Golden pomfret continues to rise. With the increase of processing scale, a large amount of wastes, such as fish head, bone and viscera are produced, accounting for about 40%-50% of the whole fish. These substances are ofen discarded, which not only pollute the environment but also make a waste of resource. The viscera is rich in protease,has a broad application prospects. At present, there are few studies on the exploitation and utilization of the visceral enzyme, therefore, the study of the internal organs of Golden pomfret is particularly important. In this study, two aspartic proteases of Golden pomfret viscera were collected and purified, and the enzymatic properties of these proteases were studied. The application in the extraction of macromolecular collagen, preparation of gelatin hydrolyzate and curd were discussed in order to provide reference for the development and application of Golden pomfret viscera. The results of the study were as follows:1. Isolation and purification of two aspartic proteases from Golden pomfret visceraThe first aspartic protease(AP-V) was isolated by pH 7.0 sodium phosphate buffer, purified by 0-55% ammonium sulfate fractionation, Sephadex G-100 and Sephadex G-50 gel chromatography from Golden pomfret viscera. The specific activity was 45.72U/mg using hemoglobin as substrate. Purification factor was 17.79.The recovery of activity was 20.95%.The second aspartic protease(AP-S) was isolated by pH 7.0 sodium phosphate buffer, purified by 0-40% ammonium sulfate fractionation, DEAE-Cellulose anion exchange chromatography and Sephadex G-100 gel chromatography from Golden pomfret stomach. The specific activity was 98.72U/mg using hemoglobin as substrate.Purification factor was 4.25. The recovery of activity was 21.5%.2. Enzymatic properties of two aspartic proteases from Golden pomfret visceraIn this paper, the molecular weight of two proteases were determined by SDS-PAGE. Secondly,the effects of pH, temperature, NaCl, inhibitors and metal ions were discussed using hemoglobin as the substrate. Finally, the hemoglobin hydrolysis capacity of two proteases was studied using the Michaelis-Menten costant as index.The study results showed that the molecular weight of purified enzyme from viscera(AP-V) and stomach (AP-S) was 18.5kDa and 36.1kDa, respectively. The optimum pH of AP-V and AP-S was 4.0 and 2.0, respectively. They are acidic protease.Compared to the AP-V, the stability of AP-S is better under acidic conditions. The optimum tempreature of AP-V and AP-S was 35 and 50℃, respectively. The results of stability experiments showed that both purified enzymes maintained high activity at 20-40℃. With the increase of NaCl concentration, the activity of AP-V gradually decreased, while the activity of AP-S was first increased after reduction. The results of protease inhibition showed that PMSF, EDTA and E-64 did not inhibit the activity of both purified enzymes, and pepstatin A had strong inhibitory effect on this two kinds of enzymes, indicating that the active centers of two kinds of enzymes contained aspartic acid, which are aspartic proteases. Na+、K+、Ca2+ and Zn2+ had very small effect on the activity of aspartic protease, and Mg2+ activated on the both enzymes, Hg2+ inhibited on both enzymes. The Km of AP-V and AP-S was 10.13 and 5.78g/L, respectively. AP-S has better ability to hydrolyze hemoglobin than AP-V.3. Application of two aspartic proteases from Golden pomfret visceraIn order to investigate AP-V and AP-S in the extraction of Tilapia fish scale collagen, two enzymes were used to extract the tilapia fish scale collagen, and porcine pepsin was as a control, the results showed that two kinds of protease were both type I -collagen. The collagen yield of AP-S was higher than AP-V, and was close to porcine pepsin.In order to investigate AP-V and AP-S in the preparation of gelatin hydrolysates,two enzymes were used to hydrolyze the tilapia fish skin gelatin, and porcine pepsin was as a control, the results showed that the proteolytic activity of AP-V was stronger than that of AP-S and porcine pepsin, suggesting that AP-V is more suitable for the preparation of small molecule collagen peptide.In order to investigate the application of AP-V and AP-S in dairy processing, we determined the milk coagulation activity of two aspartic protease, and compared with porcine pepsin. The results showed that the activity of curd activity of AP-V was not detected, while the activity of AP-S was similar to that of porcine pepsin. The results showed that AP-S could be used as a potential substitute for rennet.