一株由工研院提供的高溫菌株經長期研究證明其吸金能力極佳且專一性強，有利於在環境中以環保的程序回收貴金屬。本研究欲以蛋白質體學分析鑑定其吸金蛋白，首先使用超過濾法及 His-trap 親和性管柱分別分離胞外蛋白，再進行對金離子或奈米金吸附測試，具強吸金能力的蛋白經電泳 (SDS-PAGE) 及串聯式質譜 (Q-TOF-MS) 分析並鑑定出蛋白身分。採用 SWISS-model 模擬分析蛋白結構，推測主要吸金反應的關鍵為正負電荷間的作用力、alpha 螺旋結構提供結合位點、或具有金屬結合點。將目標蛋白以基因工程方法構建於 pET 系統並轉化至大腸桿菌 (E. coli) 中表達，然而測試金離子吸附效果僅些微提升，較原生高溫菌蛋白來的差，推測吸金機制是蛋白中特定胜肽序列的協同效應，後續欲根據 PmrA/PmrB 雙組分蛋白初步建立金屬離子檢測的響應系統直接證明吸金序列的功能性。
另外，因大腸桿菌本身亦可吸附金屬離子，本文比較六株 E. coli 的金離子吸附能力，分別是 W3110、DH5α、BL21 及三株已導入綠色螢光蛋白的 DH5α 菌株 (分別是原生 GFP、優化結構的 sfGFP 及表面展示含金結合胜肽的 FadL-GBP-GFP)。結果顯示皆具有良好的吸金效果且無明顯差異，但螢光蛋白在細胞吸金後造成螢光淬滅且 sfGFP > GFP > FadL-GBP-GFP。進一步研究金、銀及銅離子添加量對於 sfGFP 細胞螢光信號的變化，取得螢光強度 (log Y) 與感應耦合電漿放射光譜 (ICP-OES) 定量具有線性相關性，金、銀、銅的線性回歸結果分別是金：log Y = 1.502 - 0.0337 X (R2 = 0.995)；銀： log Y = 2.039 - 0.0197 X (R2 = 0.964) 及銅： log Y = 2.060 - 0.0333 X (R2 = 0.987)。測量 zeta 電位分析細胞表面在吸附金、銀、銅屬離子前後帶電荷量，金離子的電荷差異最大，達 14 mV，此與螢光淬滅的效應也吻合；金明顯影響了細胞表面的負電及 sfGFP 內部 Ser65-Tyr66-Gly67 殘基自催化環化作用的電子轉移，阻止氧化還原反應的進行而無法發出綠色螢光。本研究取得螢光信號與 ICP-OES 之間的線性方程式，提供了一種效率好且靈敏度高的生物感測新方法。

The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from this thermophilic strain. The results showed that small proteins or peptides have higher ability to adsorb gold and the binding was resulted from the negatively charged domains, alpha-helix and metal binding sites. After heterologous expressed two predicted proteins in E. coli, 65aa enhanced 41.5 % gold adsorption ability while 99aa showed no function. However, they were not better than the protein mixture from wild type. According to the results, the mechanism of Au adsorption in this strain is supposed to be a synergistic effect with specific peptide sequences. Further, we attempt to establish a responsive system to detect metal ions based on the PmrA/PmrB two-component system. Moreover, a novel approach to quantify precious metal ion concentration by fluorescent cells was established. The sfGFP intensity decreased linearly after adsorbed Au3+, Ag+, or Cu2+, thus well correlations between fluorescence intensity and ICP-OES results were obtained, which is a novel, precious, eco-friendly quantified method for metal ion concentration in the solution.

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