Division of Biotechnology, Department of Chemistry, Lund University, Lund, Sweden.
Instituto de Investigaciones Fármaco Bioquímicas, Universidad Mayor de San Andrés, La Paz, Bolivia.
Appl Environ Microbiol. 2024 Apr 17;90(4):e0222323. doi: 10.1128/aem.02223-23. Epub 2024 Mar 18.
An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from strain E-1 was identified from genomic sequence data, and the encoded enzyme, Xyn43-l, was produced in Xyn43-l (52.9 kDa) is a two-domain endo-β-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. Xyn43-l also released xylobiose from NPX with low turnover ( of 0.044 s) but was inactive on NPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, Xyn43-l from strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer.
The genome of strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-β-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.
从 E-1 菌株的基因组序列数据中鉴定出一个编码糖苷水解酶家族 43 样酶的未表征基因，所编码的酶 Xyn43-l（52.9 kDa）是一种由 C 端 CBM6 和 GH43 样催化结构域组成的双结构域内切-β-木聚糖酶。GH43 中保守的催化二联体、催化碱（Asp74）和质子供体（Glu240）的位置在包括不同亚家族已知 3D 结构的 GH43 酶的比对中被确定。Xyn43-l 在 pH7.0-9.0 下具有活性，最适温度为 65°C，在该温度下不可逆失活研究中半衰期超过 7 天。该酶水解桦木木聚糖、藜麦茎葡甘聚糖阿拉伯木聚糖和小麦阿拉伯木聚糖，主要水解产物为木三糖和木四糖。Xyn43-l 也从 NPX 中释放出低周转率（为 0.044 s）的木二糖，但对 NPX 无活性，表明聚合度为三（DP3）是最小可水解的底物。二价离子影响木聚糖底物的比活性，这取决于离子，可以增加或减少。总之，E-1 菌株的 Xyn43-l 是第一个被表征的一大组同源假设蛋白的成员，这些蛋白被注释为 GH43 样，是一种耐热内切木聚糖酶，可产生高 DP（木三糖和木四糖）的木寡糖。
E-1 菌株的基因组编码了许多假设酶，被注释为糖苷水解酶样，但未在碳水化合物活性酶数据库 (CAZy) 中分类。一种新型耐热 GH43 样酶被鉴定为内切-β-木聚糖酶，可从不同木聚糖来源生产益生元木寡糖（XO）。Xyn43-l 是一种由催化 GH43-l 结构域和 CBM6 结构域组成的双结构域酶，主要产生木三糖作为 XO 产物。该酶在许多相关菌株中有同源物，这可能表明其具有相似的功能，并且在该微生物进化谱系中是一种以前未知的内切木聚糖酶。
