数字农科院2.0

Effects of N-linked glycosylation on the enzymatic properties of GH12 bifunctional enzymes from Aspergillus terreus expressed in Pichia pastoris

文献类型： 外文期刊

作者： Dinghui Zuo;Jinjian He;Xihang Sun;Hanting Liu;Shenju Xia;Mansheng Wang;Xia Zheng;Huiqin Liu;Pengjun Shi

作者机构：

关键词： GH12 endoglucanase;N-glycosylation;Pichia pastoris;Thermalstability

期刊名称： International Journal of Biological Macromolecules

ISSN： 0141-8130

年卷期： 2025 年 304 卷

页码：

收录情况： SCIE（2025版） ; ; EI（2025版）

摘要： In industry, bioenergy, food process, and feed application, endoglucanases are highly valuable for lignocellulose degradation with high catalytic activity under high temperatures. The glycoside hydrolase family 12 endoglucanase (AtEglD) from Aspergillus terreus can efficiently hydrolyze both β-glucan and xyloglucan of barley with an optimal temperature of 55 °C under pH 5.0. To enhance the industrial potential of AtEglD, the rational design of its N-glycosylation sites is imperative. The genes encoding AtEglD (N-glycosylation site at Asn65), along with two mutants: D168S (N-glycosylation site at Asn166) and N65Q (which lacks an N-glycosylation site) were successfully expressed and characterized. AtEglD exhibits reduced activity at 60 °C whereas, the N65Q mutant exhibited enhanced activity, maintaining substantial activity even after 90 min incubation. In barley-β-glucan, its specific activity reached 3204.27 U·mg−1, representing 2.73 times increase compared to AtEglD (1175.35 U·mg−1), while the catalytic efficiency was measured at 779.00 S−1·mM−1, indicating a 74.4 % enhancement relative to AtEglD (447.34 S−1·mM−1). For xyloglucan, N65Q demonstrated a significantly greater affinity compared to AtEglD, with 36.0 % increase in catalytic efficiency. Intriguingly, the D168S mutant exhibited a marked reduction in both specific activity and catalytic efficiency across both substrates. The structure analysis of AtEglD revealed that the N65 residues are far away from the catalytic domain, while the N166 residues are close to the catalytic site. It is implied that N-glycosylation proximal to the catalytic site maybe constrict the substrate-binding channel, thereby diminishing substrate recognition. These findings underscore the pivotal role of N-glycosylation site variations of GH12 endoglucanase in modulating enzyme characteristics.

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