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首页> 外文期刊>Journal of Biotechnology >26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry
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26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

机译：大麦种子中的26kDa内切壳多糖酶：使用电喷雾电离质谱法实时监测酶促反应和底物结合实验

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摘要

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme-oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.
机译：通过电喷雾电离质谱（ESI-MS）专门对来自大麦种子的26 kDa内切几丁质酶进行了酶学表征。首先，通过ESI-MS实时监测大麦几丁质酶催化的寡糖水解。通过ESI-MS监测获得的反应时间过程与早期通过HPLC获得的数据一致，并且通过酶促水解的动力学模型成功地模拟了定量曲线。显然，通过ESI-MS进行的实时监控方法可以更快，更便宜地确定未标记底物的几丁质酶活性。此外，分析了大麦几丁质酶E67Q突变体的酶活性，并比较了在自然和变性条件下获得的酶蛋白的质谱图，讨论了Glu67的作用。然后确定观察到的E67Q中酶活性的丧失肯定是由Glu67的点突变引起的，而不是由于突变酶的部分展开而引起的。最后，从质谱获得的斯卡查德图计算酶-寡糖复合物的缔合常数。发现对于E67Q获得的结合自由能值与先前在液相中获得的结合自由能值相当，但是较少依赖寡糖的链长。据我们所知，这项研究是仅通过这种创新的ESI-MS系统对几丁质酶的首次酶促表征。

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