PDF(Pubmed)
Abstract：
Enzymes have become important tools in many industries. However, the full exploitation of their potential is currently limited by a lack of efficient and cost-effective methods for enzyme purification from microbial production. One technology that could solve this problem is foam fractionation. In this study, we show that diverse natural foam-stabilizing proteins fused as F-Tags to β-lactamase, penicillin G acylase, and formate dehydrogenase, respectively, are able to mediate foaming and recovery of the enzymes by foam fractionation. The catalytic activity of all three candidates is largely preserved. Under appropriate fractionation conditions, especially when a wash buffer is used, some F-Tags also allow nearly complete separation of the target enzyme from a contaminating protein. We found that a larger distance between the F-Tag and the target enzyme has a positive effect on the maintenance of catalytic activity. However, we did not identify any particular sequence motifs or physical parameters that influenced performance as an F-tag. The best results were obtained with a short helical F-Tag, which was originally intended to serve only as a linker sequence. The findings of the study suggest that the development of molecular tags that enable the establishment of surfactant-free foam fractionation for enzyme workup is a promising method. KEY POINTS: • Foam-stabilizing proteins mediate activity-preserving foam fractionation of enzymes • Performance as an F-Tag is not restricted to particular structural motifs • Separation from untagged protein benefits from low foam stability and foam washings.
摘要：
酶已成为许多行业的重要工具。然而,目前，由于缺乏从微生物生产中纯化酶的有效且具有成本效益的方法，因此限制了对其潜力的充分利用。可以解决该问题的一种技术是泡沫分馏。在这项研究中,我们表明，多种天然泡沫稳定蛋白融合为β-内酰胺酶的F-标签，青霉素G酰基转移酶，和甲酸脱氢酶，分别,能够通过泡沫分馏介导酶的发泡和回收。所有三种候选物的催化活性在很大程度上得以保留。在适当的分馏条件下，特别是当使用洗涤缓冲液时，一些F标签还允许目标酶与污染蛋白质几乎完全分离。我们发现，F-Tag与目标酶之间的较大距离对维持催化活性具有积极作用。然而,我们没有发现任何影响F标签性能的特定序列基序或物理参数。使用短螺旋F-Tag获得最佳结果，最初旨在仅用作接头序列。研究结果表明，开发分子标签可以建立无表面活性剂的泡沫分馏以进行酶后处理是一种有前途的方法。关键点:•稳定泡沫的蛋白质介导酶的活性保持泡沫分级分离•作为F-标签的性能不限于特定的结构基序•与来自低泡沫稳定性和泡沫洗涤的未标记蛋白质的分离益处。
