The Effect Of Nano-silica Particles Interposed In The Enzyme Activity, Casein Phosphopeptides Preparation And Bioactivity Protection

Native enzyme was limited in industrial processes as regard to the problem of its instability and inactivation as reacting in harsh environments. Many researchers had manifested that immobilized enzyme had great advantages over the native ones in the aspect of improved stability. Although the attachment of enzyme onto traditional micro-materials was more stable, they were subject to loss much activity and enzyme loading per unit mass of support was considerably low. The nanoparticles would provide the upper limits in terms of balancing the contradictory issues, including high retentive activity and effective enzyme loading. This problem could be solved by immobilizing enzyme on nanoparticles. The method could be realized in mild conditions and the process was easy to conduct, so it was of great importance to industry process.In our study, silica nanoparticles were used as support materials. Firstly, we investigated the effect of the adsorbing pH, enzyme/silica ratio and particle size of silica on the properties of immobilized enzymes (trypsin, papain,α-Amylase). Secondly, we chose trypsin as model enzyme for further study of kinetic constants after immobilization, and then used the immobilized and native trypsin to prepare casein phosphopeptides (CPPs) under different reaction conditions and compared the yield of CPPs. Finally, this nano-absorption technology was also used for the protection of CPPs to investigate the protective consequents of the nanoparticles on CPPs by measuring its ability to prevent calcium phosphate precipitation. The results obtained in our study were listed as follows:(1) The adsorbed amount of enzymes on silica was affected by the solution pH and particle size of silica, and was relative highly when the adsorbing pH was between pI of silica and enzyme. The adsorbing conditions for this study were: enzyme/silica = 1:1, and adsorbed for 3 h at room temperature. The optimal adsorbing pH for trypsin, papain andα-Amylase was pH 7.8, pH 6.0 and pH 6.0, respectively. The corresponding adsorbed amount on 20 nm silica was 69.0 mg/100mg SiO₂, 87.3 mg/100mg SiO₂ and 76.9 mg/100mg SiO₂, respectively. Under the same conditions, the adsorbed amount decreased with the increase of silica size. (2) The particle size of silica had significantly impacted on the relative activity of immobilized enzyme, which decreased with the increase of particle size. The relative activity of trypsin adsorbed on micro-silica, 300 nm, 100 nm and 20 nm silica was 40.7%,46.7%,56.3% and 61.4%, respectively. The corresponding values for papain was 47.5%,54.1%,58.8% and 73.3%, respectively. Forα-Amylase was 57.5%,61.7%,66.0% and 68.1%, respectively. In addition, the pH and thermal stabilities of the three enzymes were enhanced to different degrees and this degree was enhanced with the decrease of particle size. After the treatment of 90℃for 1 h, the residual activity of 20 nm and micro-adsorbed trypsin had increased by 19.3% and 41.6% compared with that of native ones, respectively. After the treatment of pH 2.0 for 1 h, the corresponding values forα-Amylase were 2 times and 2.8 times compared with that of native ones, respectively.(3) The surface hydrophobicity (H0) of immobilized trypsin, papain andα-Amylase had increased to different degrees. The H0 values of enzyme adsorbed on micro-silica was higher than that of adsorbed on nano-silica when adsorbing at the same conditions. After adsorbing for 3 h, the H0 values for micro-adsorbedα-Amylase and 20 nm-adsorbedα-Amylase had increased by 18.7.% and 10.8%, respectively. The corresponding values for papain were 7.2% and 4.2%, respectively. For trypsin were 25.5% and 10.5%, respectively. Overall, after adsorption on 20 nm silica particles, papain showed the least change in surface hydrophobicity, and the corresponding relative activity 73.3% was the highest among the three enzymes, so the fraction of activity lost correlated well with the unfolding content of enzymes.(4) Casein phosphopeptides (CPPs) were prepared from casein hydrolyzed with native or immobilized trypsin. In the strong acidic (pH 2.0⁵.0), strong alkaline (pH 11.0¹2.0) and high temperature (60℃⁸0℃) reaction conditions, the yield of CPPs prepared by immobilized trypsin was significantly higher than that of prepared by native ones, and 20 nm-silica immobilized trypsin was superior to mirco-ones. At pH 2.0, the yield of CPPs prepared by micro-adsorbed and 20 nm-adsorbed trypsin had increased by 2.9 times and 5.5 times compared with that of native ones. At 80℃, this value was 62.9% and 35.0%, respectively.(5) Adsorption of trypsin on silica particles had significant effect on their kinetic constants. When exposed to harsh reaction conditions (such as pH <5.0 or the temperature was higher than 60℃), immobilized trypsin showed great advantage over native ones. The structure of enzyme could be enhanced through adsorption, especially when enzyme was adsorbed on nanoparticles. This method could make enzyme maintain a relative strong affinity to the substrate and high speed of reaction in harsh environments. At 80℃,pH 7.8, the Vm of micro-adsorbed trypsin had increased by 13.35%, while the Km had decreased by 8.81% compared with that of native ones. The corresponding values for 20 nm-adsorbed trypsin were 23.61% and 13.09%, respectively. At pH 2.0,37℃, the Vm of micro-adsorbed trypsin had increased by 32.76%, while the Km had decreased by 32.26% compared with that of native ones. The corresponding values for 20 nm-adsorbed trypsin were 37.14% and 47.82%, respectively.(6) Protective effects of nanoparticles could not only act on the enzyme molecule, but also on the peptides. After acid or high temperature treatment on native and adsorbed CPPs, the ability of preventing the calcium phosphate precipitation was placed in the order as follows: nano-silica adsorbed CPPs> micro-silica adsorbed CPPs> native CPPs. After the treatment at pH 3.5 for 3 h, the time of inhibiting calcium phosphate precipitation by the micro-adsorbed and 20 nm-adsorbed CPPs had increased by 14.2% and 36.8% compared with that of native ones, respectively. After the treatment of 100℃for 1 h, these corresponding values were 8.5% and 34.2%, respectively. These results indicated that the conformation of CPPs could be maintained or protected by adsorption on support materials when CPPs were treated in harsh environments. And this protective effect was more eminent when the support material was of smaller particle size.