Functional analysis of carbohydrate-active enzymes

A2子项目旨在对海洋类杆菌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，我们发现在微藻春季水华的浮游细菌中反复检测到木聚糖特异的Puls及其蛋白功能。由于编码的酶的功能在很大程度上是未知的，我们使用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。具体地说，我们可以从生化和功能上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，由P450单加氧酶作用于6-O-甲基-D-半乳糖产生的甲醛是通过尾部途径解毒的。最后，我们发现了另一条降解乌尔万的途径，其中包括一种新的脱水酶活性。在POMPU的第三个资助阶段，我们将重点关注一种海洋PUL类型，这种类型在硅藻驱动的浮游植物水华中被大量检测到。我们的初步生物信息学分析表明，这种PUL类型是迄今为止未知的硫化木聚糖的利用的原因。为了达到这一目标，我们定义了以下目标：(I)对PUL编码的硫酸酯酶在从硅藻中分离的硫酸木聚糖上的活性进行功能分析；(Ii)新的降解木聚糖的CAZymes的生化特性；(Iii)阐明类SusD碳水化合物结合蛋白在木聚糖利用中的底物专一性。最后，我们将支持联盟合作伙伴对海洋阿拉伯半乳聚糖和β-葡聚糖特定的CAZymes进行功能分析。所有实验将包括重组表达和纯化酶/蛋白质，包括与POMPU联盟内的一系列合作伙伴一起对选定的CAZyme进行深入的生化分析和结构生物学特性。

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.