| Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method
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Author:
Date:
2017-06-20
[Abstract] α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also named Thermofluor. We have shown that Thermofluor can also be applied to predict binding of substrates and inhibitors to a yeast α-glucosidase. The methods described here in detail were used in Viigand et al., 2016.
[摘要] α-葡糖苷酶（包括麦芽糖酶和异麦芽糖酶）是从一组α-葡糖苷底物释放葡萄糖的酶。 可以使用该特征来测定其催化活性，底物特异性和热稳定性。 蛋白质的热稳定性也可以使用高通量差示扫描荧光测定法（也称为Thermofluor）来测定。 我们已经表明，Thermofluor也可以应用于预测底物和抑制剂与酵母α-葡萄糖苷酶的结合。 这里详细描述的方法用于Viigand等人，2016。
【背景】麦芽糖酶（EC 3.2.1.20）和异麦芽糖酶（EC 3.2.1.10）是根据CAZy分类属于糖苷水解酶家族13的α-葡糖苷酶（Lombard等，2014）。甲基营养酵母多形汉酵母的麦芽糖酶MAL1是非选择性的，它将产生D-葡萄糖的麦芽糖和异麦芽糖状α-葡萄糖苷水解为反应产物之一。因此，麦芽糖酶对其底物的活性可以根据葡萄糖释放来确定。该工作描述的葡萄糖液色辅助方法可以快速方便地测定麦芽糖酶的活性，底物特异性和热稳定性。重要的是，这种基于活性的方法可以适用于产生葡萄糖作为反应产物的其它酶。高通量Thermofluor方法主要用于蛋白质晶体学测量（热）稳定性蛋白质（Boivin等，2013; Ericsson等，2006）。我们使用Thermofluor ...
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| Evolution of Escherichia coli to Macrophage Cell Line
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Author:
Date:
2014-09-05
[Abstract] The genomes of species of Escherichia coli (E. coli) show an extraordinary amount of diversity, which include commensal strains and strains belonging to different pathovars. Many strains of E. coli, which can cause mild or severe pathologies in humans, have a commensal ancestor. Understanding the evolutionary changes that can lead to a transition from commensal to pathogen is an important task, which requires integration of different methodologies. One method is experimental evolution of bacteria, in controlled environments, that mimic some of the selective ...
[摘要] 大肠杆菌物种的基因组（大肠杆菌）显示出非凡的多样性，其包括共生菌株和属于不同病原体的菌株。许多菌株。大肠杆菌，其可引起人类的轻度或严重病理，具有共生祖先。了解可导致从共生转变为病原体的进化变化是一项重要任务，需要整合不同的方法。一种方法是在受控环境中的细菌的实验进化，其模拟一些选择性压力，在向发病过渡期间可能是重要的。这种转变的成功将至少部分地取决于E的能力。大肠杆菌以适应免疫系统细胞的存在。在这里，我们描述了用于进行共生应变的实验进化的方案。大肠杆菌，在充分研究的K12的衍生物，在先天免疫系统的细胞施加的恒定选择压力下，特别是RAW 264.7鼠巨噬细胞细胞系。
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| Fitness Measurements of Evolved Esherichia coli
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Author:
Date:
2014-09-05
[Abstract] Bacteria can adapt very rapidly to novel selective pressures. In the transition from commensalism to pathogenicity bacteria have to face and adapt to the host immune system. Specifically, the antagonistic interaction imposed by one of the first line of defense of innate immunity cells, macrophages, on commensal bacteria, such as Escherichia coli (E. coli), can lead to its rapid adaptation. Such adaptation is characterized by the emergence of clones with mutations that allow them to better escape macrophage phagocytosis. Here, we describe how to quantify the amount of fitness ...
[摘要] 细菌可以非常迅速地适应新的选择压力。在从共生到致病性的转变中，细菌必须面对并适应宿主免疫系统。具体地，由先天免疫细胞（巨噬细胞）的第一防御线对共生细菌如大肠杆菌（大肠杆菌）（大肠杆菌）施加的拮抗相互作用可以导致其快速适应。这种适应的特征在于具有允许它们更好地逃脱巨噬细胞吞噬的突变的克隆的出现。在这里，我们描述如何量化在巨噬细胞的恒定选择压力下，从鼠细胞系RAW 264.7进化的细菌克隆的适应增加的量。用于测量沿进化实验室实验的适应度变化的最广泛使用的测定法是竞争性健身测定法。该测定法包括确定进化的菌株在竞争中胜过祖先的速度，其中每个以相同的频率开始。菌株在进化实验的相同环境中竞争，并且如果进化菌株已经获得强的有益突变，其将在重复的竞争性健康测定中变得显着过表达。
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