Study On Immobilization Of Lipase Based On Emulsion System

Enzyme has been widely used in many fields as a kind of green and efficient biocatalyst.However,due to the many problems of free enzyme,like the poor stability and difficult to reuse,enzyme is often immobilized.Although the traditional immobilization methods can solve the problem of the application of enzyme constrainedly,there are still a lot of inadequacies.The aim of this paper is to explore a new method for the immobilization of enzyme.In this paper,Pseudomonas fluorescent lipase as a model enzyme.It was immobilized in the water-in-oil emulsions.The main purpose is to obtain immobilized lipase which is a higher activity,good stability,reusable.Moreover,the stability of water-in-oil emulsions was studied,the immobilization technology was optimized,and the enzymatic properties of immobilized lipase were determined,the morphology and structure of immobilized lipase particles were also explored.The specific research contents and results are as follows:1.In the first part,the relatively stable water in oil emulsion system was constructed.The surfactant(HLB=7)was used as emulsifier and the proportion was 25%(v/v).The oil-water ratio of emulsion system was 25:1,the emulsifying method was first ultrasonic emulsification 5 min,and then the magnetic emulsion was 5 min.The particle size of emulsion system was measured by the Zeta potential particle size analyzer,and the particle size range was between 15~40?m,and its average particle diameter was 26?m.2.Through response surface experiments lipase immobilization process was optimized.Determining the condition of the immobilized lipase preparation optimal: soybean oil emulsion system as the oil phase,the concentration of lipase was 25.0 mg/mL,immobilization temperature was 7,immobilization time was 1.8 h,the protecting agent added in an amount of aqueous phase 25%(v/v),the crosslinking agent added in an amount of aqueous phase 11%(v/v).And under these conditions the immobilized lipase prepared and measured obtaining the lipase hydrolysis activity was 9333 U/mg.Than before optimization 13.4%.3.Through the study of the enzymatic properties of immobilized lipase,It was found that: the optimum temperature was 45?,the optimum pH was 6.The activation effect of common metal ions was obviously enhanced,and the inhibitory effect was decreased.After 35 days of low temperature storage,it still retains 96% of initial activity,after 11 times of recycling,the hydrolysis activity remains approximately 78% of the initial activity.The immobilization rate of lipase was about 91%,and the recovery rate of activity was 70%.After comparing the kinetic parameters of free lipase and immobilized lipase,it was found that the affinity of immobilized lipase and substrate was smaller,but the maximum reaction rate of the immobilized enzyme was increased by about 2 times.4.After immobilization,lipase change greatly both in appearance and structure by using Fluorescence microscopy,Fourier transform infrared spectroscopy,scanning electron microscope.It was found that immobilized enzyme particles scattered,the particle size is relatively uniform in the buffer.The proportion of non regular curl after lipase immobilization changes small,the change of ?-helix and ?-folding was larger,and the ratio of ?-folding decreased by 1.1%,and the percentage of ?-helix increased by 3.8%,?-turn is reduced by 2.5%.After lyophilization immobilized lipase in the shape of an irregular sheet structure,most of the particle size of about 30 ?m.