Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 221, DK-2800, Kongens Lyngby, Denmark.
Appl Microbiol Biotechnol. 2019 May;103(10):4065-4075. doi: 10.1007/s00253-019-09797-w. Epub 2019 Apr 4.
Glucuronoyl esterases (CE15 family) enable targeted cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), particularly those linking lignin and glucuronoyl residues in xylan. A substantial challenge in characterization and kinetic analysis of CE15 enzymes has been the lack of proper substrates. Here, we present an assay using an insoluble LCC-rich lignin fraction from birch; lignin-rich pellet (LRP). The assay employs quantification of enzyme reaction products by LC-MS. The kinetics of four fungal CE15 enzymes, PsGE, CuGE, TtGE, and AfuGE originating from lignocellulose-degrading fungi Punctularia strigosozonata, Cerrena unicolor, Thielavia terrestris, and Armillaria fuscipes respectively were characterized and compared using this new assay. All four enzymes had activity on LRP and showed a clear preference for the insoluble substrate compared with smaller soluble LCC mimicking esters. End-product profiles were near identical for the four enzymes but differences in kinetic parameters were observed. TtGE possesses an alternative active site compared with the three other enzymes as it has the position of the catalytic glutamic acid occupied by a serine. TtGE performed poorly compared with the other enzymes. We speculate that glucuronoyl LCCs are not the preferred substrate of TtGE. Removal of an N-terminal CBM on CuGE affected the catalytic efficiently of the enzyme by reducing K by more than 30%. Reaction products were detected from all four CE15s on a similar substrate from spruce indicating a more generic GE activity not limited to the hardwood. The assay with natural substrate represents a novel tool to study the natural function and kinetics of CE15s.
糖醛酸基酯酶（CE15 家族）能够靶向切割木质素-碳水化合物复合体（LCC）中的酯键，特别是那些将木质素和木糖中的糖醛酸残基连接起来的酯键。在 CE15 酶的特性和动力学分析中，一个主要的挑战是缺乏合适的底物。在这里，我们提出了一种使用桦木中富含木质素的不溶性 LCC 部分的测定方法；木质素丰富的颗粒（LRP）。该测定方法通过 LC-MS 定量测定酶反应产物。四种真菌 CE15 酶，来自木质纤维素降解真菌 Punctularia strigosozonata、Cerrena unicolor、Thielavia terrestris 和 Armillaria fuscipes 的 PsGE、CuGE、TtGE 和 AfuGE 的动力学特性分别用这种新的测定方法进行了表征和比较。这四种酶在 LRP 上都有活性，与较小的可溶性 LCC 模拟酯相比，它们对不溶性底物表现出明显的偏好。四种酶的终产物谱几乎相同，但动力学参数存在差异。与其他三种酶相比，TtGE 具有替代的活性位点，因为其催化谷氨酸的位置被丝氨酸占据。与其他三种酶相比，TtGE 的表现不佳。我们推测，糖醛酸 LCC 不是 TtGE 的首选底物。CuGE 上 N 端 CBM 的去除通过将 K 值降低超过 30%，影响了酶的催化效率。来自所有四种 CE15 的反应产物都在类似的云杉底物上被检测到，这表明了一种更通用的 GE 活性，而不仅仅局限于硬木。使用天然底物的测定方法代表了一种研究 CE15 天然功能和动力学的新工具。
