Fungal β-N-acetylhexosaminidases of the CAZy family 20 of glycoside hydrolases are well-established tools for the enzymatic synthesis of a wide variety of natural and modified oligosaccharides and glycoconjugates. In order to increase their synthetic efficiency, the β-N-acetylhexosaminidase from Aspergillus oryzae (AoHex) was employed as a model enzyme for enzyme engineering aiming at shifting the reaction course from hydrolysis toward transglycosylation. Specifically, nine mutant variants of AoHex were designed by molecular modeling based on its crystal structure and molecular dynamics simulations. The selected mutation hotspots included the tyrosine residue at the active site, which stabilizes the transition state of the reaction, and two residues at the aglycone-binding site, which were replaced by tryptophan residues to increase the hydrophobicity of this subsite. Besides the individual mutants, combined double-mutant variants were also prepared and characterized. As a result, eight out of the studied new AoHex variants had transglycosidase activity, with V306W/Y445N AoHex being a superior transglycosidase with a transglycosylation-to-hydrolysis ratio greater than 110, which is entirely unique among the hypertransglycosylating glycosidase mutants including the GH20 β-N-acetylhexosaminidases.

中文翻译：

---

用于合成壳寡聚体的 GH20 β-N-乙酰己糖胺酶的超糖基化变体

糖苷水解酶的 CAZy 家族 20 的真菌 β- N-乙酰氨基己糖苷酶是用于酶促合成多种天然和修饰的寡糖和糖缀合物的成熟工具。为了提高它们的合成效率，将米曲霉的β- N-乙酰氨基己糖苷酶（Ao Hex）用作酶工程的模型酶，旨在将反应过程从水解转变为转糖基化。具体来说，Ao的九个突变变体Hex是基于其晶体结构和分子动力学模拟通过分子建模设计的。选定的突变热点包括活性位点的酪氨酸残基，它稳定了反应的过渡状态，以及糖苷配基结合位点的两个残基，它们被色氨酸残基取代，以增加该亚位点的疏水性。除了单个突变体，还制备和表征了组合的双突变体变体。结果，研究的新Ao Hex 变体中有 8 个具有转糖苷酶活性，其中 V306W/Y445N Ao Hex 是一种优良的转糖苷酶，转糖基化与水解的比率大于 110，这在包括GH20 β-N-乙酰氨基己糖苷酶。