Rapid Kinetic Characterization of Glycosyl Hydrolases Based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)
Rapid Kinetic Characterization of Glycosyl Hydrolases Based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)
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DOI：
10.1021/cb5000289
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发表时间：
2014-07-01
影响因子：
4
通讯作者：
Northen, Trent R.
中科院分区：
文献类型：
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作者：
Deng, Kai;Takasuka, Taichi E.;Northen, Trent R.
糖苷水解酶（GH）对于环境中植物生物质的循环、人类肠道微生物组对复杂多糖的消化以及诸如部署纤维素生物燃料的工业活动至关重要。高通量测序方法显示GH之间存在巨大的序列多样性，但在超过150，000种包含GH的独特结构域排列中，只有相对较少的例子得到了功能表征。在这里，我们展示了如何无细胞表达，生物共轭化学和基于表面的质谱可以用来研究糖苷水解酶与植物生物质的反应。可溶性产物的检测是通过将独特的化学探针以稳定的肟键与寡糖的还原端偶联来实现的，而使用C-13标记的单糖标准品（木糖和葡萄糖）允许对衍生化聚糖进行定量。我们应用这种基于肟的纳米结构引发剂质谱（NIMS）方法来表征由热纤梭菌（Clostridium thermocellum）分泌的GH的功能多样性。新的反应特异性被确定，个别酶的速率和产量的差异被证明在与生物质底物的反应。时间序列数据的数值分析表明，单功能和多功能GH的协同组合可以降低生物燃料生产过程中水解植物生物质所需的酶的复杂性。
Glycoside hydrolases (GHs) are critical to cycling of plant biomass in the environment, digestion of complex polysaccharides by the human gut microbiome, and industrial activities such as deployment of cellulosic biofuels. High-throughput sequencing methods show tremendous sequence diversity among GHs, yet relatively few examples from the over 150,000 unique domain arrangements containing GHs have been functionally characterized. Here, we show how cell-free expression, bioconjugate chemistry, and surface-based mass spectrometry can be used to study glycoside hydrolase reactions with plant biomass. Detection of soluble products is achieved by coupling a unique chemical probe to the reducing end of oligosaccharides in a stable oxime linkage, while the use of C-13-labeled monosaccharide standards (xylose and glucose) allows quantitation of the derivatized glycans. We apply this oxime-based nanostructure-initiator mass spectrometry (NIMS) method to characterize the functional diversity of GHs secreted by Clostridium thermocellum, a model cellulolytic organism. New reaction specificities are identified, and differences in rates and yields of individual enzymes are demonstrated in reactions with biomass substrates. Numerical analyses of time series data suggests that synergistic combinations of mono- and multifunctional GHs can decrease the complexity of enzymes needed for the hydrolysis of plant biomass during the production of biofuels.