磁性壳聚糖微球的制备及其用于甲酸脱氢酶的固定化

Preparation of magnetic chitosan microspheres and their application to immobilization of formate dehydrogenase

分别采用乳化交联法和共沉淀法制备磁性壳聚糖微球载体,并对形貌结构进行比较,结果表明,采用共沉淀法制备的磁性壳聚糖微球负载Fe3O4的效果好,故将其作为载体固定甲酸脱氢酶。最佳固定化条件:添加酶量9 U.g-1,pH=7.0,固定化时间5 h。游离酶和固定化酶的最适宜反应温度分别为50℃和30℃;游离酶的最适宜pH=7.0,固定化酶的最适宜pH=6.0;将游离酶和固定化酶分别置于60℃恒温水浴放置180 min后,游离酶和固定化酶的相对酶活力分别为0.78%和40.39%;将游离酶和固定化酶置于不同pH的缓冲液中保存1 h后,在强酸(pH=2.0)和强碱(pH=10.0)条件下,固定化酶的相对酶活力分别为11.03%和38.43%,游离酶已全部失活;固定化酶重复使用6次后,相对酶活力为73.53%,表明固定化酶具有较好的热稳定性、酸碱稳定性和操作稳定性。

Magnetic chitosan microspheres were prepared by emulsification cross-linking method and co-precipitation method, respectively. Compared with the morphology and structure of the magnetic chitosan microspheres, magnetic chitosan microspheres supported with Fe304 and prepared by co-precipi- tation method showed good effect, so they were used as the support for immobilizing formate dehydrogen- ase. The optimal immobilization condition was as follows： enzyme concentration 9 U ~ g-1, pH = 7.0, immobilization time 5 h. The optimum reaction temperature for free and immobilized formate dehydrogen- ase was 50 ℃ and 30 ℃, respectively. The optimal pH for free and immobilized formate dehydrogenase was 7.0 and 6.0, respectively. After the free and immobilized formate dehydrogenase were put in thermostatic waterbath at 60 ℃ for 180 min, the relative activity of free and immobilized formate dehydrogenase was 0.78% and g0.39%, respectively. After being put in strong acidic buffer solution （pH = 2.0） and strong alkaline buffer solution（ pH = 10.0） for 1 h ,the relative activity of immobilized formate dehydrogenase was 11.03% and 38.43%, respectively, and the relative activity of free formate dehydrogenase was about 0; After 6 consecutive operations, the immobilized formate dehydrogenase retained 73.53% residual activity. It indicated that the immobilized formate dehydrogenase had better thermal stability, acid-base stability and reusability.