Vasquez Robie, Song Ji Hoon, Lee Jae Seung, Kim Sanghoon, Kang Dae-Kyung
Department of Animal Biotechnology, Dankook University, Cheonan, Republic of Korea.
Front Bioeng Biotechnol. 2025 Mar 10;13:1564764. doi: 10.3389/fbioe.2025.1564764. eCollection 2025.
The degradation of hemicellulose, including xylan, is an important industrial process as it provides cheap and sustainable source of economically valuable monosaccharides. β-xylosidases are key enzymes required for complete degradation of xylan and are used in the production of monosaccharides, such as xylose. In this study, we characterized a novel, xylose-tolerant β-xylosidase isolated from SK152. Sequence analysis and protein structure prediction revealed that the putative β-xylosidase belongs to the glycoside hydrolase (GH) family 43 subfamily 11 and exhibits high homology with other characterised GH43 β-xylosidases from fungal and bacterial sources. The putative β-xylosidase was named Xyl43. The catalytic residues of Xyl43, which are highly conserved among GH 43 β-xylosidases, were predicted. To fully characterise Xyl43, the gene encoding it was heterologously expressed in . Biochemical characterisation revealed that the recombinant Xyl43 (rXyl43) was active against artificial and natural substrates containing β-1,4-xylanopyranosyl residues, such as -nitrophenyl-β-D-xylopyranoside (pNPX) and oNPX. Moreover, it demonstrated weak α-L-arabinofuranosidase activity. The optimal activity of rXyl43 was obtained at pH 7.0 at 35°C. rXyl43 could degrade xylo-oligosaccharides, such as xylobiose, xylotriose, and xylotetraose, and showed hydrolysing activity towards beechwood xylan. Moreover, rXyl43 demonstrated synergy with a commercial xylanase in degrading rye and wheat arabinoxylan. The activity of rXyl43 was not affected by the addition of metal ions, chemical reagents, or high concentrations of NaCl. Notably, rXyl43 exhibited tolerance to high xylose concentrations, with a value of 100.1, comparable to that of other xylose-tolerant GH 43 β-xylosidases. To our knowledge, this is the first β-xylosidase identified from a lactic acid bacterium with high tolerance to salt and xylose. Overall, rXyl43 exhibits great potential as a novel β-xylosidase for use in the degradation of lignocellulosic material, especially xylan hemicellulose. Its high activity against xylo-oligosaccharides, mild catalytic conditions, and tolerance to high xylose concentrations makes it a suitable enzyme for industrial applications.
包括木聚糖在内的半纤维素的降解是一个重要的工业过程，因为它能提供廉价且可持续的具有经济价值的单糖来源。β-木糖苷酶是木聚糖完全降解所需的关键酶，用于生产木糖等单糖。在本研究中，我们对从SK152中分离出的一种新型木糖耐受型β-木糖苷酶进行了表征。序列分析和蛋白质结构预测表明，推定的β-木糖苷酶属于糖苷水解酶（GH）家族43亚家族11，与其他已表征的来自真菌和细菌来源的GH43β-木糖苷酶具有高度同源性。推定的β-木糖苷酶被命名为Xyl43。预测了Xyl43的催化残基，这些残基在GH 43β-木糖苷酶中高度保守。为了全面表征Xyl43，编码它的基因在[此处原文缺失具体宿主]中进行了异源表达。生化表征表明，重组Xyl43（rXyl43）对含有β-1,4-木糖吡喃糖基残基的人工和天然底物具有活性，如对硝基苯基-β-D-木糖吡喃糖苷（pNPX）和邻硝基苯基-β-D-木糖吡喃糖苷（oNPX）。此外，它还表现出较弱的α-L-阿拉伯呋喃糖苷酶活性。rXyl43在pH 7.0和35°C时获得最佳活性。rXyl43可以降解木寡糖，如木二糖、木三糖和木四糖，并对山毛榉木聚糖表现出水解活性。此外，rXyl43在降解黑麦和小麦阿拉伯木聚糖时与商业木聚糖酶表现出协同作用。rXyl43的活性不受金属离子、化学试剂或高浓度NaCl添加的影响。值得注意的是，rXyl43对高木糖浓度具有耐受性，其耐受值为100.1，与其他木糖耐受型GH 族43β-木糖苷酶相当。据我们所知，这是首次从乳酸菌中鉴定出的对盐和木糖具有高耐受性的β-木糖苷酶。总体而言，rXyl43作为一种新型β-木糖苷酶在木质纤维素材料尤其是木聚糖半纤维素的降解中具有巨大潜力。它对木寡糖的高活性、温和的催化条件以及对高木糖浓度的耐受性使其成为适合工业应用的酶。
