發(fā)布時間：2019-05-23 13:25

【摘要】：鹵醇脫鹵酶HheC既能催化鄰鹵醇碳-鹵鍵斷裂生成還氧化物,也可以在多種親核試劑介導下催化還氧化物發(fā)生開環(huán)反應生成相應的β-取代醇。因此,該酶在降解環(huán)境有機鹵化物污染物和生產手性藥物中間體等領域具有重要作用。但野生態(tài)的鹵醇脫鹵酶在實際應用中存在一定弊端,對鹵醇脫鹵酶進行定向改造和突變體文庫的篩選成為該領域的重要工作。為此,一種快速、靈敏的文庫篩選方法的建立就顯得尤為重要。目前對于檢測鹵醇脫鹵酶催化鄰鹵醇脫鹵的篩選方法較為成熟,但方便可靠的開環(huán)反應檢測方法暫無報導。由于鹵醇脫鹵酶HheC在催化環(huán)氧化物開環(huán)反應中的各種親核試劑N3-(N=N=N)、CN-(C=N)、OCN-(O=C=N)、SCN-(S=C=N)具有區(qū)別于蛋白酰胺I帶的特征紅外吸收峰,原則上紅外光譜技術可以用來檢測鹵醇脫鹵酶催化環(huán)氧化物開環(huán)反應活性。基于此,本論文以HheC催化疊氮介導的CHBE(全稱及中文名)環(huán)氧化物開環(huán)反應作為模式反應體系,建立一種基于傅里葉變換紅外(FTIR)技術的鹵醇脫鹵酶的酶活檢測方法。首先在酶促反應條件下檢測游離N3-及其開環(huán)產物疊氮取代醇所對應的伸縮振動峰位置,分別在2048cm-1和2115cm-1波數(shù)處,同時分別制作兩種化合物的標準曲線,均與峰面積具有較好的線性關系;通過在酶促反應中檢測2048cm-1和2115cm-1波數(shù)處吸光度隨時間變化的曲線,得出底物NaN3及產物4-疊氮-3-羥基丁酸乙酯濃度隨時間變化的曲線,進而計算得到鹵醇脫鹵酶的比酶活為0.19 U/mg。同時我們也對其它5種環(huán)氧化物的酶活性進行了檢測,結果均與氣相色譜法(GC)所測得的值接近。表明應用FTIR方法進行鹵醇脫鹵酶酶活檢測切實可行。與傳統(tǒng)的GC方法相比,該檢測手段具有連續(xù)、直觀、實時、快速等優(yōu)勢。紅外光譜技術能靈敏感知酶與底物結合過程中引起的微小結構變化,在研究酶催化反應結構動力學的研究領域具有較大的優(yōu)勢,我們嘗試著應用該技術來研究鹵醇脫鹵酶催化反應過程中的結構變化。研究結果表明:在有和沒有N3-基團存在時,鹵醇脫鹵酶的紅外吸收峰位置及峰面積均沒有明顯變化。結合該酶前期的相關研究結果,可能是由于N3-基團的紅外吸收較弱以及結合有N3-基團的鹵醇脫鹵酶所占的比例較低造成的。通過提高底物和酶的濃度,沒有得到預期的結果,為此,我們擬通過引入其他可能的紅外探針來解決問題。目前通過非天然氨基酸標記蛋白質從而引入紅外光譜探針的手段受到廣泛關注,因為該方法無標記位點的局限。為此,我們借助基因密碼子擴展技術,通過在大腸桿菌體內轉入一種正交改造過的tRNA和與之配對的氨酰-tRNA合成酶,即可實現(xiàn)非天然氨基酸在鹵醇脫鹵酶中引入琥珀終止密碼子UAG所對應氨基酸的特定位點處的標記。結果表明:該表達體系已經構建成功,并且成功表達和純化在特定位點引入外源紅外探針標記的鹵醇脫鹵酶,為后續(xù)應用紅外光譜技術研究鹵醇脫鹵酶催化過程的結構動力學奠定基礎。
[Abstract]:The halohydrin dehalogenase HheC not only can catalyze the generation of the o-halohydrin carbon-halogen bond, but also can catalyze the ring-opening reaction of the metal halide to generate the corresponding halogen-substituted alcohol under the mediation of a plurality of nucleophiles. Therefore, the enzyme plays an important role in the fields of degrading environment organic halide pollutants and producing chiral drug intermediates. However, there are some disadvantages in the practical application of the wild-type halohydrin dehalogenase, and the orientation transformation of the halohydrin dehalogenase and the screening of the mutant library have become an important work in this field. To this end, the establishment of a fast and sensitive library screening method is particularly important. Currently, the method for screening the halogen-free alcohol dehalogenation by the halogen-free dehalogenation enzyme is more mature, but the method is convenient and reliable, and the open-loop reaction detection method is not reported. Because of the various nucleophiles N3-(N = N = N), CN-(C = N), OCN-(O = C = N), SCN-(S = C = N) in the ring-opening reaction of the catalytic epoxide, the halohydrin dehalogenase HheC has the characteristic infrared absorption peak which is different from the protein and the amine I band. In principle, the infrared spectroscopy technique can be used to detect the ring-opening reaction activity of the halohydrin dehalogenase catalytic epoxide. On the basis of this, the open-loop reaction of CHBE (full name and Chinese name), which is mediated by HheC, is used as a mode reaction system, and an enzyme activity detection method based on Fourier transform infrared (FTIR) technology is established. First, the