Background Plant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated. Results Two carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns. Conclusion We demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.

中文翻译：

---

来自土壤和肠道拟杆菌的多模块融合乙酰-阿魏酸酯酶可改善顽固生物质的木聚糖酶解聚。

背景植物生物质是一种丰富且可再生的碳源，它对化学和生化降解都有抵抗力。木聚糖是生物质中仅次于纤维素的第二丰富多糖，它具有多种碳水化合物替代物和非碳水化合物修饰物，可阻碍糖苷水解酶的酶促降解。碳水化合物酯酶能够切割酯连接的装饰，从而提高木聚糖骨架对糖苷水解酶的可及性，从而改善降解过程。先前已显示在同一多肽上包含多个催化糖苷水解酶结构域的酶在生物质降解期间表现出分子内协同作用。类似地，天然融合的碳水化合物酯酶结构域由某些细菌编码，但这些酶是否能在生物质降解中产生类似的协同促进作用，以前尚未评估过。结果从拟杆菌属物种卵形拟杆菌和约氏黄杆菌编码的木聚糖靶向多糖利用基因座 (PUL) 中选择了两种具有相似结构的碳水化合物酯酶，每一种都包含来自家族 1 (CE1) 和 6 (CE6) 的两个不同物理连接的催化结构域. 全长酶以及单个催化结构域在一系列合成模型底物、玉米芯生物质和日本山毛榉生物质上显示出活性，其中 N 末端 CE6 结构域的乙酰酯酶活性和 C 的阿魏酸酯酶活性占优势。 -终端 CE1 域。而且，几种酶构建体能够显着提高市售木聚糖酶对玉米芯生物质（接近两倍）和日本山毛榉生物质（高达 20 倍）的性能。有趣的是，在添加来自卵形芽孢杆菌的全长多结构域酶与添加其两个分离的单结构域后，观察到木聚糖酶生物量降解的显着改善，表明酯酶结构域之间存在分子内协同作用。尽管来自卵形芽孢杆菌和约氏梭菌的酯酶结构域之间具有高度序列相似性，但将它们添加到木聚糖分解反应中会导致不同的降解模式。结论 我们证明了多域碳水化合物酯酶，针对不同木聚糖多糖上的非碳水化合物修饰，可以显着促进糖苷水解酶介导的木聚糖和富含木聚糖的生物质的水解。此外，我们首次证明了多域碳水化合物酯酶的两个融合催化域之间的协同效应。