Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Center, Kurchatov Institute, Gatchina, Leningrad Region, Russia.
Kurchatov Genome Center - PNPI, Gatchina, Leningrad Region, Russia.
J Basic Microbiol. 2020 Nov;60(11-12):971-982. doi: 10.1002/jobm.202000303. Epub 2020 Oct 25.
Endo-β-xylanases are hemicellulases involved in the conversion of xylans in plant biomass. Here, we report a novel acidophilic β-xylanase (ScXynA) with high transglycosylation abilities that was isolated from the filamentous fungus Scytalidium candidum 3C. ScXynA was identified as a glycoside hydrolase family 10 (GH10) dimeric protein, with a molecular weight of 38 ± 5 kDa per subunit. The enzyme catalyzed the hydrolysis of different xylans under acidic conditions and was stable in the pH range 2.6-4.5. The kinetic parameters of ScXynA were determined in hydrolysis reactions with p-nitrophenyl-β-d-cellobioside (pNP-β-Cel) and p-nitrophenyl-β-d-xylobioside (pNP-β-Xyl ), and k /K was found to be 0.43 ± 0.02 (s·mM) and 57 ± 3 (s·mM) , respectively. In the catalysis of the transglycosylation o-nitrophenyl-β-d-xylobioside (oNP-β-Xyl ) acted both as a donor and an acceptor, resulting in the efficient production of o-nitrophenyl xylooligosaccharides, with a degree of polymerization of 3-10 and o-nitrophenyl-β-d-xylotetraose (oNP-β-Xyl ) as the major product (18.5% yield). The modeled ScXynA structure showed a favorable position for ligand entry and o-nitrophenyl group accommodation in the relatively open -3 subsite, while the cleavage site was covered with an extended loop. These structural features provide favorable conditions for transglycosylation with oNP-β-Xyl . The acidophilic properties and high transglycosylation activity make ScXynA a suitable choice for various biotechnological applications, including the synthesis of valuable xylooligosaccharides.
内切-β-木聚糖酶是参与植物生物质中木聚糖转化的半纤维素酶。在这里，我们报告了一种新型的嗜酸β-木聚糖酶（ScXynA），它具有很高的转糖基化能力，是从丝状真菌 Scytalidium candidum 3C 中分离出来的。ScXynA 被鉴定为糖苷水解酶家族 10（GH10）二聚体蛋白，每个亚基的分子量为 38±5 kDa。该酶在酸性条件下催化不同木聚糖的水解，在 pH 值 2.6-4.5 范围内稳定。通过 p-硝基苯-β-D-纤维二糖苷（pNP-β-Cel）和 p-硝基苯-β-D-木二糖苷（pNP-β-Xyl）的水解反应，确定了 ScXynA 的动力学参数，发现 k /K 分别为 0.43±0.02（s·mM）和 57±3（s·mM）。在转糖基化反应中，o-硝基苯-β-D-木二糖苷（oNP-β-Xyl）既可以作为供体也可以作为受体，从而有效地生成 o-硝基苯木低聚糖，聚合度为 3-10，o-硝基苯-β-D-木四糖（oNP-β-Xyl）为主要产物（产率 18.5%）。模拟的 ScXynA 结构显示了配体进入和 o-硝基苯基团容纳在相对开放的-3亚位点的有利位置，而切割位点被一个扩展的环覆盖。这些结构特征为 oNP-β-Xyl 的转糖基化提供了有利的条件。该酶的嗜酸特性和高转糖基化活性使其成为各种生物技术应用的理想选择，包括有价值的木低聚糖的合成。
