Bioconversion Group, Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Hiroshima, Japan.
Polymer Chemistry Group, Research Institute for Sustainable Chemistry, AIST, Ibaraki, Japan.
Appl Environ Microbiol. 2019 Oct 30;85(22). doi: 10.1128/AEM.01442-19. Print 2019 Nov 15.
Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus (Xyn30C). Xyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that Xyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4--methyl-α-d-glucuronic acid (MeGlcA) substituent. Xyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 2-(MeGlcA)-xylobiose (UX). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 2-(MeGlcA)-XOSs (X UX, ≥ 0) in the initial stages of the hydrolysis and by specific release of UX from a mixture containing X UX. Our results suggest that the unique endoxylanase activity of Xyn30C may be applicable to the production of linear and acidic XOSs. The gene was located adjacent to the putative GH62 arabinofuranosidase gene () in the genome. The expression of both genes was induced by cellulose. The results suggest that Xyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the cellulolytic system. Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase Xyn30C from was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 2-(4--methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.
糖苷水解酶家族 30 亚家族 7（GH30-7）酶包括各种类型的木聚糖酶，如葡糖醛酸木聚糖酶、内切木聚糖酶、木二糖水解酶和还原端释放木糖的外切木聚糖酶。在这里，我们对纤维素分解真菌 （Xyn30C）的 GH30-7 内切木聚糖酶的作用模式和基因表达进行了表征。Xyn30C 具有模块化结构，由 GH30-7 催化结构域和 C 端纤维素结合模块 1 组成，其纤维素结合能力已得到证实。GH30-7 木聚糖酶的序列比对表明，Xyn30C 在对应 GH30-7 葡萄糖醛酸木聚糖酶中保守的精氨酸残基的位置上具有保守的苯丙氨酸残基，这是识别 4--甲基-α-d-葡萄糖醛酸（MeGlcA）取代基所必需的。Xyn30C 以类似的动力学常数降解葡萄糖醛酸木聚糖和阿拉伯木聚糖，主要以末端方式产生 2 到 3 个聚合度的线性木低聚糖（XOSs）。值得注意的是，葡萄糖醛酸木聚糖的水解导致 2-（MeGlcA）-木二糖（UX）的积累。这种酸性 XOS 的产生可能是通过假定的葡萄糖醛酸木聚糖酶活性的多步反应进行的，该活性在水解的初始阶段产生 2-（MeGlcA）-XOSs（X UX，≥0），并通过特定的从含有 X UX 的混合物中释放 UX 来进行。我们的结果表明，Xyn30C 的独特内切木聚糖酶活性可能适用于线性和酸性 XOSs 的生产。基因 位于 基因组中假定的 GH62 阿拉伯呋喃糖苷酶基因（）的旁边。两个基因都被纤维素诱导表达。这表明 Xyn30C 可能参与了木质纤维素水解过程中 纤维素分解系统中木聚糖的去除。木低聚糖（XOSs）由β-1,4 键连接的木糖单元组成，最近作为食品和饲料工业中的益生元引起了人们的兴趣。除了线性 XOSs 之外，用甲基葡萄糖醛酸和阿拉伯糖等取代基修饰的支链 XOSs也具有潜在的应用。内切木聚糖酶是从木聚糖中生产 XOSs 的一种很有前途的工具。生成的 XOSs 的结构多样性取决于酶的底物特异性以及木聚糖中取代基的分布。因此，预计探索具有新型特异性的内切木聚糖酶将有助于提供一系列 XOSs。在这项研究中，来自 的内切木聚糖酶 Xyn30C 被表征为一种独特的糖苷水解酶，属于家族 GH30-7，它可以特异性地从硬木木聚糖中释放 2-（4--甲基-α-d-葡糖醛酸基）-木二糖。本研究为 GH30-7 内切木聚糖酶生产线性和支链 XOSs 提供了新的见解。
