Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

中文翻译：

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多糖利用基因座驱动的酶发现揭示了 BD-FAE：一种对复杂的天然木聚糖具有活性的双功能阿魏酰和乙酰木聚糖酯酶


如今，利用木质纤维素生物废物生产生物燃料和其他生物基产品的循环经济已成为一种强劲趋势。由于避免了刺激性化学品和高温，在生产过程的几个阶段（例如糖化）使用酶可以提供可持续的路线。对于新酶的发现，在细菌中针对碳水化合物降解的物理连接基因簇、多糖利用位点（PUL），在测序基因组数量呈指数级增长的时代被认为是“宝库”。我们确定了来自海狸粪便和富含杨树水解产物的驼鹿瘤胃的宏基因组 PUL 中编码的未知功能蛋白 (PUF) 的生化特性和结构。相应的新型双功能碳水化合物酯酶（CE），现在命名为BD-FAE，在简单的合成底物上表现出阿魏酸酯酶（FAE）和乙酰酯酶活性。虽然在来自桦木的乙酰化葡糖醛酸木聚糖上检测到了乙酰木聚糖酯酶 (AcXE) 活性，但在来自玉米纤维的乙酰化和阿魏酰化低聚木糖上仅观察到了 FAE 活性。 BD-FAE 200 个同源物的基因组背景表明，33 个最接近的同源物出现在 PUL 中，可能涉及木聚糖分解，而更远的同源物要么在藻酸盐靶向 PUL 中，要么在 PUL 背景之外发现。尽管BD-FAE结构采用典型的具有催化三联体（Ser-Asp-His）的α/β-水解酶折叠，但它与其他生化特征CE不同。双功能 CE BD-FAE 代表了生物质加工的新候选者，因为它能够分别从天然玉米和桦木木聚糖底物中去除阿魏酸和乙酸。 其详细的生化特征和解析的晶体结构添加到了用于生物质增值的酶工具箱以及为新CE分类提供信息的结构信息。