Classification of fungal glucuronoyl esterases (FGEs) and characterization of two new FGEs from Ceriporiopsis subvermispora and Pleurotus eryngii
Classification of fungal glucuronoyl esterases (FGEs) and characterization of two new FGEs from Ceriporiopsis subvermispora and Pleurotus eryngii
复制标题
真菌葡萄糖醛酸酯酶 (FGE) 的分类以及来自 Ceriporiopsis subvermispora 和 Pleurotus eryngii 的两种新 FGE 的表征
DOI：
10.1007/s00253-018-9318-5
复制
发表时间：
2018
影响因子：
5
通讯作者：
Katahira Masato
中科院分区：
文献类型：
--
作者：
Lin Meng-I;Hiyama Akiho;Kondo Keiko;Nagata Takashi;Katahira Masato
真菌葡糖醛酸酯酶（FGE）催化将木质素醇连接至葡糖醛酸葡聚糖的木聚糖结合的4-O-甲基-d-葡糖醛酸的酯键的裂解。因此，FGE能够降解木质素-碳水化合物复合物，并具有生物技术应用于木质生物质利用的潜力。因此，识别和表征新的FGE是至关重要的。首先，在这项研究中，我们建立了一个系统发育树，从近400个推定的FGE的BLAST分析，并确定了六个主要分支。在系统发育树中，所有子囊菌的FGE都聚在进化枝I到IV中，而大多数担子菌的FGE（B-FGE）聚在进化枝V到VI中。有趣的是，几个B-FGE被发现聚类在进化枝II中，大多数进化枝II的FGE被发现具有更高的理论等电点比其他五个进化枝。为了深入了解尚未被表征的分支中的假定FGE，我们选择了分别属于分支V和II的Ceriporiopsis subvermispora（CsGE）和Pleuporerynnelae（PeGE）的FGE。在毕赤酵母中成功表达并纯化了CsGE和PeGE的催化结构域。以苄基葡萄糖醛酸为底物，测定CsGE和PeGE的活性，并对水解产物葡萄糖醛酸进行定量。CsGE和PeGE均具有明显的酯酶活性。此外，我们证明了PeGE对几种变性剂具有高耐受性，这可能使其成为一种潜在的更适用的酶。
Fungal glucuronoyl esterases (FGEs) catalyze cleavage of the ester bond connecting a lignin alcohol to the xylan-bound 4-O-methyl-d-glucuronic acid of glucuronoxylans. Thus, FGEs are capable of degrading lignin-carbohydrate complexes and have potential for biotechnological applications toward woody biomass utilization. Therefore, identification and characterization of new FGEs are of critical importance. Firstly, in this study, we built a phylogenetic tree from almost 400 putative FGEs obtained on BLAST analysis and defined six main clades. In the phylogenetic tree, all the putative FGEs of ascomycetes cluster in clades I to IV, and most of the putative FGEs of basidiomycetes (B-FGEs) cluster in clades V to VI. Interestingly, several B-FGEs were found to cluster in clade II; most FGEs of clade II were found to have higher theoretical isoelectric points than those in the other five clades. To gain an insight into the putative FGEs in the clades that have not been characterized yet, we chose the FGEs ofCeriporiopsis subvermispora(CsGE) andPleurotus eryngii(PeGE), which belong to clades V and II, respectively. The catalytic domains of bothCsGE andPeGE were successfully expressed usingPichia pastoris, and then purified. Benzyl glucuronic acid was used as a substrate to confirm the activities of theCsGE andPeGE, and the hydrolyzed product, glucuronic acid, was quantified spectrophotometrically. BothCsGE andPeGE clearly exhibited the esterase activity. Additionally, we demonstrated thatPeGE exhibits high tolerance toward several denaturing agents, which may make it a potentially more applicable enzyme.