磁性壳聚糖微球的制备及其用于甲酸脱氢酶的固定化

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。

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

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 Fe3O4 and prepared by co-precipitation method showed good effect,so they were used as the support for immobilizing formate dehydrogenase.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 dehydrogenase 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 40.39%,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.