Tuveng Tina R, Eijsink Vincent G H, Arntzen Magnus Ø
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.
Methods Mol Biol. 2019;1871:159-177. doi: 10.1007/978-1-4939-8814-3_12.
Secretomes from microorganisms growing on biomass contain carbohydrate-active enzymes (CAZymes) of potential biotechnological interest. By analyzing such secretomes, we may discover key enzymes involved in degradation processes and potentially infer the mode-of-action of biomass conversion. Some of these enzymes may have predicted functions in carbohydrate degradation, while others may not, while yet exhibiting a similar expression pattern; these latter enzymes constitute potential novel enzymes involved in the degradation process and provide a basis for further biochemical exploration. Hence, secretomes represent an important source for the study of both predicted and novel CAZymes. Here we describe a plate-based culturing technique that allows for collection of protein fractions that are highly enriched for secreted proteins, bound or unbound to the substrate, and which minimizes contamination by intracellular proteins trough unwanted cell lysis.
生长在生物质上的微生物分泌组含有具有潜在生物技术价值的碳水化合物活性酶（CAZymes）。通过分析此类分泌组，我们可能发现参与降解过程的关键酶，并有可能推断生物质转化的作用模式。其中一些酶可能在碳水化合物降解中具有预测的功能，而其他酶可能没有，但却表现出相似的表达模式；这些后一类酶构成了参与降解过程的潜在新酶，并为进一步的生化探索提供了基础。因此，分泌组是研究预测的和新的CAZymes的重要来源。在此，我们描述了一种基于平板的培养技术，该技术能够收集高度富集分泌蛋白的蛋白质组分，这些蛋白与底物结合或未结合，并且通过避免不必要的细胞裂解将细胞内蛋白污染降至最低。
