Hameleers Lisanne, Penttinen Leena, Ikonen Martina, Jaillot Léa, Fauré Régis, Terrapon Nicolas, Deuss Peter J, Hakulinen Nina, Master Emma R, Jurak Edita
Department of Bioproduct Engineering, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Department of Bioproducts and Biosystems, Aalto University, Kemistintie 1, 02150, Espoo, Finland.
Biotechnol Biofuels. 2021 May 31;14(1):127. doi: 10.1186/s13068-021-01976-0.
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.
如今，利用木质纤维素生物废料生产生物燃料和其他生物基产品的循环经济趋势强劲。在生产过程的几个阶段（如糖化）使用酶，由于避免了使用苛刻的化学品和高温，可提供一条可持续的途径。对于新型酶的发现，在细菌中靶向碳水化合物降解的物理连接基因簇，即多糖利用位点（PULs），在测序基因组数量呈指数增长的时代被认为是“宝库”。
我们确定了来自富含杨树水解产物的海狸粪便和驼鹿瘤胃宏基因组PULs中编码的一种未知功能蛋白质（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），现命名为BD-FAE，对简单的合成底物表现出阿魏酸酯酶（FAE）和乙酰酯酶活性。虽然在桦木木聚糖的乙酰化葡糖醛酸木聚糖上检测到乙酰木聚糖酯酶（AcXE）活性，但在玉米纤维的乙酰化和阿魏酰化木寡糖上仅观察到FAE活性。BD-FAE的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可能参与木聚糖分解的PULs中，而较远的同源物则出现在靶向藻酸盐的PULs中或PULs背景之外。尽管BD-FAE结构采用具有催化三联体（Ser-Asp-His）的典型α/β水解酶折叠，但它与其他已进行生化表征的CE不同。
双功能CE，BD-FAE，因其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，代表了生物质加工的新候选酶。其详细的生化表征和解析的晶体结构为生物质增值的酶工具箱增添了内容，也为新CE的分类提供了结构信息。
