Background Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for lignocellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermostability, site-directed mutagenesis was conducted to modify the N-glycosylation sites of the thermostable β-1,4-endoglucanase CTendo45 from Chaetomium thermophilum. Results In this study, structure-based rational design was performed based on the modification of N-glycosylation sites in CTendo45. Eight single mutants and one double mutant were constructed and successfully expressed in Pichia pastoris. When the unique N-glycosylation site of N88 was eliminated, a T90A variant was active, and its specific activity towards CMC-Na and β-d-glucan was increased 1.85- and 1.64-fold, respectively. The mutant R67S with an additional N-glycosylation site of N65 showed a distinct enhancement in catalytic efficiency. Moreover, T90A and R67S were endowed with extraordinary heat endurance after 200 min of incubation at different temperatures ranging from 30 to 90 °C. Likewise, the half-lives (t 1/2) indicated that T90A and R67S exhibited improved enzyme thermostability at 80 °C and 90 °C. Notably, the double-mutant T90A/R67S possessed better hydrolysis activity and thermal stability than its single-mutant counterparts and the wild type. Conclusions This study provides initial insight into the biochemical function of N-glycosylation in thermostable endoglucanases. Moreover, the design approach to the optimization of N-glycosylation sites presents an effective and feasible strategy to improve enzymatic activity and thermostability.

中文翻译：

---

通过优化 N-糖基化位点提高超热稳定性内切葡聚糖酶的催化活性和热稳定性。

背景内切葡聚糖酶已作为木质纤维素生物质降解的关键生物催化剂广泛用于工业过程中。在高温下具有高催化活性的耐热内切葡聚糖酶在工业应用中是优选的。为了提高活性和热稳定性，进行定点诱变以修饰来自嗜热毛壳菌的热稳定 β-1,4-内切葡聚糖酶 CTendo45 的 N-糖基化位点。结果在本研究中，基于CTendo45中N-糖基化位点的修饰进行了基于结构的合理设计。在毕赤酵母中构建并成功表达了8个单突变体和1个双突变体。当 N88 独特的 N-糖基化位点被消除时，T90A 变体具有活性，其对 CMC-Na 和 β-d-葡聚糖的比活性增加 1。分别为 85 倍和 1.64 倍。具有 N65 的额外 N-糖基化位点的突变体 R67S 显示出催化效率的明显增强。此外，T90A 和 R67S 在 30 至 90 °C 的不同温度下孵育 200 分钟后具有非凡的耐热性。同样，半衰期 (t 1/2) 表明 T90A 和 R67S 在 80 °C 和 90 °C 下表现出改善的酶热稳定性。值得注意的是，双突变体 T90A/R67S 比其单突变体对应物和野生型具有更好的水解活性和热稳定性。结论 本研究提供了对热稳定内切葡聚糖酶中 N-糖基化的生化功能的初步了解。而且，