| As catalysts derived from the organisms,enzymes have the advantages of high specificity,high catalytic efficiency,high products purity and mild reaction conditions.Nevertheless,enzymes still have drawbacks such as high cost,difficulty in separation and ease of inactivation in practical application.Most of enzymes require a proper solution environment for optimal catalysis,and extreme p H or temperature will generally result in reduced enzyme activity or even inactivations.In this thesis,to address the problems above,urease and glucose oxidase(GOD)were encapsulated in calcium carbonate microspheres by biomimetic mineralization strategy,which were capable of regulating acidic/alkaline microenviroment by catalyzing the corresponding substrates.The main research contents and results are as follows.1.Urease-encapsulated calcium carbonate microspheres were prepared by biomimetic mineralization strategy,which showed improved stability against acidic environment by catalyzing the hydrolysis of urea.The successful immobilization of urease and the presence of vaterite calcium carbonate were verified by FI-TR and SEM tests.It was found that the maximum immobilization efficiency of urease was above 70%with a maximum loading amount of 18 mg/g when the concentration of enzyme ranged from 0.16-1.12 mg/m L.The immobilized urease maintained higher relative activity compared to the free enzyme over a wider temperature range,and the relative activity also exceeds 20%under extreme conditions such as p H range of 3-4,which is higher than that of the free enzyme.The immobilized enzyme has a K_mconstant of 0.023 mol/L,which is lower than the free enzyme and has a better affinity to the substrate.After the immobilized enzyme was immersed in urea solutions at p H 2,3 and 3.5 for a certain period of time,all of the calcium carbonate microspheres carrier were not completely dissolved and the urease still retained part of activity.The microspheres retained 40%of the original weight after immersed in the p H=3solution for more than 120 min,while the non enzyme-loaded calcium carbonate microspheres were completely dissolved.Furthermore,40%of the original activity of immobilized enzyme was retained after immersed in the p H=3.5 solution for 240 min,while that of free enzyme was only 10%.2.GOD-encapsulated calcium carbonate microspheres were prepared by biomimetic mineralization strategy and dissolution of calcium carbonate microspheres in weakly acidic solutions(p H 5-6)was achieved in the presence of glucose.It was found that the maximum immobilization efficiency of GOD was 95%with a maximum loading ratio of 23 mg/g when the concentration of enzyme ranged from 0.25-1.25 mg/m L.The immobilized GOD possessed a higher relative activity than the free enzyme in the range of 20-70°C.The relative activity exceeded 20%at p H=3 and 80%at p H=8,both of which were also higher than that of the free enzyme.After the immobilized GOD was immersed in glucose solutions at p H 5-6 for a period of time,the calcium carbonate microspheres all showed a certain degrees of dissolution.After 120min immersion in the glucose solution at p H=5,the weight of remaining microspheres was only 70%of the original.3.Based on above results,a new method for preparing acid-resistant and glucose-sensitive calcium carbonate microspheres was developed.The urease and GOD co-encapsulated calcium carbonate microspheres prepared by the biomimetic mineralization strategy still had more than 40%weight of the calcium carbonate microspheres after 60 min of immersion in urea solution at p H=3.Followed,their weight was reduced by more than 50%after 60 min of immersion in glucose solution at p H=5,.This work has potential applications in the field of intelligent drug delivery system. |
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