| Laccase (EC1.10.3.2, size of 6.5nm×5.5nm×4.5nm), an oxidoreductase containing 500 amino acids (64-140 kDa, depending on the type), is widely distributed in plants, fungi and bactierial strains. However, the use of laccase in its native form is often hindered by several limitations, such as the sensitivity to environment, low operational stability and difficulties in recovery and reutilization. Immobilization technology has been proven to be an effective method for implementing efficient and continuous applications of an enzyme. In the present study, zirconium and copper were selected as the central metal ion to synthesize mesoporous Metal-Organic-Frameworks (Cu-MOF and Zr-MOF) with carboxyl on the surface. The laccase was immobilized onto the MOFs via physical adsorption. Furthermore, the graphene aerogel-mesoMOFs was synthesized to remove hydroquinone from aqueous phase after the immobilization of laccase. The main results obtained in this paper are as follows.1). A generation of Cu-metal organic framework (Cu-MOF) was synthesized by utilizing surfactant-templating method with different mass ratios of surfactant/chelating agent. The Brunauer-Emmett-Teller (BET) surface area and pore diameter of the nanoscale Cu-MOF were 365.6-1153.8m2/g and 15.2-20.7nm, respectively. It can bear high temperature up to 350tyxy. The conditions of 5mg/mL laccase and 1h were chosen as the optimal adsorption concentration and adsorption time. This immobilized system exhibited a ultrahigh adsorption capacity (502.29±5.84mg/g), comparing to similar supports under the optimal adsorption conditions. The activity recovery rate was 95.18±0.29%. The optimum reaction conditions for the immobilization of laccase on Cu-MOF were pH4.0 and 50tyxy. The immobilized laccase showed better stability under extreme conditions than free laccase. According to the Linewaever-Burk plot, the Km of this immobilized laccase was 0.157mmol/L, which means that the affinity of immobilized enzyme to its substrate was higher than that of free laccase (0.290mmol/L). The activity of immobilized laccase after reusing for 7 and 10 times remained approximately 50%a nd 6%, respectively, which means that the stability of the immobilized laccase still need to be improved.2). Zr-metal organic framework (Zr-MOF, MMU(Bimodal mesoporous UIO-66)) was synthesized by similar method to improve the stability and reusability of Cu-MOF. The BET surface area of the nanoscale MMU was 453.8m2/g, and the pore diameter was 3.5 and 7nm. This material exhibited good thermal stability to resist 450℃. The immobilization prepared in a 5mg/mL laccase solution for lh led to the highest adsorption amount (221.83±8.74 mg/g) and favorable activity recovery (95.90±0.28%). The optimum reaction conditions for the immobilization of laccase on Zr-MOF were pH4.0 and 40tyxy. The immobilized laccase showed broader pH and temperature profiles, better stability and repeatability than free laccase. According to the Linewaever-Burk plot, the Km of this immobilized laccase was 0.166mmol/L, which was similar to that of Cu-MOF. The activity of immobilized laccase after utilization 10 times remained at approximately 50%, and it kept 55.4% of its initial activity at the end of 3 weeks of storage in an aqueous phase. The good stability and retention of enzymatic activity indicated that this bimodal mesoporous Zr-MOF is a good support for the immobilization of laccase.3). To improve the operability, the graphene aerogel-Zr-MOF was sythysized and utilized as membranes in solid phase extraction device to remove hydroquinone after immobilization of laccase. The adsorption of laccase on this membrane was 73.84mg/g, which is mainly due to the adsorption effect of Zr-MOF. The membrane showed good efficiency in removing hydroquinone (78.98%) at different flow rate (0-3.5L/h). After recycling 5 times, the removal rate of hydroquinone remained an acceptable level (70%), which demonstrated the good operability of graphene aerogel-Zr-MOF for immobilization. |
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