应用红外光谱检测卤醇脱卤酶催化活性及初步研究其催化过程的结构基础

发布时间：2019-05-23 13:25

【摘要】：卤醇脱卤酶HheC既能催化邻卤醇碳-卤键断裂生成还氧化物,也可以在多种亲核试剂介导下催化还氧化物发生开环反应生成相应的β-取代醇。因此,该酶在降解环境有机卤化物污染物和生产手性药物中间体等领域具有重要作用。但野生态的卤醇脱卤酶在实际应用中存在一定弊端,对卤醇脱卤酶进行定向改造和突变体文库的筛选成为该领域的重要工作。为此,一种快速、灵敏的文库筛选方法的建立就显得尤为重要。目前对于检测卤醇脱卤酶催化邻卤醇脱卤的筛选方法较为成熟,但方便可靠的开环反应检测方法暂无报导。由于卤醇脱卤酶HheC在催化环氧化物开环反应中的各种亲核试剂N3-(N=N=N)、CN-(C=N)、OCN-(O=C=N)、SCN-(S=C=N)具有区别于蛋白酰胺I带的特征红外吸收峰,原则上红外光谱技术可以用来检测卤醇脱卤酶催化环氧化物开环反应活性。基于此,本论文以HheC催化叠氮介导的CHBE(全称及中文名)环氧化物开环反应作为模式反应体系,建立一种基于傅里叶变换红外(FTIR)技术的卤醇脱卤酶的酶活检测方法。首先在酶促反应条件下检测游离N3-及其开环产物叠氮取代醇所对应的伸缩振动峰位置,分别在2048cm-1和2115cm-1波数处,同时分别制作两种化合物的标准曲线,均与峰面积具有较好的线性关系;通过在酶促反应中检测2048cm-1和2115cm-1波数处吸光度随时间变化的曲线,得出底物NaN3及产物4-叠氮-3-羟基丁酸乙酯浓度随时间变化的曲线,进而计算得到卤醇脱卤酶的比酶活为0.19 U/mg。同时我们也对其它5种环氧化物的酶活性进行了检测,结果均与气相色谱法(GC)所测得的值接近。表明应用FTIR方法进行卤醇脱卤酶酶活检测切实可行。与传统的GC方法相比,该检测手段具有连续、直观、实时、快速等优势。红外光谱技术能灵敏感知酶与底物结合过程中引起的微小结构变化,在研究酶催化反应结构动力学的研究领域具有较大的优势,我们尝试着应用该技术来研究卤醇脱卤酶催化反应过程中的结构变化。研究结果表明:在有和没有N3-基团存在时,卤醇脱卤酶的红外吸收峰位置及峰面积均没有明显变化。结合该酶前期的相关研究结果,可能是由于N3-基团的红外吸收较弱以及结合有N3-基团的卤醇脱卤酶所占的比例较低造成的。通过提高底物和酶的浓度,没有得到预期的结果,为此,我们拟通过引入其他可能的红外探针来解决问题。目前通过非天然氨基酸标记蛋白质从而引入红外光谱探针的手段受到广泛关注,因为该方法无标记位点的局限。为此,我们借助基因密码子扩展技术,通过在大肠杆菌体内转入一种正交改造过的tRNA和与之配对的氨酰-tRNA合成酶,即可实现非天然氨基酸在卤醇脱卤酶中引入琥珀终止密码子UAG所对应氨基酸的特定位点处的标记。结果表明:该表达体系已经构建成功,并且成功表达和纯化在特定位点引入外源红外探针标记的卤醇脱卤酶,为后续应用红外光谱技术研究卤醇脱卤酶催化过程的结构动力学奠定基础。
[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.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.33;O643.3

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