Preparation And Catalytic Performance Of Secondary Immobilized Lipase In Supramolecular Assembly

Enzymes play an important role in the field of biocatalysis.The nature and state of the enzyme directly affect the efficiency and cost of the catalytic reaction.In the initial stage of biocatalysis application,the enzymes separated from organism are directly applied,which not only has high production cost,but also faces problems of difficulty in separation in the later stage.In order to adapt to the industrial application of the enzyme,the technology of immobilized enzymes has been continuously developed and improved,and to some extent,the problem of poor stability of the enzyme and difficulty in recycling is alleviated.With the rapid rise of nanotechnology,a new type of enzyme immobilization technology was born.Enzyme-inorganic hybrid nanoflowers are favored for their excellent catalytic activity and simple preparation methods.However,its own nano or micron size increases the difficulty of later separation.Therefore,it is extremely urgent to design an immobilization method that can easily and quickly separate nanoflowers.In this paper,the copper chloride solution and Pseudomonas cepacii lipase were self-assembled in phosphate buffer solution to obtain activated lipase-Cu₃(PO₄)₂ hybrid nanoflowers with similar chrysanthemum shape.The hybrid nanoflower enzyme activity was2.54 times of the free enzyme.Then the nanoflower was mingled in the polyvinyl alcohol-chitosan composite gel,and the gel was crosslinked by freeze-thaw method,and the nanoflower was firmly embedded in the porous hydrogel grid.The structure and composition of the immobilized enzyme were determined by SEM,FT-IR,XRD,EDS.By detecting the enzyme activity of the composite gel immobilized enzyme,the enzyme activity reached to146.12±6.54 U/g when the mass ratio of nanoflowers in hydrogel was 3.00%,which was 1.50times that of free enzyme.The catalytic activity of 41.59%of the free enzyme was maintained after repeated use for 7 times.In addition,nanoflowers are combined with magnetic materials to prepare lipase nanoflowers(lipase@MNF)that can be separated by an external magnetic field.Firstly,Fe₃O₄nanoparticles were prepared by thermal decomposition method and silylated to form magnetic nanoflowers.The complex of lipase Fe₃O₄-Cu₃(PO₄)₂·3H₂O was determined by FT-IR,XRD,EDS and other detection methods.The enzyme activity is the highest when the initial enzyme concentration was 0.5 mg/m L,which is 1.10 times of free enzyme.It has good stability and magnetic properties,which can achieve rapid separation and continuous catalysis.The lipase@MNF was applied to the transesterification of benzyl alcohol and vinyl acetate to produce benzyl acetate.The catalytic products were characterized by FT-IR and NMR.The molecular structure of the product was pure benzyl acetate and the rate reached 39.06%when reaction was carried out 48 h.