Xylanases, capable of hydrolyzing xylans which are abundant in nature, have been employed as important biocatalyst in many industrial processes. Xylanases with advantageous properties, especially excellent thermostability, are in high demand in industry. In this study, we aim to improve the thermostability of XynCDBFV, a fungal GH11 xylanase. To achieve this aim, we discovered residues 87-QNSS-90 with pronounced flexibility based on B-factor analysis, identified highly conserved residues 87-RGHT-90 in GH11 xylanases by multiple sequence alignment, and constructed four single mutants by substituting residues from 87 to 90 by site-directed mutagenesis. Temperature stability measurements showed promising enhancement of thermostability for all four single mutants, and the thermal tolerant ability from strong to weak is N88 G, S90 T, S89H, Q87R, XynCDBFV. Four single mutants all retained higher than 50% activities after incubation at the optimal temperature 60℃ for 1 h, while the retained activity for XynCDBFV was only 20.94% at the same condition. N88 G retained greater than 60% residual activity after incubation at 65℃ for 1 h, while the residual activity of XynCDBFV decreased rapidly, losing all activity after 45 min of incubation. Molecular dynamics simulations and structural analysis were applied to explore the heat-resistant mechanisms for mutants: novel hydrogen bonding interaction were discovered and accounted for the improved thermostability. Enzyme activity of the single mutants compromised with their thermostability and combined mutations displayed antagonistic effect due to the closed contact of the mutated residues. This study confirms that combining B-factor analysis and multiple sequence alignment is an effective strategy for obtaining a thermostable enzyme, and the negative findings help to recognize limitations in xylanase engineering for preferable properties.

中文翻译：

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通过 B 因子分析和多序列比对提高真菌 GH11 木聚糖酶的耐热性

木聚糖酶能够水解自然界中丰富的木聚糖，已被用作许多工业过程中的重要生物催化剂。工业上对具有优势特性，尤其是优异的热稳定性的木聚糖酶有很高的需求。在本研究中，我们旨在提高真菌 GH11 木聚糖酶 XynCDBFV 的热稳定性。为了实现这一目标，我们基于 B 因子分析发现了具有显着灵活性的残基 87-QNSS-90，通过多序列比对鉴定了 GH11 木聚糖酶中高度保守的残基 87-RGHT-90，并通过替换 87通过定点诱变达到 90。温度稳定性测量显示所有四个单突变体的热稳定性都有希望增强，耐热能力从强到弱依次为 N88 G、S90 T、S89H、Q87R、XynCDBFV。4个单突变体在最适温度60℃孵育1 h后均保留了50%以上的活性，而XynCDBFV在相同条件下的保留活性仅为20.94%。N88 G 65℃孵育1 h后残留活性保持在60%以上，而XynCDBFV的残留活性迅速下降，45 min后活性全部丧失。应用分子动力学模拟和结构分析来探索突变体的耐热机制：发现了新的氢键相互作用并解释了热稳定性的提高。由于突变残基的紧密接触，单个突变体的酶活性受到其热稳定性的影响，并且组合突变显示出拮抗作用。