Abstract：
Endo-β-N-acetylglucosaminidase (ENGase) is an enzyme that hydrolyzes the chitobiose core of N-glycans and is widely used for glycan analysis on glycoproteins and preparation of precursors for glycosylated compounds. While most of the ENGases that can hydrolyze complex-type glycans are derived from eukaryotes, their production by heterologous expression using Escherichia coli is insufficient, making the production process expensive. From an industrial perspective, there is a need for a less expensive enzyme with higher activity and stability. In this study, we identified a novel ENGase gene from a thermophilic fungus, Rhizomucor pusillus, and named it Endo-Rp. Characterization of the recombinant Endo-Rp showed that the enzyme had maximum hydrolytic activity at 60 °C and hydrolyzed high-mannose-type and biantennary complex-type glycans, but not (2,4)-branched triantennary complex-type or fucosylated glycans. Endo-Rp also hydrolyzed N-glycans attached to RNase B and human transferrin. In summary, we consider Endo-Rp to be a valuable enzyme in various scientific and industrial applications.
摘要：
内切-β-N-乙酰葡糖胺糖苷酶（ENGase）是一种水解N-聚糖的几丁糖核心的酶，广泛用于糖蛋白的聚糖分析和糖基化化合物前体的制备。虽然大多数可以水解复合型聚糖的酶来自真核生物，它们通过使用大肠杆菌异源表达的生产是不够的，使生产过程昂贵。从工业的角度来看,需要具有较高活性和稳定性的较便宜的酶。在这项研究中,我们从嗜热真菌中鉴定出一个新的ENGase基因,根瘤菌，并命名为Endo-Rp.重组Endo-Rp的表征表明，该酶在60°C下具有最大的水解活性，并且水解了高甘露糖型和双触角复合型聚糖，但不是（2,4）-分支三触角复合型或岩藻糖基化聚糖。Endo-Rp还水解与RNA酶B和人转铁蛋白连接的N-聚糖。总之,我们认为Endo-Rp在各种科学和工业应用中是一种有价值的酶。
