Functional analysis of carbohydrate-active enzymes

碳水化合物活性酶的功能分析

• 批准号: 320431848
• 负责人: Professor Dr. Uwe T. Bornscheuer
• 金额: --
• 依托单位: Arbeitskreis Biotechnologie und Enzymkatalyse
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/320431848?language=en
• 关键词: Functional; analysis; carbohydrate; active; enzymes

Subproject A2 aims to functionally characterize carbohydrate-active enzymes of marine Bacteroidetes that specialize in polysaccharide degradation. In the first POMPU phase, we found that xylan-specific PULs and their protein functions are repeatedly detectable in the bacterioplankton of microalgal spring blooms. Since the function of the encoded enzymes was largely unknown, we used the second funding phase to investigate PULs related to marine xylan degradation and their corresponding enzyme cascades. Specifically, we could biochemically and functionally characterize two different xylan-specific PULs of a marine Bacteroidetes strain. Furthermore, we could show that formaldehyde produced by the action of a P450 monooxygenase on 6-O-methyl-D-galactose is detoxified via the RuMP pathway. Finally, we discovered an alternative pathway for the degradation of ulvan, which includes a novel dehydratase activity. In the third funding phase of POMPU we will focus on a marine PUL type, which was abundantly detected in diatom-driven phytoplankton blooms. Our preliminary bioinformatic analyses indicated that this PUL type is responsible for the utilization of so far unknown sulfated xylans. In order to reach this goal, we have defined the following objectives: (i) functional analysis of the PUL encoded sulfatases for their activity on sulfated xylans isolated from diatoms, (ii) biochemical characterization of new xylan-degrading CAZymes, (iii) elucidation of the substrate specificity of SusD-like carbohydrate binding proteins in xylan utilization. Finally, we will support the consortium partners in the functional analysis of marine arabinogalactan- and ß-glucan-specific CAZymes. All experiments will include recombinant expression and purification of the enzymes/proteins including in-depth biochemical analyses and structural biological characterizations of selected CAZymes together with a range of partners within the POMPU consortium.

子项目A2旨在对海洋拟杆菌门中专门降解多糖的碳水化合物活性酶进行功能表征。在第一个POMPU阶段，我们发现木聚糖特异性PULs及其蛋白质功能在微藻春季华的浮游细菌中反复检测到。由于编码酶的功能在很大程度上是未知的，我们使用了第二个资助阶段来研究与海洋木聚糖降解相关的PULs及其相应的酶级联反应。具体来说，我们可以生化和功能表征海洋拟杆菌菌株的两种不同的木聚糖特异性PULs。此外，我们可以证明P450单加氧酶作用于6- o -甲基- d -半乳糖产生的甲醛通过RuMP途径解毒。最后，我们发现了另一种降解ulvan的途径，其中包括一种新的脱水酶活性。在POMPU的第三个资助阶段，我们将重点研究一种海洋PUL类型，这种类型在硅藻驱动的浮游植物繁殖中大量发现。我们的初步生物信息学分析表明，这种PUL类型负责利用迄今为止未知的硫酸盐木聚糖。为了实现这一目标，我们确定了以下目标：(i) PUL编码的硫酸盐酶对硅藻中分离的硫酸木聚糖活性的功能分析，（ii）新的木聚糖降解酶的生化表征，（iii）阐明木聚糖利用中susd样碳水化合物结合蛋白的底物特异性。最后，我们将支持联盟合作伙伴进行海洋阿拉伯半乳聚糖和ß-葡聚糖特异性CAZymes的功能分析。所有实验将包括酶/蛋白质的重组表达和纯化，包括深入的生化分析和所选CAZymes的结构生物学特征，以及POMPU联盟中的一系列合作伙伴。