| Lignocellulose is one of the most abundant renewable resources in the world,and biological enzymatic saccharification is a key link to convert it into ethanol and other biofuels.Cellulase plays an important role in the development and utilization of biomass,but it has some shortcomings such as relatively high cost of use,poor stability,difficulty in recycling,etc.Immobilized cellulase is a measure with development potential to break through these shortcomings.In recent years,magnetic nanomaterials represented by magnetic Fe3O4 nanoparticles have been widely used in immobilization of enzymes due to their strong practicability,low toxicity and easy of recycling.In this paper,the functionalized magnetic nano Fe3O4 was used as the carrier to immobilize the cellulase by a "two-step method".The immobilization process was optimized by designing single factor experiment and orthogonal experiment,the optimal immobilization process was obtained.The enzymatic properties of immobilized cellulase were studied.The saccharification effect of the enzymatic hydrolysis steam explosion poplar by immobilized cellulase was explored,and the kinetics was studied.The main results were as follows:1.The magnetic nano Fe3O4 was prepared by the co-precipitation method,the magnetic nano Fe3O4@SiO2 was synthesized by the improved Stober method and functionalized modification with APTES to obtain A-Fe3O4@SiO2.The morphology,particle size,composition and properties were characterized by SEM,TEM,VSM and FTIR.It was proved that magnetic nano Fe3O4 and Fe3O4@SiO2 nanospheres have been successfully prepared,with particle sizes of 10-20 and 20-40 nm,respectively.They have good magnetic properties and can be separated and recovered effectively by magnetic field.2.The A-Fe3O4@SiO2 was used to immobilize cellulase,and the immobilization process was optimized by single factor and orthogonal experiments.The optimal immobilized conditions were obtained:When the cellulase was 100 u,the immobilized temperature was 20℃,and the immobilized pH was 4.0,the activity of the immobilized cellulase was 3097 u/g.SDS-PAGE gel electrophoresis was used to analyze the changes of each component of cellulase before and after immobilization.It was found that magnetic nanoparticles Fe3O4@SiO2 had no significant selective specificity for cellulase.3.The enzymatic properties of immobilized cellulase were studied.The optimal reaction temperature of immobilized cellulase was 60℃,and the optimal pH was 4.5,Meanwhile,the immobilized cellulase showed good activity,stability and adaptability in a wide range of temperature and pH.Stored at 4℃ for 180 d,85%of the initial enzyme activity was maintained.After 6 consecutive cycles,the relative enzyme activity of the immobilized cellulase decreased to 37%,and there was some leakage problem.The Km of the immobilized cellulase was 0.015 mg/mL,which was higher than the free cellulase(0.0125 mg/mL),indicating that the affinity to the substrate was reduced.4.Immobilized enzyme was used for enzymatic saccharification of steam explosion poplar.The optimal parameters were mixing rate 200 r/min,substrate concentration 10%,immobilized enzyme was 40 IU,the saccharification rate can reach more than 40%.The Michaelis-Menten model and Chrastil model were used to perform kinetic fitting on the enzymatic hydrolysis saccharification. |
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