Preparation Of Poly(Acrylonitrile-co-acrylic Acid) Nanofibrous Membranes And Lipase Immobilization

Poly(acrylonitrile-co-acrylic acid) (PANCAA) membrane, possessing reactive carboxyl groups, can be used as support for enzyme immobilization. PANCAA nanofibrous membrane was fabricated by electrospinning process and used as support for lipase immobilization to increase the amount of enzyme loading, due to the higher specific area of the nanofibrous membrane. However, as synthetic polymer materials, the relatively poor biocompatibility of this membrane probably causes to some nonbiospecific interactions between enzymes and the membrane surface, therefore, resulting in the partial denaturation of enzyme protein and the loss of enzyme activity. To depress these unfavorable interactions, the membrane surface was modified respectively with ESM (Egg shell membrane) hydrolysate and collagen to create a biofriendly microenvironment for immobilized lipase (from Candida rugosa). Through the use of both these two methods, it was expected to get immobilized enzyme with both high enzyme loading and high activity. The relative experiments and results are summarized as follow.PANCAA nanofibrous membrane was fabricated by electrospinning process. The morphologies of nanofibrous membrane were analysized with field emission scanning electro microscopy (FESEM). It was then used as support for lipase immobilization, on which the carboxyl groups were actived with 1-ethyl-3-(dimethyl-aminopropyl) carbodiamine (EDC)/N-hydroxyl succinimide (NHS). The properties of immobilized lipase were assayed and compared with those of the free one and the hollow fiber membrane. It was found that, compared to those of the hollow fiber membrane, the enzyme loading increased from 2.36 mg/g to 15.2 mg/g. But compared to free one, the enzyme activity decreased a lot. Result of thermal stability indicated that the residual activity of the immobilized lipase is 63% at 50°C for 2 h.ESM hydrolysate was produced from ESM use Ba(OH)₂·8H₂O as catalyst. It was then tethered on the PANCAA nanofibrous membrane surface in the presence of EDC/NHS. The chemical change of the membrane surface was characterized by Fourier transform infrared spectroscopy (FT-IR). Lipase was immobilized on the membrane by covalent binding using glutaradehyde (GA) as coupling agent. Results on the basis of the enzyme loading amount, activity, kinetic parameters and stabilitywere compared with those of the nascent nanofibrous membrane. It was found that, due to the improvement of biocompatibility on the membrane surface, the activity retention of the immobilized lipase on the ESM hydrolysate-modified membrane increased to 20.4%, which was higher than that of the nascent nanofibrous membrane. The amount of enzyme loading and thermal, reuse stability were nearly the same as that of the nascent one.Collagen was also tethered on the PANCAA nanofibrous membrane surface in the presence of EDC/NHS. The chemical change of the membrane surface was characterized by FT-IR. Lipase was immobilized on this membrane using GA as coupling agent. Results on the basis of the enzyme loading amount, activity, kinetic parameters and stability were compared with those of the nascent nanofibrous membrane. It was found that, due to the great improvement of biocompatibility on the membrane surface, the activity retention of the immobilized lipase on the collagen-modified membrane increased to 61.7%, which was nearly 4 times as that of the nascent one. The amount of enzyme loading decreased a lot, was only 9.07 mg/g. Thermal and reuse stability were also nearly the same as that of the nascent one.