Role of N-linked glycosylation in the enzymatic properties of a thermophilic GH 10 xylanase from Aspergillus fumigatus expressed in Pichia pastoris.
Role of N-linked glycosylation in the enzymatic properties of a thermophilic GH 10 xylanase from Aspergillus fumigatus expressed in Pichia pastoris.
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N 连接糖基化在巴斯德毕赤酵母中表达的烟曲霉嗜热 GH 10 木聚糖酶的酶特性中的作用
DOI：
10.1371/journal.pone.0171111
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Yao B
中科院分区：
文献类型：
--
作者：
Chang X;Xu B;Bai Y;Luo H;Ma R;Shi P;Yao B
N-糖基化是真菌中普遍存在的一种翻译后修饰，在多种酶功能中发挥作用。本研究在毕赤酵母中异源生产了烟曲霉糖苷水解酶(GH)家族10的木聚糖酶(Af-XYNA)，该酶含有3个潜在的N-糖基化位点(N87、N124和N335)。N-糖基化的Af-XYNA(WT)具有良好的温度和最适pH(75℃，pH 5.0)和良好的热稳定性(在60℃下保持稳定)。为了揭示N-糖基化在Af-XYNA上的作用，用Endo-β-N-乙酰氨基葡萄糖苷酶H(DE)脱糖或在N124(N124T)上进行定点突变。脱糖DE和突变体N124T的pH适应范围较窄，比活力较低，pH和热稳定性较差。进一步的热力学分析表明，N-糖基化程度越高的酶热稳定性越好。本研究表明，Af-XYNA在不同程度和不同部位的糖基化作用是不同的，其中与N124连接的糖链对Af-XYNA的pH和热稳定性起着关键作用。
N-Glycosylation is a posttranslational modification commonly occurred in fungi and plays roles in a variety of enzyme functions. In this study, a xylanase (Af-XYNA) of glycoside hydrolase (GH) family 10 from Aspergillus fumigatus harboring three potential N-glycosylation sites (N87, N124 and N335) was heterologously produced in Pichia pastoris. The N-glycosylated Af-XYNA (WT) exhibited favorable temperature and pH optima (75°C and pH 5.0) and good thermostability (maintaining stable at 60°C). To reveal the role of N-glycosylation on Af-XYNA, the enzyme was deglycosylated by endo-β-N-acetylglucosaminidase H (DE) or modified by site-directed mutagenesis at N124 (N124T). The deglycosylated DE and mutant N124T showed narrower pH adaptation range, lower specific activity, and worse pH and thermal stability. Further thermodynamic analysis revealed that the enzyme with higher N-glycosylation degree was more thermostable. This study demonstrated that the effects of glycosylation at different degrees and sites were diverse, in which the glycan linked to N124 played a key role in pH and thermal stability of Af-XYNA.