磁性壳聚糖微球固定化黄嘌呤氧化酶及其酶学性质的研究

Immobilization of Xanthine Oxidase on Magnetic Chitosan Microspheres and its Effect on Enzymatic Properties

摘要:

本文利用化学共沉淀法制备Fe3O4磁性颗粒，并以此为磁核通过乳化交联法制备磁性壳聚糖微球，以环氧氯丙烷对微球表面进行活化，用于黄嘌呤氧化酶的固定化研究。以微球表面的环氧基密度为活化指标，确定了活化过程的最适工艺条件：环氧氯丙烷体积分数为40%，NaBH4浓度为0.60 g/L，NaOH浓度为1.20 mol/L。对微球进行结构表征，结果表明：壳聚糖成功包裹了Fe3O4磁性粒子，且已活化微球的表面具有环氧基活性基团；Fe3O4磁性粒子、未活化和已活化磁性壳聚糖微球的中径分别为2.16、20.30和24.69 μm。活化结束后，将黄嘌呤氧化酶固定在磁性微球上。以酶活为指标，确定最适固定化工艺为：时间1 h，温度30 ℃，pH 8.0。对固定化黄嘌呤氧化酶的酶学性质研究，结果表明：酶的最适作用温度为48 ℃，最适作用pH为8.5，酶具有良好的热稳定性、pH稳定性及操作稳定性。

Abstract:

Iron oxide magnetic particles were prepared by chemical co-precipitation and the particles were cross-linked to magnetic chitosan microspheres. Afterwards, the magnetic chitosan microspheres were activated by surface modification with epoxy groups for subsequent xanthine oxidase immobilization. Using the epoxy density of the microsphere surface as an activation marker, optimal processing conditions were 40% (V/V) epichlorohydrin, 0.60 g/L sodium borohydrite, and 1.20 mol/L sodium hydroxide. Microsphere structural characterization showed that the iron oxide magnetic particles were successfully wrapped in chitosan and the surfaces of activated microspheres were surrounded by epoxy reactive groups. The diameter of iron oxide magnetic particles, the unactivated magnetic chitosan microspheres, and the activated magnetic chitosan microspheres were 2.16, 20.30, and 24.69 μm, respectively. After activation, xanthine oxidase was immobilized on the magnetic microspheres. Using enzyme activity as an indicator, the optimal immobilization process was conducted at 30 ℃ and pH 8.0 for 1 h. The optimum temperature and pH of the enzyme were 48 ℃ and 8.5, respectively. Moreover, the enzyme possessed good thermal, pH, and operating stability.