Department of Science, Systems, and Models (NSM), Biomaterials, Roskilde University, DK-4000 Roskilde, Denmark.
Anal Biochem. 2011 Mar 1;410(1):19-26. doi: 10.1016/j.ab.2010.11.001. Epub 2010 Nov 11.
In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.
在用于生物质转化的木质纤维素原料中，半纤维素构成了很大一部分，其中木聚糖是主要成分。尽管许多预处理方法可以减少木聚糖的含量或改变其分布，但降解残留的木聚糖以提高整体产率很重要。通常，通过化学定量测定还原末端来测量木聚糖酶反应的速率。使用等温滴定量热法（ITC），可以连续测量水解的热流，反应速率与热流成正比。碳水化合物水解的反应焓通常低于 5kJ/mol，这是使用当前 ITC 技术直接对酶促反应速率进行量热定量的限制因素。为了增加表观反应焓，我们采用碳水化合物氧化酶和过氧化氢酶对水解产物进行后续氧化。在这里，我们展示了用碳水化合物氧化酶和过氧化氢酶偶联测定法来测定来自 Aspergillus aculeatus 的 GH10 木聚糖酶在桦木木聚糖和小麦阿拉伯木聚糖上的酶动力学。结果根据对四种使用经过充分表征的底物的化学还原末端定量方法的灵敏度的严格分析进行了讨论。
