| Saccharification Protocol for Small-scale Lignocellulosic Biomass Samples to Test Processing of Cellulose into Glucose
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Author:
Date:
2016-01-05
[Abstract] Second generation biofuels are derived from inedible lignocellulosic biomass of food and non-food crops. Lignocellulosic biomass is mainly composed of cell walls that contain a large proportion of cellulosic and hemicellulosic polysaccharides. An interesting route to generate biofuels and bio-based materials is via enzymatic hydrolysis of cell wall polysaccharides into fermentable sugars, a process called saccharification. The released sugars can then be fermented to fuels, e.g., by use of yeast.
To test the saccharification efficiency of lignocellulosic biomass on a lab-scale, a manual saccharification protocol was established that uses only small amounts of biomass and a low concentration of enzyme. This protocol can be used for different plant species like Arabidopsis ...
[摘要] 第二代生物燃料衍生自食物和非食物作物的不可食用的木质纤维素生物质。木质纤维素生物质主要由含有大比例的纤维素和半纤维素多糖的细胞壁组成。生成生物燃料和生物基材料的有趣途径是通过将细胞壁多糖酶水解成可发酵的糖，这一过程称为糖化。释放的糖然后可以例如通过使用酵母发酵成燃料。
为了在实验室规模上测试木质纤维素生物质的糖化效率，建立了仅使用少量生物质和低浓度酶的手动糖化方案。该方案可以用于不同的植物物种，例如拟南芥（Arabidopsis thaliana），烟草，玉米和杨树。低的酶浓度使得可以检测糖化产量的微妙改进并分析水解的速度。虽然包括特定的酸和碱预处理，但是糖化步骤可以在任何其它预处理之后。由于不需要先进的设备，该方案可以在许多实验室进行以分析糖化产量。该方案最初在Van Acker等人（2013）中描述。
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| Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra
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Author:
Date:
2013-11-20
[Abstract] Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building blocks differ in their degree of methoxylation and their biosynthetic pathway is catalyzed by more than 10 enzymes. HCT plays a crucial role by channeling the phenylpropanoids towards the production of coniferyl and sinapyl alcohols. Interestingly, HCT has been reported to be implicated in the pathway both upstream and downstream of the 3-hydroxylation of the aromatic ring of p-coumaroyl shikimate (Figure 1) (Hoffmann et al., ...
[摘要] 木质素是沉积在二次增厚的植物细胞壁中的复杂酚类聚合物。聚合物主要衍生自当偶联时产生对羟基苯基，愈创木基和紫丁香基单元（分别为H，G和S单元）的三种主要单独的木质素：p - 香豆酰基，松柏基和芥子醇进入聚合物。结构单元的甲氧基化程度不同，它们的生物合成途径被超过10种酶催化。 HCT通过将苯丙素类导向松柏醇和芥子醇的生产来发挥关键作用。有趣的是，据报道，HCT涉及p - 香豆酰基莽草酸的芳环的3-羟基化的上游和下游途径（图1）（Hoffmann等， 2003; Hoffmann等人，2004; Vanholme等人，2013b）。这些特征突出了开发在植物中可靠地测量HCT活性的测定法的重要性。在这里，我们描述了基于UPLC-MS的方法，用于分析黑曲霉的木质部总蛋白提取物中的HCT活性，其可以适用于其他木本和草本植物物种。该方案最初在Vanholme等人（2013a）中描述。
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| High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates
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Author:
Date:
2013-07-20
[Abstract] β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a ...
[摘要] β-半乳糖苷酶和β-葡糖醛酸糖苷酶通常用作从基因启动子 - lacZ/em或uidA/em融合（分别）的基因表达的报道分子。本文所述的方案是利用荧光底物（Fiksdal等人，1994）和96孔板形式的通常使用的Miller测定法（Miller，1972）的高通量替代物。荧光底物4-甲基伞形基β-D-半乳糖苷（用于β-半乳糖苷酶测定）（Ramsay等人，2013）或4-甲基伞形基β-D-葡萄糖醛酸苷（用于β-葡糖醛酸糖苷酶测定）（ Ramsay等人，2011）切割以产生荧光产物4-甲基伞形酮。通过冷冻 - 融化和溶菌酶使细胞透化，并且通过荧光微板读数器作为动力学测定连续监测4-甲基伞形酮的产生。然后计算荧光增加的速率，从中推断相对基因表达水平。由于高灵敏度基于荧光的4-甲基伞形酮的检测和所收集的高密度时间点，该测定可以在低水平基因表达的定量中提供增加的准确度。该测定需要小的样品体积和最小的制备时间。本方案中概述的透化条件已经针对革兰氏阴性细菌（特别是大肠杆菌和沙雷氏菌）进行了优化，但是可能适合于具有最小优化的其他生物体。
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