提升BsPPS安定性應用於天然多酚磷酸化之研究

Abstract

多酚類磷酸酯合成酶(Phenolic phosphate synthetase, BsPPS)為本實驗室先前鑑定出之新穎雙激酶，能以ATP作為磷酸供體進行多酚類化合物之磷酸酯化反應，其磷酸酯產物展現出顯著之水溶性與生物可利用率提升。BsPPS具廣泛之基質催化能力，涵蓋黃酮類、查耳酮、二苯乙烯類、薑黃素類、蒽醌類、香豆雌酚類以及香豆素類等近40種化合物，顯示其在天然物磷酸酯化合物開發上的高度潛力。本實驗室先前以BsPPS搭配ATP再生酵素ErPPK建立一套耦合雙酵素多酚類磷酸酯轉化系統，實際應用於木犀草素磷酸酯之合成。然而兩者最適催化溫度及安定性之差異使該系統受限於37°C下操作。有鑒於此，本研究嘗試透過定向演化(directed evolution)與理性設計(rational design)之蛋白質工程策略提升BsPPS之熱安定性，並降低熱安定性與催化活性取捨(trade-off)。期望獲得一株兼具良好熱安定性與催化活性之BsPPS突變株，進一步拓展該耦合雙酵素系統於高溫操作條件下之應用潛力，並提升整體磷酸酯化之效能。實驗第一部分我們首先針對定向演化之實務需求，以具螢光特性之umbelliferone (UMB)建立一套具高靈敏度與再現性之高通量活性篩選平台。實驗證實BsPPS可於UMB之7-OH位點進行單磷酸酯化，生成umbelliferone 7-O-phosphate (U7P)，其結構由LC-ESI-MS/MS與1H-31P HSQC NMR鑑定。由於磷酸酯化後U7P無法去質子化，而可藉由螢光減少量推估BsPPS磷酸酯化活性。實驗第二部分系統性評估最適UMB螢光量測條件，確立於pH 7.5、50% (v/v)甲醇環境下，以360/460 nm作為激發/放射波長進行螢光之量測。所建立之螢光定量方法於1~5 µM範圍內具良好線性(R² = 0.994)，且在同日間與異日間分析中皆展現良好準確度與精密度(CV < 10%)。基質效應測試亦顯示反應體系中ATP、Mg²⁺與DMSO等成分不會對螢光訊號造成干擾。實驗第三部分結合該平台，建構一套完整之BsPPS定向演化流程，涵蓋error-prone PCR隨機突變庫建立、96孔盤突變株重組蛋白表現、粗酵素液製備與突變株篩選。於低突變率條件下建立之突變庫大小約為2.82 × 10⁴ transformants，突變率為3.48 mutations/kb。透過優化lysozyme破菌條件，並評估孔間變異與流程重複性，確認該平台具良好之再現性，並已累計篩選3,360株突變株。

Phenolic phosphate synthetase (BsPPS) is a novel dikinase previously identified by our lab that catalyzes the phosphorylation of polyphenolic compounds using ATP as the phosphate donor. The phosphorylated polyphenol exhibit significantly improved solubility and bioavailability. In addition, BsPPS displays broad substrate promiscuity, catalyzing the phosphorylation of flavonoids, chalcones, curcuminoids, anthraquinones, coumestans and coumarins up to 40 different polyphenolic compounds, highlighting its potential for the development of phosphate prodrugs derived from natural products. Previously, our lab established a coupled bienzymatic polyphenol phosphorylation system by combining BsPPS with an ATP regeneration enzyme, ErPPK, and successfully applied to the synthesis of luteolin phosphate. However, due to the distinct optimal catalytic temperatures and thermostability of BsPPS and ErPPK, the coupled system is currently restricted to operation at 37°C. To address this limitation, our study aimed to enhance the thermostability of BsPPS through directed evolution and rational design strategies, while minimizing the trade-off between stability and catalytic activity, in order to obtain a mutant with improved stability and retained enzymatic function. In the first part of this study, a high-throughput activity screening platform was established, using the fluorescent substrate umbelliferone (UMB). Experimental results demonstrated that BsPPS catalyzes the phosphorylation of UMB at the 7-OH position to form umbelliferone 7-O-phosphate (U7P), whose structure was confirmed by LC-ESI-MS/MS and 1H–31P HSQC NMR analyses. Phosphorylation at the 7-OH position suppresses deprotonation of UMB, resulting in a pronounced decrease in fluorescence intensity, which enables quantification of BsPPS activity based on fluorescence reduction. In the second part, the fluorescence measurement conditions for UMB were optimized. Optimal conditions were determined to be pH 7.5 in 50% (v/v) methanol, with excitation and emission wavelengths at 360 and 460 nm, respectively. The established fluorescence assay exhibited good linearity over a concentration range of 1–5 µM (R² = 0.994), as well as satisfactory accuracy and precision in both intra-day and inter-day analyses (CV < 10%). Matrix effect tests further confirmed that ATP, Mg²⁺, and DMSO present in the reaction system did not affect the fluorescence signal. In the third part, the screening platform was integrated to construct a directed evolution workflow for BsPPS, using error-prone PCR to generate the mutant library, recombinant protein expression in 96-well plates, crude enzyme preparation, and mutant screening. Under low mutation-rate conditions, a mutant library of approximately 2.82 × 10⁴ transformants was established, with an average mutation rate of 3.48 mutations/kb. Following optimization of cell lysis conditions and evaluation of variability and process reproducibility, the platform was confirmed to be robust and reliable, enabling the screening of a total of 3,360 mutants.