Ni^(2+)螯合的琼脂糖凝胶载体用于重组酶SUMO-Hep I-His的固定化

Immobilization of an Sumo-Heparinase I-His Fusion Enzyme on Ni^(2+) Chelating Agarose Gel Carrier

以Ni^(2+)螯合的琼脂糖凝胶(GLK-Gel Ni)作为固定化载体,对融合肝素酶Sumo-Hep I-His的固定化进行了研究,实现了融合酶的一步纯化和固定化。通过对固定化条件的优化,得到较优的固定化体系:载体与粗酶质量比为30∶1,固定化pH为7.0,吸附时间为6 h,该条件下获得的固定化酶酶活10.07±0.35 IU/mL载体。酶学性质研究结果表明:固定化酶在30℃的热稳定性相对于游离酶显著提高,固定化酶半衰期是游离酶的8倍,最适反应温度提高了3℃,反应pH的耐受性也有了明显的提高。此外,与游离酶相比,固定化酶的储藏稳定性和可重复利用性良好,在4℃下放置60 d能保持76%的初始活性;固定化酶重复使用8次后,酶活仍剩余75.8%;而且GLK-Gel Ni载体本身具有良好的重复利用性,重复固定5次Sumo-Hep I-His后,载体对酶的活性吸附仍能达到88.9%。整体而言,固定化Sumo-Hep I-His显现了较好的工业应用潜能。

In this study,immobilization of Sumo-Hep I-His was investigated using Ni2+chelating agarose gel as carrier.Under optimal conditions,i.e.,weight ratio of barrier to rude enzyme 30∶1,pH 7.0 and immobilized time 6 h,Sumo-Hep I-His was successfully immobilized with an apparent enzymatic activity of 10.07±0.35 IU/mL carrier.Compared with free enzyme,immobilized Sumo-Hep I-His showed great improvement in thermo stability(the half-live was 8 times of free Hep I at 30℃as well as pH stability.The storage and operation stability of the immobilized enzyme was also significantly improved,as 76%relative enzymatic activity was remained after storing for 60 days at 4℃and 75.8%relative enzymatic activity was remained after 8 reaction cycles.GLK-Gel Ni gel also showed good recycling stability that 88.9%adsorptive capacity was remained after 5 cycles immobilization.Conclusively,immobilized Hep I showed great potential in industrial application.