Ye Peng, Xu Zhi-Kang, Che Ai-Fu, Wu Jian, Seta Patrick
Institute of Polymer Science, Zhejiang University, Hangzhou 310027, PR China.
Biomaterials. 2005 Nov;26(32):6394-403. doi: 10.1016/j.biomaterials.2005.04.019.
A protocol was used to prepare a dual-layer biomimetic membrane as support for enzyme immobilization by tethering chitosan on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) ultrafiltration hollow fiber membrane in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxylsuccin-imide (NHS). The chemical change of the chitosan-modified PANCMA membrane surface was confirmed with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic membrane using glutaraldehyde (GA), and on the nascent PANCMA membrane using EDC/NHS as coupling agent. The properties of the immobilized enzymes were assayed and compared with those of the free one. It was found that both the activity retention of the immobilized lipase and the amount of bound protein on the dual-layer biomimetic membrane (44.5% and 66.5 mg/m2) were higher than those on the nascent PANCMA membrane (33.9% and 53.7 mg/m2). The kinetic parameters of the free and immobilized lipases, Km and Vmax, were also assayed. The Km values were similar for the immobilized lipases, while the Vmax value of the immobilized lipase on the dual-layer biomimetic membrane was higher than that on the nascent PANCMA membrane. Results indicated that the pH and thermal stabilities of lipase increased upon immobilization. The residual activity of the immobilized lipase after 10 uses was 53% on the dual-layer biomimetic membrane and 62% on the nascent PANCMA membrane.
采用一种方案，通过在1-乙基-3-(二甲基氨基丙基)碳二亚胺盐酸盐(EDC)/N-羟基琥珀酰亚胺(NHS)存在的情况下，将壳聚糖 tethering在聚(丙烯腈-共-马来酸)(PANCMA)超滤中空纤维膜表面，制备双层仿生膜作为酶固定化的载体。用傅里叶变换红外光谱和X射线光电子能谱证实了壳聚糖改性PANCMA膜表面的化学变化。使用戊二醛(GA)将皱褶假丝酵母脂肪酶固定在该双层仿生膜上，并使用EDC/NHS作为偶联剂将其固定在新生的PANCMA膜上。测定了固定化酶的性质，并与游离酶的性质进行了比较。结果发现，双层仿生膜上固定化脂肪酶的活性保留率(44.5%)和结合蛋白量(66.5 mg/m2)均高于新生PANCMA膜上的(33.9%和53.7 mg/m2)。还测定了游离和固定化脂肪酶的动力学参数Km和Vmax。固定化脂肪酶的Km值相似，而双层仿生膜上固定化脂肪酶的Vmax值高于新生PANCMA膜上的。结果表明，脂肪酶固定化后pH稳定性和热稳定性均有所提高。双层仿生膜上固定化脂肪酶10次使用后的残余活性为53%，新生PANCMA膜上为62%。
