Characterization Of Like-Cystatin From Silver Carp Egg And Its Bactericidal Action To Pseudomnas J-4 From Refrigeration Silver Carp Sliced Meat

In this study, four low molecular weight cysteine proteinase inhibitors (CPIs) were purified from silver carp egg and their biochemical properties were characterized. Moreover, the antibacterial effects of the low molecular weight CPIs to prevent the growth of pseudomonas, the dominant spoilage organisms in fish during cold storage, were investigated.The ultrafiltrated and concentrated extract of CPIs from silver carp egg were used for the purification by Octyl Sepharose 4 Fast Flow chromatography. Four active peaks, peak III, IV, V and peak VI, were obtained and made for further identification.Molecular weight identified by electrophoresis:For the reduction Tricine-SDS-PAGE, peak III gave two bands of 17.5 KDa and 6.3 KDa, and named 17.3 KD band Cystatin-I. Peak V gave two bands of 19.9 KD and 8.2 KDa, and then named 19.9 KD band Cystatin-II; Peak IV and VI gave 14.5 KDa and 14.2 KDa, separately.Inhibition stripes identified by electrophoresis:Comparison of three electrophoretic methods:Geltain-substrate-SDS-PAGE method (method A), Native-PAGE reverse zymography method (method B) and SDS-PAGE method after reaction with papain (method C). And the final selection of papain reaction after SDS-PAGE electrophoresis inverting enzymes was identified. The amount of Cystatins was 2.5μg, and that of papain (6.05 μmol/L) was 0.625 μg. After their reaction at 37℃ for more than 5 hours, Tricine-SDS-PAGE electrophoresis was made to identify the inhibitors stripes. The results showed that all the stripes from Cystatin-I to Cystatin-IV were cysteine protease inhibitor.Purity and hydrophobicity identified by RP-HPLC:C18 reversed-phase HPLC was used to identify the purity of Cystatin-I, Cystatin-III, Cystatin II, and Cystatin-IV. The results indicated that Cystatin-I(peak area:93.7%,10% trailing factor:1.48), Cystatin-II(peak area:93.5%,10% trailing factor:1.941), Cystatin-III (peak area:82.37%,10% trailing factor:0.841) all exhibited a high purity single peak at 14.2 min. At the same time, Cystatin-III exhibited a high hydrophobic single peak at 92.3 min(peak area:15.5%,10% trailing factor.0.891). However, Cystatin-IV exhibited such peak at 92.3 min(peak area:93.1%,10% trailing factor:1.074).Peak IV was applied to RP-HPLC for several times, the results identified that the hydrophobic property was not stable and the peak time was moved from 92.3 min to 14.2 min slowly. Then, the protein in two peaks was recycled, and the activity was detected. The results showed that the inhibitory activity descended as the peak time shortened, the activity degraded over time and lost almost half during 14.2 min.Identification of Cystatin-Ⅱ’s bioactivity properties:Because of the highest purity and activity, Cystatin-Ⅱ was used to identify its bioactivity properties. We found that Cystatins-Ⅱ was stable under a wide range of pH 3.0-pH 11.0, and at pH 11.0, it possessed 70% residuary activities. The thermal stability experiments showed that the inhibitive activities of Cystatin-Ⅱ were little damaged within the scope from 4℃ to 60℃. At 80℃, it could retain the inhibitive activities about 60%. Cystatins-II was a competitive inhibitor with inhibition constant of 0.1341 nmol when tested with the Dixon-plots method.Antibacterial effects on pseudomonas:The dominant spoilage organisms in fish was identified during storage at 4℃.The research showed that they were the gram-negative bacillus strains without spores, and did the biochemical identification by the oxidase enzyme, catalase, arginine dehydrolase experiments and oxidation of glucose fermentation experiment.They were positive bacillus strains. Further,16s rDNA sequence analysis showed that the strain and the registration number for NR024901, NR0433425, NR0433422 pseudomonas genus had as much as 99% of homology. The sequence was submitted to NCBI database, and we gained the login KF056821, in addition, phylogenetic tree further showed the strain belonged to pseudomonas genus. Then we named it pseudomonas J-4 in our lab.Using the titration of Cystatins-II, selected Azocasein as the substrate,45.375 mol papain could be fully inhibited by 7.57 μg Cystatins-Ⅱ, which is a unit of inhibitory activity.The nutrient broth experiments showed that when the the amount of Cystatin-Ⅱ was 135 units/mL, there were obvious bacteriostatic action, compared with 80 units/mL and 105 units/mL group. In the filter experiment, when 300 or 660 unit s/slice of the purified Cystatins-Ⅱ were added, the bacteriostatic circle appeared. The former bacteriostatic circle diameter was small and only 8 mm. Nevertheless, when adding the quantity of 660 unit/slice, bacteriostatic effect was the best and the circle diameter was the biggest, it could reach 15 mm.