標題: 發展用於監測動態生物催化反應之質譜分析方法
Development of Mass Spectrometric Methods for the Monitoring of Biocatalytic Processes
作者: 丁煦
Ting, Hsu
Pawel Urban
關鍵字: 酵素;監測;動態;自動化;氣相層析儀;質譜儀;enzyme;monitoring;dynamic;automation;gas chromatography;mass spectrometry
公開日期: 2013
摘要: 在生物催化反應中,酵素的參與可使化學反應在有限的時間內加速生成並且增加產率。 酵素在細胞及生物流體中無所不在,並且於所有的代謝過程中有著重要的地位. 而它們也被運用在工業合成各式化學品及生物燃料等。在學術界或是工業用途中,為了研究或使用酵素的催化能力,找到適合的分析方法對酵素系統進行化學性質分析是重要且需要的。質譜技術能夠偵測分析物在時間中的變化,對分析物有相當高的特異性;對於監測酵素反應系統的過程相當重要,所以質譜技術在這份論文中可作為一個理想的偵測工具。在第一部分的研究中,我開發了一個質譜分析方法對於假設的化學訊號傳導現象做探討與解釋,此反應中使用的兩種酵素分別為肌激酶和丙酮酸激酶。藉由大氣壓力游離法的配置,呈現在一高長寬比的液體區間中,化學波的傳遞速度超越如對流與擴散等自然傳輸所造成的現象。而當使用同位素標記的受質當作啟動催化的反應物時,可以同時展現及監測到兩種類型的化學傳遞。在第二部分的研究中,我開發了一個自製的自動化設備,包含了開放式資源的電子操作裝置和數個低成本的機械配備,用於監測固定化之脂解酶對轉酯化反應之催化。整套裝置的運行由一套使用Linux系統以及輸入C程式語言的單板機微電腦所操控。由於技術上的改進,在一小體積反應腔中隨著時間變化的樣品可以被自動注射至氣相層析質譜儀做分析。這套裝置可以用來監測由單一顆粒(< 1 毫米) 固定化脂解酶所催化之反應,而這類固態生物催化劑擁有的催化異質性展現於分析結果。此外,這個分析方法也能夠使用於監測植物組織在液相萃取中的動態過程。在此研究中,轉酯化反應中的產物 - 乙酸正丁酯與在植物組織中出現的三種萜類運用此法完成了定量分析。R2 的值在0.944-0.989之間,而偵測極限則是在亞微莫耳之間。簡而言之,兩種不同的質譜分析法分別已被建立,一可用於觀察藉由酵素反應隨者時間與空間所產生之傳輸變化,另一可用於監測工業應用有關的酵素催化反應隨時間所產生的動態變化。
Biocatalysis encompasses the use of enzymes to speed up chemical reactions thus increasing reaction yields within limited time periods. Enzymes are omnipresent. They are involved in all metabolic processes occurring in cells and biofluids. They also find applications in industry, in the synthesis of fine chemicals and biofuels. In order to utilize the catalyzing ability of enzymes in academic research or industry, suitable analytical methods are required to determine their activities. Mass spectrometry is an ideal tool for monitoring the progress of enzymatic processes because it allows one to follow temporal concentration profiles of reactants while ascertaining high specificity. In the first part of the project, I developed a mass spectrometric method to demonstrate a hypothesized phenomenon of chemical signal transduction aided by a set of two enzymes: adenylate kinase and pyruvate kinase. By implementing an atmospheric pressure ionization technique, it was possible to obtain spatiotemporal characteristics of chemical wave propagating in a high-aspect-ratio volume of liquid with a speed exceeding that of natural processes such as convection and diffusion. By using an isotopically labeled substrate (13C10 adenosine triphosphate) as the triggering reagent, the two types of chemical propagation could be revealed and monitored simultaneously. In the second part of the project, I implemented a home-built automated setup – incorporating open-source electronic control unit and a number of inexpensive actuators – to monitor transesterification catalyzed by immobilized lipase. The analysis was guided by a program in C language, deployed on a single-board Linux-based microcomputer. Thanks to this technological improvement, the temporal samples, collected from a low-volume reaction chamber, were automatically injected to the gas chromatography – mass spectrometry apparatus. The proposed system enabled monitoring reactions catalyzed by single microbeads (< 1 mm) of immobilized lipase, and drawing conclusions on catalytic heterogeneity of this solid-state biocatalyst. Furthermore, it allowed one to monitor the progress of liquid extraction of fresh plant tissue. Quantitative analysis of 1-butyl acetate formed in the course of transesterification, and three terpenes – extracted from plant tissue – was achieved. The R2 of the calibration plot was in the range 0.944 – 0.989, and the limits of detection of the target compounds were in the submicromolar range. In summary, two methods have been developed which can assist observation of spatiotemporal phenomena involving enzymes, and temporal screening of dynamic processes relevant to industrial applications.
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