Immobilization Of Several Lipases And Their Application

Both Aspergillus niger lipase(ANL)and porcine pancreatic lipase(PPL)have Sn-1,3-bit specificity and have broad'application prospects in various industrial fields including oil processing.In this study,the immobilized enzyme was prepared based on the optimized Aspergillus niger lipases ANL1,ANL2 and porcine pancreatic lipase PPL which were applied in previous experiment.The optimal immobilized carriers and methods,the best immobilization conditions,and enzymatic properties were studied.Then the immobilized enzyme was used to catalyze the transesterification of fish oil to enrich EPA and DHA.The results are as follows:1.Nano-magnetic nanoparticles(MNPs)were synthesized by chemical coprecipitation.The nanoparticles were coated with SiO2 by Storber method and then aminated with APTES.A-MNPs was characterized by XRD,FT-IR,TEM and TG,which proved that the magnetic nanoparticles were successfully prepared.Using A-MNPs,macroporous resin and chitosan as carriers,ANLI,ANL2 and PPL were immobilized by adsorption,cross-linking and covalent bonding methods.Using the enzyme activity recovery rate as an indicator,it was determined that the A-MNPs was used as the carrier,and-CHO covalent bonding method had the best immobilization effect.The enzyme activity recovery rates were 46.42%,45.67%,and 47.63%,respectively.2.A-MNPs were used as carriers and-CHO covalent binding method was applied to prepare immobilized enzymes.Then Five factors and four levels of orthogonal experiments were designed for optimization.The results are as follows:Immobilized ANL1:The concentration of glutaraldehyde was 3%,the enzyme amount was 4 mL,immobilized pH was 8.0,immobilization temperature was 30 ?,immobilization time was 2 h,and the enzyme activity recovery rate was 49.58%;Immobilized ANL2:The concentration of glutaraldehyde was 3%,the enzyme amount was 2.5 mL,immobilized pH was 9.0,immobilization temperature was 25?,immobilization time was 1.5 h,and the enzyme activity recovery rate was 50.13%;Immobilized PPL:The glutaraldehyde concentration was 4%,the enzyme amount was 2.5 mL,the immobilization pH was 7.0,the immobilization temperature was 40?,and the immobilization time was 3 h,and the enzyme activity recovery was 5 1.30%.3.The enzymatic properties of immobilized ANL1,ANL2,and PPL were studied.The optimum temperature of immobilized ANL1,ANL2 and PPL was 50 ?,which was 10 ?higher than that of free enzyme.The optimum pH was 7.0,6.0,and 7.0 respectively,which were consistent with that of free enzyme.The temperature stability was significantly improved.After storage at 70 °C for 2 h,the residual enzyme activity of immobilized ANL1 was 65.98%,whereas that of free ANL1 was only 41.16%.When stored at 60 °C for 2 h,the residual enzyme activities of free ANL2 and PPL were 24.19%and 36.22%,respectively.The immobilized ANL2 and PPL reached 54.89%and 53.23%,respectively.The pH stability was also significantly improved,and the residual enzyme activities of free ANL1 and ANL2 were 79.92%and 80.31%,respectively,and the immobilized ANL1 and ANL2 were 93.90%and 92.09%,respectively.The immobilized PPL was improved on the basis of good stability of the free enzyme.The activation energy of immobilized ANL1,ANL2 and PPL was lower than that of free enzyme,and the storage stability was good.4.The immobilized ANL1,ANL2 and PPL were used as catalysts to catalyze the transesterification of fish oil to produce high EPA and DHA glyceride fish oil products.The results showed that the total EPA and DHA content of glyceride-type fish oil obtained by immobilized ANLD was 48.68%.The total content of EPA and DHA catalyzed by immobilized ANL2 was 44.08%.The total content of EPA and DHA catalyzed by immobilized PPL was 47.20%.After repeated use for 6 times,the enzymatic activity of immobilized ANL1,ANL2,and PPL remained around 50%,and the reusability of the immobilized enzyme was good.