Design And Synthesis Of UCST Temperature-sensitive Block Polymer And Its Application In One-step Immobilization Of ?-glucosidase In Crude Enzyme Solution

Enzyme is a kind of efficient and specific biocatalyst.However,the free enzyme has many disadvantages,such as high cost,easy inactivation and difficult recovery,which hinder its large-scale application in industry.Immobilization of enzymes can effectively solve this dilemma.However,most of the immobilized enzyme carriers are insoluble,and it is difficult to achieve homogeneous catalysis,thus reducing their catalytic efficiency.Based on this,a water-soluble high critical solution temperature(UCST)temperature-sensitive block polymer was designed and synthesized in this paper,and its characterization and performance were studied.It was used as a soluble immobilized carrier to immobilize?-glucosidase in crude enzyme solution in one step.Specific research contents are as follows:(1)Sixteen UCST temperature-sensitive block polymers with different molecular weights and acrylonitrile contents were synthesized by reversible addition broken chain transfer polymerization(RAFT)using acrylamide and acrylonitrile as monomers.The composition,degree of polymerization and molecular weight of UCST temperature-sensitive block polymer m PEG-b-p(AAm-co-AN)were characterized by ~1H NMR,FT-IR and gel permeation chromatography.Among them,the molecular weight was determined by ~1H NMR.The UV transmittance and temperature curves were used to determine the UCST of the polymer in deionized water,which ranged from 0?to 50?.Transmission electron microscopy(TEM)and dynamic light scattering(DLS)were used to characterize the phase transition behavior of the polymer during high temperature dissolution and low temperature precipitation.Then,the influence of molecular weight of m PEG,acrylonitrile content,polymer concentration and electrolyte(sodium sulfate,sodium chloride and sodium thiocyanate)on UCST were studied.Among them,sodium sulfate will significantly increase UCST,while sodium chloride had the least effect on UCST,a small decline.The UCST of P5 and P13 could not even be detected when the concentration of sodium thiocyanate reached 200 m M.At the same time,sodium sulfate,sodium chloride and sodium thiocyanate had the same effect on different concentrations of polymer solutions.Finally,the effect of urea on UCST was investigated,and the results showed that urea could significantly reduce the UCST of the polymer.(2)In this experiment,the crude enzyme solution of Glu-Lyta was successfully cultured,and the temperature sensitive polymer P6 with UCST of 33.3?was selected to conduct a one-step immobilization study on the crude enzyme solution of Glu-Lyta,and the immobilized enzyme P6@Glu-Lyt A was prepared.The P6 polymer was activated with 30 mg/m L glutaraldehyde at 45?.Then,the Glu-Lyta crude enzyme was immobilized.The immobilization conditions were optimized.At 50?,2.5 mg/m L Glu-Lyta was immobilized in phosphate buffer solution(50 m M,p H 5.0)for 3 h,and the optimal content of the enzyme reached 48.7 mg/g.The P6@Glu-Lyt A was characterized by infrared spectroscopy,scanning electron microscopy(SEM),laser confocal microscopy(CLSM)and dynamic light scattering(DLS).After immobilized,the optimum reaction p H of Glu-Lyta was shifted to 4.5,and the optimum reaction temperature was 40?.The p H and temperature of the P6@Glu-Lyt A were applicable to a wider range,and the storage time was longer.The stability of the immobilized enzyme was significantly improved.P6@Glu-Lyt A could be repeatedly hydrolyzed p-nitrobenzene-?-D-glucoside(p-NPG)by low temperature centrifugation.The relative enzyme activity was still about 50%after 10 cycles.Moreover,the results of immobilized crude enzyme solution of Glu-Lyta catalyzing cellobiose showed that in a certain concentration range,with the increase of enzyme concentration,when the same substrate was catalyzed,the obtained glucose content was also higher.By comparing the immobilized crude enzyme solution of Glu-Lyta with the immobilized pure?-glucosidase,it was found that the catalytic efficiency of immobilized Glu-Lyta crude enzyme solution for cellobiose was 14.5%higher than that of immobilized pure?-glucosidase.