| In this study, two low molecular weight cysteine proteinase inhibitors (CPIs) were purified from silver carp egg and their biochemical properties were characterized. Moreover, the effects of the two CPIs to prevent gel weakening were investigated.First, the optimization condition of initial speed for the papain assay with the azocasein as substance was established. And the effective morgan concentration of papain(1.25mg/mL) was titrated as 6.05μmol/L with E-64. Base on it, the comparison of the inhibitive activities to papain of CPIs in silver carp egg (stage IV) and skin was conducted using both azocasein and Z-Phe-Arg-MCA. The result with azocasein showed that silver carp egg have higher total inhibitive activities (90.03unit/ml) than that in silver carp skin(26.96 unit/ml).But the specific activity of skin is 14.19unit/mg, higher than that of egg (3.98unit/mg); Inhibitive activity in egg homogenate could not be detected using Z-Phe-Arg-MCA as substance, although it was of higher sensitivity. The activities of CPIs in silver carp egg was extracted by way of homogenization, acidic treatment, ammonium sulfate fractionation, dialysis, ultrafiltration and concentration. During this stage, both the assay of azocasein and Z-Phe-Arg-MCA was carried out for monitoring the inhibitive activities. And it was found that after the acidic treatment, the inhibitive activities could be detected again with Z-Phe-Arg-MCA.The ultrafiltrated and concentrated extract of CPIs from egg was then used for the purification by chromatography. During this period, the inhibitive activity was assaied using Z-Phe-Arg-MCA. Four active peaks (Ⅰ,Ⅱ,Ⅲ,Ⅳ) were obtained after Sp Sepharose Fast Flow cation-exchange chromatography. Peak I and peak III with higer inhibitive activity and lower absorbtance at 280nm were selected for the next step.CPI in peak I was purified by way of Sephcryl S-100 gel filtration and LH-20 reversed phase chromatography orderly. And the result showed that CPI-Ⅰwas purified to 25.25 folds and with 1.26% yield. CPI-I gave one band of 10.5KD on reduction SDS-PAGE, that was coincident with the preliminary judgement of 11KDa by the standard elution curve of SephacrylS-100. The result of reversed-phase enzymogram showed that the CPI-Ⅰhad the inhibitive activity to papain. Moreover, a trait of unglycosylation was characterized by the Periodic Acid-Schiff(PAS) staining. CPI-Ⅰwere stable under a wide range of pH 3.0-pH 7.0 and possessed 60% residuary activities, and below 60℃, it could retaine the inhibitive activities about 80%. CPI-Ⅰwas a competitive inhibitor with inhibition constant of 96.97 nM when tested with the Dixon-plots method.CPIs in peakⅢwas partly purified after a serial of chromatography of Blue Sepharose 6 Fast Flow dye affinity, Sephcryl S-200 gel filtration and ConA Sepharose4B affinity. The final results showed that CPI-Ⅲwas purified to 331.66 fold and 0.78% yield. The molecular weight of CPI-Ⅲwas estimated as about 14KDa by the method of standard elution curve of SephacrylS-200. Similarly two bands of 16.5KDa and 12KDa were observed on reduction SDS-PAGE. It was proved that the protein band of 16.5KDa in CPI-Ⅲwas of the inhibitive activity to papain by reversed-phase enzymogram. The result of Periodic Acid-Schiff staining showed that the CPI-Ⅲwas not glycoprotein.The experiment of CPIs preventing the weakening of surimi gel was performed. When the purified CPI-Ⅰand partially purified CPI-Ⅲwere added to the silver carp surimi at the same level of 3units/g (azocasein), it was observed that both CPIs had inhibited the weakening of surimi obviously. The addition of CPI-Ⅰand CPI-Ⅲled to the increase of 9.8%(p>0.05),115.6%(0.01<p<0.05) in breaking strength and 28.96% (p<0.01),32.36%(p<0.01) in gel strength of surimi respectively. Accordantly, the degradation of myosin in surimi gel was significantly inhibited after addition of CPI-Ⅰand CPI-Ⅲbased on the result of SDS-PAGE. Silver carp egg CPIs could be applied to silver surimi to prevent the gel weakening. |
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