Immobilization Of High Temperature Resistant Enzymes Based On Magnetic Chitosan Composites

High-temperature resistant enzymes are mainly proteins with high efficiency and specific catalytic function isolated from thermophilic organisms.Compared with ordinary biological enzymes,high-temperature resistant enzymes can not only withstand high temperature,but also resist denaturants under extreme acidic and alkaline conditions.High-temperature resistant enzymes need to react at very high temperatures.Currently,the immobilization mechanism matching high-temperature resistant enzymes is not fully developed.The commonly used immobilized carriers in industry have low thermal stability and are difficult to use at high temperatures,which limits the development and application of high-temperature resistant enzymes.In this paper,chitosan and sodium alginate were used as raw materials,and chitosan composite hydrogels with excellent mechanical properties and high temperature resistance were prepared by the method of dissolution and hydrogen bond rearrangement under the condition of alkaline solution freezing.Further,the immobilized high temperature resistant enzyme was prepared by chemical crosslinking method.In order to obtain an excellent immobilized enzyme carrier,chitosan hydrogel was modified in this experiment,and the immobilization conditions were optimized by taking the industrial high-temperature resistant enzyme immobilization as the evaluation standard.In order to obtain the optimal process of the high-temperature resistant enzyme,the thermal resistance characteristics of the immobilized enzyme were studied by thermograys analysis（TGA）,which provided theoretical support for the research of high-temperature resistant enzyme immobilization.The main contents and results are as follows:（1）Chitosan composite hydrogels with excellent mechanical properties and high temperature resistance were prepared by using KOH/urea alkaline system to dissolve chitosan and hydrogen bond rearrangement under freezing conditions.Then chitosan hydrogel prepared by KOH/urea alkaline system was modified as the matrix.By adding a small amount of sodium alginate and Fe₃O₄magnetic nanoparticles,magnetic chitosan/sodium alginate blended hydrogel beads were prepared.The carrier could be directional separated by external magnetic field,and the strength and toughness of the material were greatly improved.The mechanical strength and thermal stability of chitosan hydrogel were further improved by metal ion complexation.According to TG,the weight loss at 500℃decreased from 43.46%before modification to 37.53%,and the thermal stability of immobilized enzyme was improved.By enzymic properties analysis,the immobilized enzyme was dried in a vacuum oven at 85℃for 2 h and boiled in hot water at 100℃for 0.5 h.The maximum enzyme activity remained at 90%and 85%,respectively,and the thermal stability of the immobilized enzyme was improved.（2）Using heat-resistant alpha-amylase as an example,the immobilized effect of the magnetic chitosan complex hydrogel was investigated.The effects of chitosan concentration,sodium alginate concentration,type and concentration of metal ions,and concentration of cross-linking agent EGDE on immobilization enzyme activity were investigated,and optimal immobilization conditions were determined.Chitosan mass fraction 5%,sodium arformate mass fraction 0.5%,Zn SO₄solution mass concentration 20 g·L^-1,ethylene glycol dicondensation glycerin ether（EGDE）volume fraction 0.8%.Even after 16 repetitions,the activity was maintained at 58.28%,showing stable operation.Measurements of the moisture content and expansion rate of imitated enzymes showed that the hydrogel with chitosan compound had a good moisture content and absorbability,with a moisture content of about80%and an expansion rate of about 136%.（3）Taking high temperature resistantα-amylase as an example,the enzymatic properties of immobilized enzyme under optimal conditions were studied.The optimum reaction temperature of free enzyme and immobilized enzyme were 90℃and 95℃,respectively,and the optimum reaction p H was 6.5.However,immobilized enzyme had higher stability than free enzyme under alkaline condition.At the concentration of 10 mmol·L^-1,the immobilized enzyme showed better tolerance to Co²⁺,Ni²⁺and other metal ions.After repeated use for 5times,the immobilized enzyme retained 80.52%maximum enzyme activity,after repeated use for 16 times,the immobilized enzyme still retained 58.28%maximum enzyme activity,indicating that the operation stability of the immobilized enzyme is good,has a certain industrial application prospect.