Immobilization Of Carbonic Anhydrase On The Surface Of Polymer Microporous Membrane

In recent years, the application of carbonic anhydrase（CA） in CO₂ removal has attracted great interest. However, as an expensive biocatalyst, solute CA is not easy to recycle and the activity of free enzyme is susceptible to a variety of the environmental factors that leads to low stability. Therefore, enzyme immobilization is necessary. In this study, CA was immobilized on polyvinylidene fluoride（PVDF） flat membrane and polyethylene（PE） hollow fiber membrane by means of covalent binding. The enzymatic activity was investigated using a p-NPA assay. Immobilization conditions were optimized and the storage stability of membrane-attached enzyme was examined. By measuring the formation rate of CaCO₃, the role of immobilized CA in CO₂ absorption was studied.PVDF was firstly activated by plasma technology in order to introduce hydroxyl groups on the surface of membrane, and then 3-aminopropyl triethoxysilane（KH550） was used for further modification, finally, CA was immobilized on the membrane surface using glutaraldehyde（GA） as crosslinking agent. The results showed that with KH550 concentration increased, grafting ratio and surface animo groups density of membrane increased and then reached saturation at KH550 concentration of 12.16 wt% and 6.67 wt%, respectively. The optimal immobilization conditions were animo groups density of 6.048×10-8 mol/cm2, CA concentration of 0.8 mg/mL, GA concentration of 0.2 wt% and 20 h reaction at pH 7.5. The maximum activity recovery of immobilized CA was 60.12%. Instead of KH550, γ-（2,3-epoxypropoxy）propyl trimethoxysilane（KH560） as the modifier of hydroxyl-groups containing PVDF flat membrane was also studied, and CA was linkded to the membrane surface by the reaction between epoxy and amino groups. The results showed that with KH560 concentration increased, grafting ratio and surface epoxy groups density of membrane increased, and then reached saturation at KH560 concentration of 10.01 wt% and 6.26 wt%, respectively. The optimal immobilization conditions were epoxy groups density of 4.48×10-7 mol/cm2（1.06×10-3 mol/100g）, CA concentration of 1.0 mg/mL and 12 h reaction at pH 8.0. The maximum activity recovery of immobilized CA was 33.02%.PE hollow fiber membrane as carrier was firstly surface-modified via the polymerization of acrylic acid（AA） under ultraviolet light irradiation, and then CA was attached on the surface of membrane with carbodiimide as an activator. The optimal immobilization conditions were grafting ratio of 9.51%, activator concentration of 0.075 g/m L, CA concentration of 0.8 mg/m L, 22 h reaction at pH 7.5 and 45 ℃. The maximum enzyme loading amount and activity recovery of immobilized CA were 18.9 mg/g and 11.78%. Comparing the storage stability of three kinds of immobilized CA in phosphate buffer, it was observed that the half-lifes of immobilized CA were 82, 78, 75 days at 4 ℃, respectively, as compared to 32 days for free CA. At 25 ℃, the half-lifes for immobilized and free CA were 50, 37, 35 and 20 days, respectively. In the presence of immobilized CA, generation rates of CaCO₃ were 76%, 65% and 57% of free CA in CO₂ absorption experiments, 6, 2.5, 1.67 times of the system without CA, respectively.