| In this work, Candida rugosa lipase was encapsulated via a two-step procedure including surface vinylation and redox polymerization, and its catalytic properties in aqueous solution and organic phase were investigated. Then the influence of different ligands inducing encapsulated lipase on the hydrolysis activities was researched after the immobilization of lipase with the substrate induction.The preparation of encapsulated lipase:at first, vinyl groups on the lipase surface were generated by a mild vinylation with N-acryloxysuccinimide (NAS) in alkali buffer. When the mole ratio of lipase and NAS was about 1:114 and the modification time was 45 min, the maximum degree of vinylation=94.8% was obtained. Then the vinylated lipase was encapsulated into polyethyleneglycol composite material by redox polymerization with the monomer including PEG200-dimethacrylate ester (A), PEG200-diacrylate ester (B), PEG400-dimethacrylate ester (C), and PEG400-diacrylate ester (D), and corresponding encapsulated lipase was produced. The proportion of reagents was the critical factor to the effect of encapsulation. And under the optimum conditions of lipase solution/monomer=2.5/1 (V:V), the best activity yield=41.6%, protein loading of four encapsulated lipase>96%.During the hydrolysis reaction, the activity yield of four encapsulated lipase (LIP-A, LIP-B, LIP-C and LIP-D) was 29.8%,32.3%,39.2% and 41.6%, respectively. Moreover, the encapsulated lipase exhibited higher pH, thermal and storage stability than the free form because of multipoint covalence connection to the carrier. In the condition of pH=2.4, pH=11.5 or 80℃, the residual activity of four encapsulated lipase was higher than 19.4%,42.0% and 10%, while the native lipase was almost entirely inactivation. Furthermore, the encapsulated lipase could retain above 22.2% and 17.5% of the original activity after immersed in the precipitation denaturant and dissolution denaturant for 30 d. And the activity of the encapsulated lipase remained more than 89.1% of its initial activities after storage for 60 days in 4℃。 In the esterification reaction, the optimum temperature of LIP-D was 40℃. Under this temperature, the esterfication reached the balance after 24 h and the conversion rate of LIP-D was 37.4%. The water could effectively improved the esterfication activity of LIP-D, and under the conditions of water content=2%(V/V), the maximum conversion rate of encapsulated lipase was gotten, while the activity reached up to 1.29-fold of the waterless form. LIP-D retained 91.25% of its initial activity after five recyles.The activities of encapsulated enzymes with substrate induction had been improved in the different extents, and the activity yield was significantly increased to 161.4% and 237.3% of non-substrate form with the existence of lauric acid +CAB35 or lauric acid+imidazoline. What's more, the substrate induction could also enhance the immobilized enzyme' stabilities of pH, temperature and storage. In the condition of strong acid(pH=2.4) and strong alkaline (pH=12.0), the encapsulated lipase with lauric acid +CAB35 inducement showed the best residual activity as high as 71.6% and 72.1%. And the encapsulated lipase with lauric acid inducement exhibited good stability i.e. the residual activity retained 39.7% under 100℃, and the 60 days storage of this enzyme revealed only 2.9 percentage point decrease in the residual activity. |
PDF Full Text Request