Study On The Immobilization Of Aspergillus Niger Lipase Onto Magnetic Metal-biomolecule-frameworks (BioMOFs) And Its Application

Enantiopure epoxides have been proven to be high additional value intermediates in organic synthesis,it has been widely used in medicine,pesticide,fine chemical and food industry.It can be used as a raw material for the synthesis of many valuable compounds,such as optically active materials,chiral drugs and pesticides etc.Among various synthetic methods,epoxidation of olefins catalyzed by lipases became a new research hotspot in recent years,because it has the advantages of high efficiency,economy,green,mild reaction conditions and high enantioselectivity etc.However,the free ANL has some drawbacks,such as unstable,easy to be deactivated,high cost,difficult to recover and reuse,which limits its application.In order to solve the above problems to improve the stability of the enzyme and reduce its catalytic cost,this study uses magnetic metal-biomolecule-frameworks?ZnGlu-MNPs?as a carrier for immobilization of Aspergillus nige lipase,which were structured by a biocompatible metal organic framework material?ZnGlu?and Fe₃O₄nanoparticles?MNPs?,and then,this immobilized Aspergillus nige lipase was used to catalytic the epoxidation reactiion of cyclooctene.In this dissertation,the immobilization of ANL on ZnGlu-MNPs and the epoxidation of cyclooctene catalyzed by the prepared ANL@ZnGlu-MNPs were for the first time investigated.Moreover,this study rationally designed and prepared a novel nano enzyme preparation?ANL@ZnGlu-MNPs?with high activity,good stability,high enzyme loading and convenient separation and recovery,which is helpful to rich immobilization technology of enzyme and increase the the researchers' understanding of the intereaction of enzymes and carriers.Additionally,this study also provides a new ideas to preparation of enantiomeric purity cyclooctene oxide.In this study,MNPs were firstly synthesized via the conventional co-precipitation method.Then MNPs were coated by ZnGlu via simple electrostatic-self-assembly technique to prepare magnetic metal-biomolecule-frameworks?ZnGlu-MNPs?.Then the scanning electron microscopy?SEM?,fourier transformed infrared spectroscopy?FTIR?,X-ray diffraction?XRD?,thermogravimetric analysis?TGA?,vibrating sample magnetometer?VSM?were applied to characterize the structure and composition of ZnGlu-MNPs.The results indicated that the ZnGlu-MNPss were successfully prepared and had good magnetic performance.Next,ANL was immobilized onto ZnGlu-MNPs via acylation reaction between amino groups of ZnGlu and free carboxyl group of ANL,and a novel magnetic nanoscale immobilized enzyme?ANL@ZnGlu-MNPs?with high activity,high stability and easeseparation and recovery was successfully prepared.The effects of different immobilization conditions on the activity recovery and of ANL@ZnGlu-MNPs were optimized.The results indicated that,the optimal conditions are as following: enzyme/support mass ratio of 7:10,immobilization pH of 7.0,immobilization time of 3.5 h.And under the optimal conditions,the protein loading of 118.0 mg/g and enzyme activity recovery of 82.0%.The characteristics of free and immobilized ANL were investigated using p-nitrophenyl palpitate as a model substrate.The results indicated that,the optimum pH of free ANL and ANL@ZnGlu-MNPs were 6.0 and 7.0,respectively;the optimum temperatures of free ANL and ANL@ZnGlu-MNPs were both obtained at 35?.In addition,the ANL@ZnGlu-MNPs was superior to its free counterpart in terms of thermal stability,pH stability and organic solvent tolerance,due to the increase of structural rigidity and the decrease of conformation variability of ANL after enzyme immobilization.Moreover,the kinetic study showed that the catalytic efficiency?Vmax/Km?of ANL@ZnGlu-MNPs was lower than that of free ANL(0.018min^-1 vs 0.014 min^-1).These results demonstrated that the ANL immobilized on the surface of ZnGlu-MNPs had improved adaption of pH and temperature,higher stability and higher catalytic efficiency.In this study,the epoxidation of olefins catalyzed by the as-prepared ANL@ZnGlu-MNPs was performed.Among all the selected olefins,satisfactory result was obtained at epoxidation of cyclooctene,and the yield is above 50%.The effects of different conditions on epoxidation of cyclooctene catalyzed by ANL@ZnGlu-MNPs were optimized.The results indicated that,the optimal substrate concentration of 150 mM,the optimal temperature of 40°C,the optimum enzyme adding amount of 100 mg,the optimum H2O2 dosage of 4.4 mmol.Under the above optimal conditions,the initial reaction rate,the product yield and product e.e.value was 16.3 m M?h,56.8% and 84.1%,respectively.Besides,ANL@ZnGlu-MNPs retained over 51.6% of its initial activity after 10 cycles of reuse,and the regioselectivity ANL@ZnGlu-MNPs showed almost no significant effect by its reuse.