Construction And Orderly Immobilization Of Multi-Enzyme Cluster Mircoparticles

The multi-enzyme co-immobilization technology based on the multi-enzyme cascade reaction system can reduce the cost of the carrier effectively and minimize the consumption of material and energy in the reaction system.In recent years,carrier-free multi-enzyme immobilization technology has received widespread attention in multi-enzyme immobilization because of it can avoid the problems of space resistance and enzyme active site shielding caused by the carrier.In order to fully investigate carrier-free multi-enzyme immobilization technology,in situ immobilization and layer-by-layer immobilization was designed to immobilize the ternary enzymes ofβ-glucosidase（β-G）,glucose oxidase（GOD）and horseradish peroxidase（HRP）in this study.The enzymatic properties of immobilized enzymes under the two methods were compared and the optimization conditions in the process of carrier-free immobilization were determined.The main contents are as follows:（1）Prepare the multi-enzyme co-immobilization templates.Calcium chloride,sodium carbonate,poly（allylamine hydrochloric acid）（PAH）and poly（sodium 4-styrene sulfonate）（PSS）were added to prepare calcium carbonate（Ca CO₃）with different structures,PAH-Ca CO₃and PSS-Ca CO₃ microspheres,respectively.Scanning electron microscopy was used for morphological characterization and X-ray diffractometer was used for structural analysis to confirm the successful construction of templates.（2）Perform in situ immobilization and layer-by-layer immobilization.The effects of the concentrations and addition sequence of calcium chloride and sodium carbonate,crosslinking agent concentrationand template removal agent concentration on the immobilized enzyme loading and relative enzyme activity were investigated.The highest total enzyme loading obtained by in situ immobilization of the multi-enzyme was 72.57±3.63%.12 different self-assembled structures of layer-by-layer immobilization were prepared using PAH-Ca CO₃and PSS-Ca CO₃ microspheres.The PSS-Ca CO₃--PAH--β-G--HRP--GOD structure showed the highest enzyme loading and relative enzyme activity.Enzyme loadings were 75.16±1.97%,87.70±4.39%and 85.46±3.24%ofβ-G,GOD and HRP,respectively,and relative enzyme activities were 81.80±2.69%,93.59±4.68%and 90.64±3.13%ofβ-G,GOD and HRP,respectively.（3）Study the enzymatic properties and kinetic parameters of the immobilized enzymes.The effects of p H and temperature on the activity of immobilized enzymes were investigated.It was found that the acid resistance of HRP increased after immobilization.At p H 5,the relative enzyme activity of HRP in layer-by-layer immobilization and in situ immobilization was 1.48and 1.50 times that of free enzyme,respectively.The thermostability ofβ-G and GOD increased after immobilization.In layer-by-layer immobilization,the relative enzyme activity ofβ-G remained 100%50-60°Cand the optimum temperature of GOD was improved in the in situ immobilization at 50°C.Furthermore,the kinetic parameters of the immobilized enzymes were studied.The affinity and catalytic efficiency of the enzymes for substrates in layer-by-layer immobilization were higher than those of in situ immobilization The K_m value of HRP in the layer-by-layer immobilization was greater than that of free enzyme,and the K_cat/K_m valuewas1.49 times of free enzyme.These results indicated the immobilized enzymes have good catalytic performance.（4）Study of the reusability of immobilized enzymes.After 8 repeated reactions,the residual enzyme activities ofβ-G in in situ immobilization and layer-by-layer immobilization was96.35±4.82%and 96.69±4.84%,respectively.GOD and HRP were 69.17±3.46%and66.68±3.33%,65.18±3.26%and 62.63±3.13%,respectively.The immobilized enzymes showed good reusability.After 6 repeated reactions of immobilized enzymes in layer-by-layer immobilization,the absorbance value was finally 0.29,which was 1.40 times of in situ immobilization on the absorbance value.This result proved the good potential application of multi-enzyme layer-by-layer immobilization.