以質譜法及化學發光法偵測多重酵素反應

Monitoring Multi-Enzyme Reactions by Mass Spectrometry and Chemiluminescence Detection

陳亭汝(Ting-Ru Chen)

指導教授 ： 帕偉鄂本

國立交通大學/理學院/應用化學系所/碩士(2016年)

摘要

在生物催化領域中，建構一個穩健的生物化學系統能夠幫助研究者了解基本的生物催化間相互合作的機制並且藉此發展出一個新型的生物催化應用。運用生物分子建構一個體外的生化反應或是設計出一個生化網絡是一件創新的基礎應用研究。在生物催化反應中，酵素無所不在且酵素的參與可使生物體中的許多化學反應加速生成。例如，生物體中的生物合成、能量代謝以及生理時鐘都有酵素的參與。在目前的研究中，運用質譜法以及簡單且低成本的化學發光法結合數位相機和微孔盤來進行研究；我們開發出酵素的放大系統可應用於合成生物學以及生物工程領域。第一部份的研究，我們利用兩種不同的酵素(丙酮酸激酶和腺苷酸激酶)組成一個化學能量緩衝系統。生物催化反應通常需要提供化學能以及以三磷酸腺苷(adenosine triphosphate, ATP)的形式提供磷酸根。事實上，在合成生物學中要穩定且持續的提供能量到一個合成系統是很有挑戰性的；低產率往往都是因為能量的短缺(例如:ATP)而造成的。因此，一個穩定且持續提供能量到(體外)生物合成系統是必須的。藉由即時質譜法，在生物合成過程中多重酵素的反應條件可以最佳化以確保高產率。第二部分的研究，我們開發了一個生化計時系統並且以小分子(例如:ATP或二磷酸腺苷(adenosine diphosphate, ADP))來觸發反應。這些核苷酸也會隨之自動放大；當ATP的濃度達到一定的範圍內後，就會產生化學發光，此時的時間點與核苷酸(如ATP或ADP)的初始濃度有關。實驗上所觀察到的現象¬¬-發光時間與觸發核苷酸的初始濃度有關-已經以動力學模型做驗證。由於此方法仰賴的是時間測量，所以利用簡單且低成本的設備就可以有精確的實驗結果；且因為有光的產生，實驗結果也可以直接用眼睛觀察。雖然我們的研究是屬於比較基礎方面的，但結果亦對於應用科學(生物合成、生物感測)上面有一定的重要性。這類的放大系統在合成生物學上可以用於穩定一定程度的能量；在生物感測方面可以利用其發光時間去定量小分子的濃度。

關鍵字

核苷酸 ； 酵素 ； 生物催化 ； 非線性化學 ； 質譜法 ； 化學發光

並列摘要

Constructing robust biochemical circuits can help researchers to understand fundamental mechanisms of biocatalytic co-operativity, and to develop new applications of biocatalysis. Replicating chains of biochemical reactions in vitro or redesigning biochemical networks – using biomolecular building blocks – is a new avenue of exciting fundamental and applied research. Enzymes are omnipresent. They speed up numerous reactions in the living organisms. For example, they are involved in biosynthetic pathways, energy metabolism, and circadian clocks. In the present work, we have developed enzymatic amplification circuits, which could be used in synthetic biology and bioengineering. We have implemented mass spectrometry (MS) as well as a simple low-cost chemiluminescence detection system integrating a digital reflex camera and a microtiter plate to study these circuits. In the first part of the project, we developed a chemical energy buffer system using two different enzymes (pyruvate kinase and adenylate kinase). Biocatalytic reactions often require the supply of chemical energy and phosphate groups in the form of adenosine triphosphate (ATP). In fact, supplying chemical energy to cell-free biosynthetic systems is one of the biggest challenges of synthetic biology. Shortage of energy-rich substrates (such as ATP) leads to low reaction yields. Therefore, a continuous supply of energy-rich substrates to biosynthetic reactions, carried out in vitro, has to be secured. By employing the real-time MS approach, conditions of multi-enzyme reactions could be optimized ensuring high yields of the target biosynthetic processes. In the second part of the project, we developed a biochemical timer reaction. The timer is triggered by small amounts of ATP or adenosine diphosphate (ADP). The nucleotides are spontaneously amplified. When the concentration of ATP reaches a certain level, light is emitted. The time from the start of the reaction to the appearance of light is related to the initial concentration of adenosine nucleotides (e.g. ATP or ADP). Validity of the observed dependence of the luminescence raise time on the concentration of the trigger nucleotide has been verified with kinetic models. Using the simple and low-cost apparatus, precise measurements could be carried out at optimal conditions. Because this method relies on time measurement (not light intensity measurement), it does not require calibration of the optical detector. Due to the emission of light, the effect of the reaction can easily be observed by eye. The proposed timer reaction concept can be used in the bioengineered systems, in which time of response needs to be linked with the concentration of a biomolecule. Although our work has fundamental aspects, its outcomes can also have implications on the applied science (biosynthesis, biosensing). The amplification circuits can be used to stabilize levels of energy-rich nucleotides in biosynthetic protocols, or to determine their concentrations based on time-of-luminescence.

並列關鍵字

Nucleotides ； Enzymes ； Biocatalysis ； Non-linear chemistry ； Mass spectrometry ； Chemiluminescence

參考文獻

3. Manchester K. L., Trends Biotechnol., 1995, 13, 511-515.

6. Buchner, E., Nobel Laureate Biography., Nobelprize.org., 1907.

7. Henri, V., Lois générales de l'action des diastases, Hermann., Paris, 1903.

10. Knowles, J. R., Ann. Rev. Biochem., 1980, 49, 877-919.

11. Kholodenko, B. N., Nat. Rev. Mol. Cell Biol., 2006, 7, 165-176.