【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(dǎo)下催化還氧化物發(fā)生開(kāi)環(huán)反應(yīng)生成相應(yīng)的β-取代醇。因此,該酶在降解環(huán)境有機(jī)鹵化物污染物和生產(chǎn)手性藥物中間體等領(lǐng)域具有重要作用。但野生態(tài)的鹵醇脫鹵酶在實(shí)際應(yīng)用中存在一定弊端,對(duì)鹵醇脫鹵酶進(jìn)行定向改造和突變體文庫(kù)的篩選成為該領(lǐng)域的重要工作。為此,一種快速、靈敏的文庫(kù)篩選方法的建立就顯得尤為重要。目前對(duì)于檢測(cè)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(kāi)環(huán)反應(yīng)檢測(cè)方法暫無(wú)報(bào)導(dǎo)。由于鹵醇脫鹵酶HheC在催化環(huán)氧化物開(kāi)環(huán)反應(yīng)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(shù)可以用來(lái)檢測(cè)鹵醇脫鹵酶催化環(huán)氧化物開(kāi)環(huán)反應(yīng)活性�；诖�,本論文以HheC催化疊氮介導(dǎo)的CHBE(全稱(chēng)及中文名)環(huán)氧化物開(kāi)環(huán)反應(yīng)作為模式反應(yīng)體系,建立一種基于傅里葉變換紅外(FTIR)技術(shù)的鹵醇脫鹵酶的酶活檢測(cè)方法。首先在酶促反應(yīng)條件下檢測(cè)游離N3-及其開(kāi)環(huán)產(chǎn)物疊氮取代醇所對(duì)應(yīng)的伸縮振動(dòng)峰位置,分別在2048cm-1和2115cm-1波數(shù)處,同時(shí)分別制作兩種化合物的標(biāo)準(zhǔn)曲線,均與峰面積具有較好的線性關(guān)系;通過(guò)在酶促反應(yīng)中檢測(cè)2048cm-1和2115cm-1波數(shù)處吸光度隨時(shí)間變化的曲線,得出底物NaN3及產(chǎn)物4-疊氮-3-羥基丁酸乙酯濃度隨時(shí)間變化的曲線,進(jìn)而計(jì)算得到鹵醇脫鹵酶的比酶活為0.19 U/mg。同時(shí)我們也對(duì)其它5種環(huán)氧化物的酶活性進(jìn)行了檢測(cè),結(jié)果均與氣相色譜法(GC)所測(cè)得的值接近。表明應(yīng)用FTIR方法進(jìn)行鹵醇脫鹵酶酶活檢測(cè)切實(shí)可行。與傳統(tǒng)的GC方法相比,該檢測(cè)手段具有連續(xù)、直觀、實(shí)時(shí)、快速等優(yōu)勢(shì)。紅外光譜技術(shù)能靈敏感知酶與底物結(jié)合過(guò)程中引起的微小結(jié)構(gòu)變化,在研究酶催化反應(yīng)結(jié)構(gòu)動(dòng)力學(xué)的研究領(lǐng)域具有較大的優(yōu)勢(shì),我們嘗試著應(yīng)用該技術(shù)來(lái)研究鹵醇脫鹵酶催化反應(yīng)過(guò)程中的結(jié)構(gòu)變化。研究結(jié)果表明:在有和沒(méi)有N3-基團(tuán)存在時(shí),鹵醇脫鹵酶的紅外吸收峰位置及峰面積均沒(méi)有明顯變化。結(jié)合該酶前期的相關(guān)研究結(jié)果,可能是由于N3-基團(tuán)的紅外吸收較弱以及結(jié)合有N3-基團(tuán)的鹵醇脫鹵酶所占的比例較低造成的。通過(guò)提高底物和酶的濃度,沒(méi)有得到預(yù)期的結(jié)果,為此,我們擬通過(guò)引入其他可能的紅外探針來(lái)解決問(wèn)題。目前通過(guò)非天然氨基酸標(biāo)記蛋白質(zhì)從而引入紅外光譜探針的手段受到廣泛關(guān)注,因?yàn)樵摲椒o(wú)標(biāo)記位點(diǎn)的局限。為此,我們借助基因密碼子擴(kuò)展技術(shù),通過(guò)在大腸桿菌體內(nèi)轉(zhuǎn)入一種正交改造過(guò)的tRNA和與之配對(duì)的氨酰-tRNA合成酶,即可實(shí)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(duì)應(yīng)氨基酸的特定位點(diǎn)處的標(biāo)記。結(jié)果表明:該表達(dá)體系已經(jīng)構(gòu)建成功,并且成功表達(dá)和純化在特定位點(diǎn)引入外源紅外探針標(biāo)記的鹵醇脫鹵酶,為后續(xù)應(yīng)用紅外光譜技術(shù)研究鹵醇脫鹵酶催化過(guò)程的結(jié)構(gòu)動(dòng)力學(xué)奠定基礎(chǔ)。
[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(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O657.33;O643.3

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