Data_Sheet_3_Molecular and Biochemical Characterization of a Bimodular Xylanase From Marinifilaceae Bacterium Strain SPP2.PDF

In this study, the first xylantic enzyme from the family Marinifilaceae, XynSPP2, was identified from Marinifilaceae bacterium strain SPP2. Amino acid sequence analysis revealed that XynSPP2 is a rare Fn3-fused xylanase, consisting of a signal peptide, a fibronectin type-III domain (Fn3), and a C-terminal catalytic domain belonging to glycoside hydrolase family 10 (GH10). The catalytic domain shared 17–46% identities to those of biochemically characterized GH10 xylanases. Structural analysis revealed that the conserved asparagine and glutamine at the glycone −2/−3 subsite of GH10 xylanases are substituted by a tryptophan and a serine, respectively, in XynSPP2. Full-length XynSPP2 and its Fn3-deleted variant (XynSPP2ΔFn3) were overexpressed in Escherichia coli and purified by Ni-affinity chromatography. The optimum temperature and pH for both recombinant enzymes were 50°C and 6, respectively. The enzymes were stable under alkaline condition and at temperature lower than 50°C. With beechwood xylan as the substrate, XynSPP2 showed 2.8 times the catalytic efficiency of XynSPP2ΔFn3, indicating that the Fn3 module promotes xylanase activity. XynSPP2 was active toward xylooligosaccharides (XOSs) longer than xylotriose. Such a substrate preference can be explained by the unique −2/−3 subsite composition in the enzyme which provides new insight into subsite interaction within the GH10 family. XynSPP2 hydrolyzed beechwood xylan into small XOSs (xylotriose and xylotetraose as major products). No monosaccharide was detected by thin-layer chromatography which may be ascribed to putative transxylosylation activity of XynSPP2. Preferring long XOS substrate and lack of monosaccharide production suggest its potential in probiotic XOS manufacture.

本研究首次从 Marinifilaceae 菌株 SPP2 中鉴定出该家族的首个木聚糖酶——XynSPP2。氨基酸序列分析表明，XynSPP2 是一种罕见的 Fn3 融合型木聚糖酶，由信号肽、纤连蛋白类型 III 结构域（Fn3）以及属于糖苷水解酶家族 10（GH10）的 C 端催化结构域组成。催化结构域与已生化鉴定的 GH10 木聚糖酶在 17–46% 的序列上具有同源性。结构分析显示，GH10 木聚糖酶中位于糖苷 -2/−3 位点的保守天冬酰胺和谷氨酰胺在 XynSPP2 中分别被色氨酸和丝氨酸所取代。全长 XynSPP2 及其 Fn3 结构域缺失变异体（XynSPP2ΔFn3）在大肠杆菌中过表达，并通过 Ni亲和层析纯化。两种重组酶的最适温度和 pH 值分别为 50°C 和 6。在碱性条件下及低于 50°C 的温度下，酶均保持稳定性。以山毛榉木木聚糖为底物，XynSPP2 表现出比 XynSPP2ΔFn3 高 2.8 倍的催化效率，这表明 Fn3 结构域促进了木聚糖酶的活性。XynSPP2 对比洛糖（XOSs）的活性优于对三聚糖的活性。这种底物偏好性可以归因于酶中独特的 −2/−3 位点组成，这为 GH10 家族中位点间相互作用提供了新的见解。XynSPP2 将山毛榉木木聚糖水解为小 XOSs（以木三糖和木四糖为主要产物）。通过薄层色谱未检测到单糖，这可能归因于 XynSPP2 的潜在转木糖基化活性。偏好长 XOS 底物以及缺乏单糖的产生，暗示其在益生菌 XOS 制造中的潜在价值。