| Lipase was a kind of biological catalyst, which had good catalytic property. However,free enzyme(FL) was easy become inactivation because of its poor stability. Enzyme immobilization was a good way to improve enzyme catalytic capacity and extensive the range of industry application, which including the research of new enzyme immobilization materials and the design of the immobilization schemes. In this work, a new method of enzyme immobilization was designed base on the bionic structure of plant cell. Lipid coated lipase(LL) was used as the core solution of the coaxial electrospinning machine, and sodium alginate was used to coated on the surface of liposome. Porous gelatin microcapsule with liposome-encapsulated lipase(GLL) was prepared after calcification.In this paper, ethanol was used as the organic solvent to prepare homogeneous solution for the accurately measurement of lipase activity. Lipase catalytic activity in this system was tested and the condition for lipase activity measurement was optimized. For the purpose of establish a bio-mimetic microenvironment liposome was use to capsule lipase. LL with proper size was prepared and lipase activity in LL was also measured. As far as our research, there had no report on the application of coaxial electrospinning technic in lipase encapsulation. In this paper, coaxial electrospinning technic was used to coat the alginate calcium gel on the surface of LL. Encapsulation effect of the microcapsule was used to optimize prepared parameters. In order to research the release property of porous gel, FL directly encapsulated in alginate calcium gel(GL) was also prepared as control group. The optimum catalytic condition and the enzymatic property of FL, LL, GL and GLL was researched respectively.Furthermore, thermal stability, p H stability and reusability of these systems were researched.Based on the determination of free and immobilized lipase catalytic activity and the theories of fluid flow rule and mass transfer kinetics, the models of surface adsorption lipase and internal particles embedding lipase on or in microspheres was established respectively.The results shown, ethanol was a kind of good organic solvent to prepare homogeneous water-based solution. This test method could correctly determined lipase activity in micro-pores. Under the optimum p H of the catalytic condition for FL was 5.5 and the temperature was 45 ℃ with the reaction time of 5min. This reaction satisfied with Arrhenius-equation and the activation energy value was 31.72 k J/mol. The catalysis property of free lipase according with Michaelis-Menten equation with the Vm value of 1.99±0.116 U.mg-1 and Km value of 5.17±0.176 m M.In order to achieve high efficient of enzyme immobilization, the property of liposome was researched. The preparation condition of liposome with the size of 100 nm was list as follow: total lipid concentration of 2.0 mg/ml, lecithin/cholesterol mass ratio of 4:1, and hydration time for 3 h. LL encapsulate efficiency(EE) of liposome was 81.27 % under optimum preparation condition. What’s more, the optimal catalytic condition(T:50℃,p H:6.5) of immobilized lipase was differ from free lipase. Under this condition, the relative activity of immobilized lipase was 165 %.GL and GLL microcapsule was prepared via coaxial electrospinning technic, theparameters of the preparation process were listed as follows: total lipid content of 2.0 mg/m L,ALG-Na concentration of 2.0 wt %, Ca Cl2 concentration of 0.10 mol/L, voltage of 8 KV,core-shell velocity ratio of 1:2(Vi: Vo). Under this method, the EE value of GLL was 57.45%,and the EE value of GL under the same condition was 63.42 %. The optimal catalytic environments of LL, GL and GLL compared with FL were changed. Their thermal stability,p H stability and storage stability were better than that of the FL. The residual activity of FL was less than 20 %, while the residual activity of GL and GLL was respectively 40 % and50 % in 65℃ for 1 h. What’s more, the storage stability of the immobilized enzyme was also improved. As for storage time of 30 days, the remaining activity of FL was less than 5 %, this value of LL, GL and GLL were 28 %, 46 % and 67 %, respectively. GL and GLL have reusability, GL and GLL residual activity was 47 % and 7 2 % respectively after 10 repetitions.Based on the measurement release rate of enzyme from porous gels, the reality enzyme molecule activity was 85 % and 92 %, respectively.The diffusion coefficient of the designed homogeneous solution was 4.13×10-13 m2/s.Under the condition of external diffusion effect only considered, the catalytic efficiency factor(outer diffusion efficiency factor,ηout) of lipase immobilized on the surface of microspheres was 41.6 %. On the contrary, the catalytic efficiency factor of lipase encapsulated in the nanoparticles of microspheres( η in) was 96.7 % under the condition of considering the diffusion effect. The embedding of the catalytic enzyme activity was 1.72×10-2 and the type of this reaction meet zero order reaction. This theoretical calculation could provide theoretical basis for the industrial application of immobilized lipases.In conclusion, coaxial electrospinning technic was an effective method for liposome encapsulation. According to the research result of this paper, encapsulated lipase with high affection was prepared after combine with appropriate materials. What’s more, the new measuring method of lipase activity has the advantages of accuracy, easy operation and the test area was wide. Moreover, the theoretical analysis of immobilized lipase catalyze models may provide theoretical basis for lipase industrial application. |
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