Suppr超能文献

大麦种子中 26kDa 内壳素酶:使用电喷雾电离质谱实时监测酶反应和底物结合实验。

26kDa endochitinase from barley seeds: real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry.

机构信息

Analytical Research Group, Chair of Biopolymer Chemistry, Department for Basic Life Sciences, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising-Weihenstephan, Germany.

出版信息

J Biotechnol. 2009 Sep 25;143(4):274-83. doi: 10.1016/j.jbiotec.2009.08.003. Epub 2009 Aug 7.

Abstract

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.

摘要

大麦种子中的一种 26 kDa 内切几丁质酶仅通过电喷雾电离质谱(ESI-MS)进行了酶学特性分析。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先前通过 HPLC 获得的数据一致,并且通过酶水解的动力学建模成功模拟了定量图谱。显然,通过 ESI-MS 进行的实时监测方法可以更快、更便宜地测定未标记底物的几丁质酶活性。此外,还分析了大麦几丁质酶 E67Q 突变体的酶活性,并通过比较天然和变性条件下获得的酶蛋白质谱,讨论了 Glu67 的作用。然后确定在 E67Q 中观察到的酶活性丧失肯定是由于 Glu67 的点突变引起的,而不是由于突变酶的部分展开引起的。最后,通过质谱获得的 Scatchard 图计算了酶-寡糖复合物的结合常数。对于 E67Q,获得的结合自由能值与先前在液相中获得的值相当,但对寡糖链长的依赖性较小。据我们所知,这项研究是首次通过这种创新的 ESI-MS 系统对几丁质酶进行酶学特性分析。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验