β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. In this study, BxlB—a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites—was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

中文翻译：

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重新设计 GH3 β-木糖苷酶中的 N-糖基化位点可提高酶效率

β-木糖苷酶是糖苷水解酶 (GH)，可将低聚木糖和/或木二糖切割成较短的低聚糖和木糖。构巢曲霉是一种既定的遗传模型，也是碳水化合物活性酶 (CAZymes) 的良好来源。大多数真菌酶是 N-糖基化的，这会影响它们的分泌、稳定性、活性、信号传导和蛋白酶保护。对 N-糖基化过程的更深入了解将有助于更好地解决当前在获得用于工业应用的真菌蛋白的高分泌产量方面的瓶颈。在这项研究中，BxlB（一种来自构巢曲霉的高度分泌的 GH3 β-木糖苷酶，具有高活性和多个 N-糖基化位点）被选择用于 N-糖基化工程。设计了几种糖突变体来研究 N-聚糖对 BxlB 分泌和功能的影响。与野生型 (BxlBwt) 相比，非糖基化突变体 (BxlBnon-glyc) 表现出相似水平的酶分泌和活性，而部分糖基化突变体 (BxlBN1;5;7) 表现出更高的活性。此外，尽管转录水平很高，但通过引入四个新的 N-糖基化位点 (BxlBCC) 改变了 N-糖基化环境的突变体中没有酶分泌。BxlBwt、BxlBnon-glyc 和 BxlBN1;5;7 形成了相似的二级结构，尽管与野生型相比，突变体的熔解温度较低。基于 BxlBN1;5;7 设计了另外六种糖突变体，以更好地了解其增加的活性。其中，分别仅保留两个 N-糖基化位点的两种糖突变体（BxlBN1；5 和 BxlBN5；7）显示出提高的催化效率，而其他四种突变体的催化效率降低。N-糖基化位点 N5 对于提高 BxlB 催化效率很重要，但需要由 N1 和/或 N7 补充。BxlBnon-glyc 和 BxlBN1;5 的分子动力学模拟表明，催化袋附近结构元素的迁移模式在 N1 和 N5 N-糖基化位点发生变化，增强了底物结合特性，这可能是观察到的催化效率差异的基础BxlBnon-glyc 和 BxlBN1；5。这项研究证明了 N-糖基化对构巢曲霉 BxlB 产生和功能的影响，强调蛋白质糖工程是增强热稳定性、分泌和酶活性的有前途的工具。我们的报告还可能支持其他 CAZymes 的 N-糖基化修饰的生物技术应用。