Endo-β-1,4-xylanase from Thermotoga maritima, TmxB, is an industrially attractive enzyme due to its extreme thermostability. To improve its application value, four variants were designed on the basis of multiple sequence and three-dimensional structure alignments. Wild-type TmxB (wt-TmxB) and its mutants were produced via a Pichia pastoris expression system. Among four single-site mutants, the tyrosine substitution of a threonine residue (T74Y) at putative -3/-4 subsite led to a 1.3-fold increase in specific activity at 40 °C - 100 °C and pH 5 for 5 min, with beechwood xylan as the substrate. T74Y had an improved catalytic efficiency (kcat/Km), being 1.6 times that of wt-TmxB. Variants DY (two amino acid insertions) and N68Q displayed a slight increase (1.2 fold) and dramatic decline (1.7 fold) in catalytic efficiency, respectively. Mutant E67Y was totally inactive under all test conditions. Structural modeling and docking simulation elucidated structural insights into the molecular mechanism of activity changes for these TmxB variants. This study helps in further understanding the roles of the non-catalytic amino acids at the glycone subsites of xylanases from glycoside hydrolase family 10.

中文翻译：

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通过糖基亚位点非催化残基的诱变提高海栖热袍菌热稳定木聚糖酶的催化活性

来自海栖热袍菌的内切-β-1,4-木聚糖酶，TmxB，由于其极端的热稳定性，是一种具有工业吸引力的酶。为了提高其应用价值，在多序列和三维结构比对的基础上设计了四个变体。野生型 TmxB (wt-TmxB) 及其突变体是通过毕赤酵母表达系统产生的。在四个单点突变体中，在假定的 -3/-4 亚位点处苏氨酸残基 (T74Y) 的酪氨酸取代导致在 40 °C - 100 °C 和 pH 5 下比活性增加 1.3 倍，持续 5 分钟，以山毛榉木木聚糖为基材。T74Y 具有改进的催化效率 (kcat/Km)，是 wt-TmxB 的 1.6 倍。变体 DY（两个氨基酸插入）和 N68Q 分别显示催化效率略有增加（1.2 倍）和急剧下降（1.7 倍）。突变体 E67Y 在所有测试条件下都完全不活跃。结构建模和对接模拟阐明了对这些 TmxB 变体活性变化的分子机制的结构见解。这项研究有助于进一步了解非催化氨基酸在来自糖苷水解酶家族 10 的木聚糖酶的糖苷亚位点上的作用。