PDF(Pubmed)
Abstract：
Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues. The active site availability is similar for all four structures. The split enzymes are comparable in stability with their native analogues, meaning that they can be used for further biotechnology applications. Also, the tertiary structures of the two peroxidases are similar. However, differences that might help in choosing one system over another for biotechnology applications were noticed. The main difference between the two systems is glycosylation which is not present in the case of APX/sAPEX2, while it has a high impact on the HRP/sHRP stability. Further differences are calcium ions and cysteine bridges that are present only in the case of HRP/sHRP. Finally, computational results identified sAPEX2 as the systems with the smallest structural variations during molecular dynamics simulations showing its dominant stability comparing to other simulated proteins. Taken all together, the sAPEX2 system has a high potential for biotechnological applications due to the lack of glycans and cysteines, as well as due to high stability.
摘要：
过氧化物酶是许多生物技术应用中的基本要素。一个特别有趣的概念涉及裂解酶，其中酶被分成两个较小的无活性的蛋白质，可以二聚成一个完全活性的酶。已经为辣根过氧化物酶（HRP）和抗坏血酸过氧化物酶（APX）开发了这种分裂形式。两种过氧化物酶在生物技术应用中都具有很高的潜力。在本研究中,我们对这两种过氧化物酶及其分裂类似物进行了生物物理比较。所有四个结构的活动站点可用性相似。裂解酶的稳定性与其天然类似物相当，这意味着它们可以用于进一步的生物技术应用。此外，两种过氧化物酶的三级结构相似。然而,人们注意到，可能有助于为生物技术应用选择一个系统而不是另一个系统的差异。两种系统之间的主要区别是糖基化,其在APX/sAPEX2的情况下不存在,而其对HRP/sHRP稳定性具有高度影响。其他差异是仅在HRP/sHRP的情况下存在的钙离子和半胱氨酸桥。最后,计算结果表明，在分子动力学模拟过程中，sAPEX2是结构变化最小的系统，显示出其相对于其他模拟蛋白质的主要稳定性。把所有放在一起，由于缺乏聚糖和半胱氨酸，sAPEX2系统具有很高的生物技术应用潜力，以及由于高稳定性。
