铁蛋白介导的地衣多糖酶胞内自发聚集及其高效制备

In vivo self-aggregation and efficient preparation of recombinant lichenase based on ferritin

摘要:

酶分离纯化、固定化及催化性能提升一直是生物催化领域的研究热点和前沿,也是众多研究者致力解决的难点。研究和开发新型的纯化、固定化及提升催化性能的方法,降低纯化及储存等设备的要求及生产成本,对酶大规模应用具有重要意义。文中将铁蛋白(ferritin)与目标酶(地衣多糖酶)基因融合,经高效表达后,在细胞内自发形成无载体固定化酶,且可经低速离心分离纯化,纯度可达90%以上,酶活回收率为61.1%,在30℃储存608 h后,依然能够保持活力,且比初始时活力略有增加。在重复使用10次以后,仍保持初始酶活力的50.0%。该微米级固定化酶可自发复溶,形成直径约为12 nm的可溶寡聚地衣多糖酶,其比活力是游离地衣多糖酶的12.48倍,催化效率则是后者的7.11倍,在50℃时,酶活性降低为初始值一半所需时间也延长了11.09倍。这表明铁蛋白有望成为一种全新的分离纯化目标酶并大幅提升其催化性能的标签。

Abstract:

Enzyme separation,purification,immobilization,and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis.Thus,the development of novel methods for enzyme purification,immobilization,and improvement of their catalytic performance and storage are of great significance.Herein,ferritin was fused with the lichenase gene to achieve the purpose.The results showed that the fused gene was highly expressed in the cells of host strains,and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo.Through low-speed centrifugation,the purity of the enzymes was up to>90%,and the activity recovery was 61.1%.The activity of the enzymes after storage for 608 h was higher than the initial activity.After being used for 10 cycles,it still maintained 50.0% of the original activity.The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm.The specific activity of them was 12.09 times that of the free lichenase,while the catalytic efficiency was 7.11 times and the half-life at 50℃ was improved 11.09 folds.The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo,which can simplify the preparation of the target enzymes,improve their catalysis performance,and facilitate their storage.