| In biomass utilization,cellulase can catalyze the multi-step hydrolysis of cellulose into glucose,and then produce high value-added chemicals such as bioethanol.However,its application in industry is greatly limited because of its poor stability and difficult recovery.Immobilization of cellulase is considered as a feasible solution.In this study,carrier-free and carrier-based immobilization techniques were used to modify cellulase,to explore the effect of preparation conditions on the catalytic activity of immobilized cellulase,and the enzymatic properties of immobilized cellulase such as the optimal temperature,optimal pH,reusability and storage stability were systematically studied,and a complete system of cellulase from immobilization to enzymatic hydrolysis performance evaluation was constructed.The main findings are as follows:（1）Cellulase was immobilized by carrier-free immobilization technology,and the preparation process was optimized.It was found that ammonium sulfate had the best precipitation effect on cellulase.The best immobilization effect was obtained when the dialdehyde concentration,cross-linking time and cross-linking temperature were 95%（w/v）,50 min,50 mg/mL,2%（v/v）,35℃ and 3.5 h,respectively.By analyzing the secondary structure content of cellulase before and after cross-linking,it was found that the rigidity of the molecular structure of cellulase was enhanced by cross-linking.Compared with free cellulase,the optimum temperature（70℃）of cross-linked cellulases（C-CLEAs）increased by 20℃,and the optimum pH（3.0）shifted to an acidic environment,and its tolerance to temperature and pH was improved.After 10 cycles of use and storage for 56 days,C-CLEAs retained 65%and 63%of their activities,respectively.In addition,the average t1/2 and Ed of C-CLEAs were 5.98 and 1.93 times higher than those of free cellulase,respectively,indicating that the former has higher thermotolerance.The Km of C-CLEAs was also found to be increased,indicating that its affinity for the substrate decreased.（2）Cellulase@MIL-88B（Fe）was prepared by using MIL-88B（Fe）as the carrier.When the immobilization temperature,time,cellulase concentration and pH were 30℃,6 h,1.2 mg/mL and 5.0,respectively,the immobilization effect was the best.Through FT-IR,XRD and TGA characterization analysis,it was found that the cellulase was successfully immobilized on MIL-88B（Fe）without destroying the integrity of the lattice structure of the carrier.Compared with free cellulase,the optimum pH of Cellulase@MIL-88B（Fe）was unchanged,but the optimum temperature was increased by 10℃,and it can maintain higher activity in a wider pH and temperature range.Cellulase@MIL-88B（Fe）maintained 78%and 85%of the activity after 10 cycles of use and 49 days of storage,respectively,and its t1/2 and Edvalues were higher than those of free cellulase,indicating that it has more high thermal stability and thermal inactivation.In addition,the Km（1.46 mg/mL）of Cellulase@MIL-88B（Fe）was slightly higher than that of free cellulase（1.41 mg/mL）,and Cellulase@MIL-88B（Fe）can be applied to actual corn cob enzymatic hydrolysis middle.（3）The influence of preparation conditions on the immobilization of cellulase by NH2-MIL-88B（Fe）was optimized.The best preparation conditions were:1.2 mg/mL cellulase solution in pH 5.0 buffer solution at 30℃ Medium hair should be 10 h.By FT-IR,XRD and TGA characterization,it was proved that the cellulase was successfully immobilized on NH2-MIL-88B（Fe）without affecting the lattice structure of the carrier,and Cellulase@NH2-MIL-88B（Fe）structural rigidity enhancement.Compared with free cellulase,the optimum temperature and pH of Cellulase@NH2-MIL-88B（Fe）did not change,but showed a wider adaptation condition,and its thermal stability,organic solvent tolerance,reusability and storage stability and other enzymatic properties have been significantly improved.Cellulase@NH2-MIL-88B（Fe）can also be used in the enzymatic hydrolysis of CMC and corn cob because it is less affected by the outside world. |
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