Poly(Acrylonitrile-co-maleic Acid) Membranes For Lipase Immobilization

Poly(acrylonitrile-co-maleic acid) (PANCMA) hollow fiber membrane, possessing reactive carboxyl groups, can be used as support for enzyme immobilization. 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 partical denaturation of enzyme protein and the loss of enzyme activity. In this paper, to depress these unfavorable interactions, the membrane surface was modified respectively with chitosan and gelatin to creat a biofriendly microenvironment for immobilized lipase (from Candida rugosa). On the other hand, PANCMA nanofiber 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 nanofiber membrane. Based on the above work, the PANCMA nanofiber membrane was modified with chitosan and gelatin respectively, and used as support for lipase immobilization. The relative experiments and results are summarized as follow.PANCMA hollow fiber membrane was prepared and then used as support for liapse immobilization, on which the carboxyl groups were actived with 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide (EDC)/N-hydroxyl succinimide (NHS). Using the hydrolysis reaction of p-nitrophenyl palmitate in aqueous media and organic media, the properties of immobilized lipase were assayed and compared with those of the free one. It was found that in aqueous media the enzyme activity decreases, while in heptane it increases upon immobilization. The activity retention of the immobilized lipase is 33.9 % in aqueous and 114 % in heptane. The amount of enzyme loading is 53.7 ± 1.4 mg/m~2. Result of thermal stability indicates that the residual activity of the immobilized lipase is 62% at 50 °C for 2 h.Chitosan was tethered on the PANCMA hollow fiber membrane surface in the presence of EDC/NHS. The chemical changes of the membrane surface were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR/FT-IR) and X-ray photoelectron spectroscopy (XPS). Lipase was immobilized on the membrane by adsorption and covalent bindingusing glutaradehyde (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 hollow fiber membrane. It was found that the activity retention of the immobilized lipase increases to 54.1 % by adsorption and 44.5 % by GA, for the chitosan-modified membrane, due to the improvement of biocompatibility on the membrane surface. The amount of enzyme loading is 31.2 ± 1.6 mg/m2 by adsorption and 66.5 ± 1.8 mg/m2 by GA. Result of thermal stability indicates that the residual activity of the immobilized lipase is 38 % by adsorption and 65 % by GA.Gelatin was also tethered on the PANCMA hollow fiber membrane surface in the presence of EDC/NHS. The chemical changes of the membrane surface were characterized by ATR/FT-IR and XPS. 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 hollow fiber membrane. It was found that the activity retention of the immobilized lipase on the gelatin-modified membrane increases to 49.2 %, which is higher than that on the nascent one, due to the improvement of biocompatibility on the membrane surface. The amount of enzyme loading is 54.6 ±1.5 mg/m2. Result of thermal stability indicates that the residual activity of the immobilized lipase is 66 %.PANCMA nanofiber membrane was fabricated by electrospinning process. The morphology and fiber diameter were analysized with field emission scanning electro microscopy (SEM). Lipase was immobilized on the nanofiber membrane using EDC/NHS as coupling agent. Results on the basis of the enzyme loading amount, activity, kinetic parameters and stability were compared with those of the hollow fiber membrane. It was found that the enzyme loading and activity retention of the immobilized lipase on the nanofiber membrane increase to 21.2 ± 0.71 mg/g and 37.6 %, compared to those of the hollow fiber membrane. Result of thermal stability indicates that the residual activity of the immobilized lipase is 63 %.Chitosan and gelatin was respectively tethered on the PANCMA nanofiber membrane surface in the presence of EDC/NHS. Lipase was immobilized on the membrane using GA as coupling agent. Results on the basis of the enzyme loading amount, activity, kinetic parameters and stability were compared withthose of the nascent nanofiber membrane. It was found that the enzyme loading and activity retention of the immobilized lipase are 22.5 ± 0.75 mg/g and45.6 % on the chitosan-modified nanofiber membrane, 20.7 ± 0.75 mg/g and49.7 % on the gelatin-modified nanofiber membrane. Result of thermal stability indicates that the residual activity of the immobilized lipase is 66 % on the chitosan-modified nanofiber membrane and 62 % on the gelatin-modified nanofiber membrane.