Background Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity. Results Five N-glycosylation sites with variable high mannose-type oligosaccharides (Man3-9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in T m ) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity. Conclusion The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

中文翻译：

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来自 Kluyveromyces cicerisporus 的重组外切菊粉酶的独特 N-糖基化及其对酶活性和热稳定性的影响。

背景技术菊粉酶可将聚果聚糖水解成高果糖浆和低聚果糖，广泛应用于食品、医药工业和菊芋生物精炼。在本研究中，来自 Kluyveromyces cicerisporus CBS4857 的重组外切菊粉酶 (rKcINU1) 被证明是一种 N-连接糖蛋白，并且 N-连接聚糖链的去除导致活性降低。结果在 rKcINU1 中确认了五个具有可变高甘露糖型寡糖 (Man3-9GlcNAc2) 的 N-糖基化位点。结构建模显示所有五个糖基化位点（Asn-362、Asn-370、Asn-399、Asn-467 和 Asn-526）均位于 C 端 β-夹心结构域，已被证明更有利于比N-末端结构域发生糖基化修饰。获得了Asn被Gln取代的单位点N-糖基化突变体，构建了去除所有5个N-糖基化位点的Mut，导致所有酶活性丧失。有趣的是，N362Q导致对菊粉的比活性增加18%，而热稳定性显着下降（T m 下降2.91°C），其他单突变导致比活性不同程度的降低，其中N467Q的酶活性最低。结N-聚糖带来刚性。同时，其他四个位点的 N-糖基化通过微调 β-夹心结构域结构正向调节由底物亲和力改变引起的酶活性。这可能促进了底物的捕获和转移到酶活性腔，其方式与碳水化合物结合模块 (CBM) 的方式非常相似，即赋予 β-夹心结构域的链具有 CBM 的功能。本研究发现了一个独特的更利于糖基化的C末端序列，从而为真菌酶的糖工程改造开辟了新的视角，即通过重新设计C末端结构域的氨基酸序列，优化酶学性质。其他四个位点的 N-糖基化通过微调 β-夹心结构域结构正向调节由改变底物亲和力引起的酶活性。这可能促进了底物的捕获和转移到酶活性腔，其方式与碳水化合物结合模块 (CBM) 的方式非常相似，即赋予 β-夹心结构域的链具有 CBM 的功能。本研究发现了一个独特的更利于糖基化的C末端序列，从而为真菌酶的糖工程改造开辟了新的视角，即通过重新设计C末端结构域的氨基酸序列，优化酶学性质。其他四个位点的 N-糖基化通过微调 β-夹心结构域结构正向调节由改变底物亲和力引起的酶活性。这可能促进了底物的捕获和转移到酶活性腔，其方式与碳水化合物结合模块 (CBM) 的方式非常相似，即赋予 β-夹心结构域的链具有 CBM 的功能。本研究发现了一个独特的更利于糖基化的C末端序列，从而为真菌酶的糖工程改造开辟了新的视角，即通过重新设计C末端结构域的氨基酸序列，优化酶学性质。以与碳水化合物结合模块 (CBM) 非常相似的方式，即赋予 β-三明治结构域以 CBM 功能的链。本研究发现了一个独特的更利于糖基化的C末端序列，从而为真菌酶的糖工程改造开辟了新的视角，即通过重新设计C末端结构域的氨基酸序列，优化酶学性质。以与碳水化合物结合模块 (CBM) 非常相似的方式，即赋予 β-三明治结构域以 CBM 功能的链。本研究发现了一个独特的更利于糖基化的C末端序列，从而为真菌酶的糖工程改造开辟了新的视角，即通过重新设计C末端结构域的氨基酸序列，优化酶学性质。