Studies On Preparation Of Protease Inhibitors And Their Application In Gel Weakening Inhibition Of Alaska Pollock Surimi

Surimi is a minced and washed fish muscle consisting of salt-soluble myofibrillar proteins. The endogenous proteases in surimi cause an irreversible destruction of the gel structure of surimi and result in decreasing the quality of surimi-based product and its market value. Protease inhibitors have been used to prevent the degradation of surimi gel. Chum salmon egg and plasma were used as raw materials for preparation of protease inhibitors. A cystatin was purified from chum salmon egg with a series of acidic treatment, ultrafiltration and papain affinity chromatography. The recovery, specific inhibitory activity and purification fold were 0.4%, 1.54 U/mg and 192.6, respectively. Its molecular mass was 13 kDa based on the result of SDS-PAGE. Its N-terminal was determined to be N-Gly-Leu-Ile-Gly-Gly-Pro-Met-Asp-Ala-Asn-C, which was identical to that of chum salmon cystatin. A kininogen was purified from chum salmon plasma with one step of papain affinity chromatography. The recovery, specific inhibitory activity and purification fold were 0.94%, 3.84 U/mg and 30.36, respectively. Its molecular mass was 70 kDa based on SDS-PAGE and FPLC. Only under non reducing condition can this CPI be detected by inhibitory activity staining. The result of Periodic acid-Schiff staining showed that it was a glycoprotein.Chum salmon egg cystatin showed the highest inhibitory activity against papain. The inhibitory activity of chum salmon plasma kininogen against papain was higher than that of glassfish egg inhibitor I, pond smelt inhibitor and chum salmon egg inhibitor. For cathepsins L, chum salmon egg cystatin showed inhibitory activity only lower than that of glassfish egg inhibitor II. Chum salmon plasma kininogen showed higher activity than did glassfish egg inhibitor I and chum salmon egg inhibitor. Chum salmon egg cystatin and plasma kininogen were stable under a wide range of pH. Nearly no loss of its activity was determined under pH 5-8. Chum salmon plasma kininogen possessed higher than 60% activity at pH 6-9. 50% of chum salmon egg cystatin activity could be retained after heating for 30 min at 70°C, while 50% of that of chum salmon plasma kininogen could be retained. Chum salmon egg cystatin and plasma kininogen showed activity mainly at 20-50°C. The Dixon-plots showed that chum salmon cystatin was a competitive inhibitor with inhibition constant of 2.12 nM. Chum salmon plasma kininogen was a noncompetitive inhibitor with inhibition constant of 105.32 nM.Recombinant chum salmon cystatin (RC) was overexpressed in Saccharomyces cerevisiae YPH 499 incorporating pYES2NT/C vector. After cultivation and induction, RC could be purified by nickel select affinity chromatography or alcohol precipitation. The recovery, specific inhibitory activity and purification fold for nickel affinity chromatography were 61.23%, 7.45 U/mg and 5.60, respectively. Molecular mass of RC was determined to be 35 kDa based on the result of SDS-PAGE and interlinking of recombinant cystatin was supposed to be taken place for uncertain reason. RC showed lower activity and acidic stability, but higher thermal stability than that of natural chum salmon egg cystatin. The culture condition for the production of RC by S. cerevisiae YPH 499 was optimized in shake flasks using Response Surface Methodology (RSM). The highest amount of RC production in shake flask, 0.57 U/mL, was obtained at 5.7 of medium pH, 6.7 h of inducing time, and 5.6 g/L of inducing assistant, which was consistent with the result of validation experiment. Thereafter, based on the results of shake flask, the effects of agitation and aeration rates on RC production by S. cerevisiae YPH 499 were determined for scale-up in fermentor. The highest RC production in fermentor, 0.56 U/mL, was obtained at 350 rpm of agitation rate and 1.0 vvm of aeration rate, which was consistent with the result of validation experiment.RC was added to Alaska pollock surimi to determine its inhibition of gel weakening. RC purified by affinity chromatography at 100μg/g showed the highest inhibition rate of 90% against the autolysis of surimi. The addition of RC resulted in increase of 4.5 folds gel strength and 30% of deformation. The expressible drip of surimi gel decreased from 17.85% to 6.27% and whiteness increased from 49.78 to 52.60 at 100μg/g of RC. The degradation of myosin in surimi gel was significantly inhibited after addition of RC based on the result of SDS-PAGE. RC prevented the degradation of proteins in Alaska pollock surimi better than did egg white (EW). Crude RC purified by alcohol precipitation could also inhibit the gel weakening of Alaska pollock surimi. The gel strength and deformation increased 2.7 folds and 10%, respectively, at 500μg/g of crude RC. Therefore, RC could be applied to Alaska pollock surimi to prevent the gel weakening.