磁性琼脂糖微球固定化血管紧张素转化酶的制备及性质

Preparation and Properties of Magnetic Agarose Microsphere Immobilized Angiotensin Converting Enzyme

摘要:

采用反相悬浮包埋法制备磁性琼脂糖微球，然后以环氧氯丙烷为活化剂固定血管紧张素转化酶(ACE)。探讨了ACE固定化的影响因素，确定了固定化最适条件：酶溶液蛋白质量浓度为8g/L，pH为7.8，温度为50℃，固定化时间为2h，所得的固定化酶的活力达到0.128U/g；对磁性固定化ACE的性质进行了研究，固定化ACE最适温度为42℃ ,最适pH为8.3。同时，比较了磁性固定化与游离ACE对pH的耐受力和热稳定性，在 pH =5 的缓冲液中放置1h后，固定化ACE和游离ACE酶活力保留率分别为62.1%和40.7%，当pH= 9，两者酶活力保留率分别为95.7%和89.2%；60℃时，两者酶活力保留率分别为50.2%和20.7%；-20℃储存30d后, 两者酶活力保留率分别为90.3%和43.0%；连续操作10次后,固定化ACE活力仍保持53.0%。研究表明，磁性固定化ACE在外加磁场的作用下可快速重复回收利用，具有良好的应用前景。

Abstract:

Magnetic agarose microsphere was prepared by reverse-phase microemulsion method, and its surface was modified with epoxy groups to immobilize angiotensin converting enzyme (ACE). The factors that influence the immobilization of ACE were investigated. The optimum immobilization conditions were: ACE concentration of 8 mg/mL，pH=7.8，temperature 50℃, immobilization time of 2h, with a optimal specific activity of 0.128 U/g spheres. The properties of magnetic immobilized ACE were studied. The optimal catalytic pH of the immobilized ACE was 8.3 and the optimal temperature was 42℃. The pH tolerance and thermal stability of the magnetic immobilized ACE and free ACE were also evaluated, the enzyme activity retention rate after keeping at pH=5 for 1h were 62.1% and 40.7%, respectively, and the retention rate were 95.7% and 89.2% at pH=9. The immobilized enzyme still kept 50.2% of the original activity at 60 ℃, as the compared with the free enzyme was 20.7%. After 30 days storage at -20℃, the immobilized ACE activity remained 90.3%. 53.0% of the initial activity remained after recycled for 10 times. The result shows that magnetic immobilized ACE has a good application prospect due to its quick repeated recycling under an additional magnetic field.