position of the free N3-and its open-loop product azido-substituted alcohol is detected under the condition of enzymatic reaction, and the standard curves of the two compounds are respectively prepared at the positions of 2048 cm-1 and 2115 cm-1, and the linear relationship with the peak area is good. The curve of the concentration of the substrate NaN3 and the product 4-azido-3-hydroxybutyrate over time was obtained by detecting the curve of the absorbance over time in the enzymatic reaction of 2048 cm-1 and 2115 cm-1, and then the specific enzyme activity of the halohydrin dehalogenase was calculated to be 0.19 U/ mg. At the same time, we also tested the enzyme activity of five other epoxides, and the results were close to those measured by gas chromatography (GC). It is shown that it is feasible to use the FTIR method to carry out the enzyme activity detection of the halohydrin dehalogenase. Compared with the traditional GC method, the detection method has the advantages of continuous, intuitive, real-time, fast and the like. In order to study the changes of the microstructural changes caused by the binding of the enzyme with the substrate, the infrared spectroscopy has a great advantage in the study of the kinetics of the catalytic reaction of the enzyme, and we try to apply the technology to study the structural changes of the halohydrin dehalogenase catalytic reaction. The results show that there is no significant change in the infrared absorption peak position and the peak area of the halohydrin dehalogenase in the presence of and without the N3-group. In combination with the relevant research results of the prophase of the enzyme, it is possible that the ratio of the N3-group to the infrared absorption of the N3-group is weak and the proportion of the halohydrin dehalogenase which is bound to the N3-group is low. By increasing the concentration of the substrate and the enzyme, no expected results are obtained, and for this reason, we intend to solve the problem by introducing other possible infrared probes. The means to introduce infrared spectroscopy probes by non-native amino acid-labeled proteins are now widely concerned, as the method is free of the limitation of the marker sites. To this end, by means of the gene codon extension technique, by transferring to an orthogonal modified tRNA in the E.coli body and an ammonia-tRNA synthetase that is paired with it, And the mark at the special positioning point of the amino acid corresponding to the amber stop codon UAG is introduced in the halogen alcohol dehalogenation enzyme by the non-natural amino acid. The results show that the expression system has been successfully constructed and successfully expressed and purified the halohydrin dehalogenase which is marked by the exogenous infrared probe at the specific site, and lays the foundation for the subsequent application of the infrared spectroscopy to study the structural dynamics of the halohydrin dehalogenation enzyme catalysis process.
【學位授予單位】：電子科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.33;O643.3

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