適用於含氟化合物生物降解的細胞表面展示氟乙酸脫鹵酶的載體蛋白篩選

Abstract

將目標蛋白質表達在膜上不僅可以防止受質受到外膜的阻礙，還有助於評估酵素的可重複使用性，這對於酵素的回收至關重要。在這項研究中，我們利用不同的跨膜系統 (包括IgA、Ag43和eCPX) 優化大腸桿菌 (E. coli) 膜上目標蛋白質的表面展示。比較分析顯示，Ag43是最佳的表達系統，具有優越的蛋白質表達能力，並且在蛋白質表達在膜外時仍能保持酵素活性。為了驗證這一概念，我們以紅色螢光蛋白 (RFP) 和4,5多巴雙加氧酶為例，展示Ag43系統在表面展示中的效率。這些發現對未來的酵素表達實驗尤其在生物降解應用中具有潛力，本研究篩選用不同系統表達的氟乙酸脫鹵酶 (FAcD)，結果顯示IgA系統具有較高的活性。並且通過MALDI-TOF MS驗證FAcD降解受質後的產物，結果與預期一致。此外，證明IgA-FAcD系統具有良好的重複使用性，以及FAcD的高度穩定性。最後，探討表達於膜外的FAcD在生物降解應用中的潛力，為未來的研究提供重要的參考價值。Expressing the target protein on the membrane prevents substrates from encountering barriers posed by the outer membrane. This approach not only broadens the range of reacting substrates but also facilitates the assessment of enzyme reusability, essential for enzyme recovery. In this study, we optimized the surface presentation of target proteins on the Escherichia coli (E. coli) membrane, employing various transmembrane systems, including IgA, Ag43, and eCPX. Comparative analysis revealed Ag43 as the optimal expression system, showcasing its superior protein expression capability and the ability to maintain enzyme activity when proteins are expressed outside the membrane. To validate the concept, we utilized Red Fluorescent Protein (RFP) and DOPA-dioxygenase as examples, demonstrating the efficiency of the Ag43 system in surface presentation. The findings hold promise for future enzyme expression experiments, particularly in biodegradation applications. We screened for fluoroacetate dehalogenase expressed using different systems, with results indicating higher activity in the IgA system. Additionally, we confirmed the products of FAcD substrate degradation using MALDI-TOF MS, which matched expectations. Furthermore, we demonstrated the excellent reusability of the IgA-FAcD system and the high stability of FAcD. Finally, we discussed the potential of extracellularly expressed FAcD in biodegradation applications, providing valuable insights for future research.