N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4–(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs. The gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12–35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min−1 mg−1, 0.50 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3–(GlcNAc)7) from (GlcNAc)2–(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

中文翻译：

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一种来自梅园几丁质分解菌的新型细菌β-N-乙酰氨基葡萄糖苷酶，具有转糖基化和逆向水解活性。

N-乙酰氨基葡萄糖（GlcNAc）和N-乙酰壳寡糖（N-Acetyl COSs）具有多种生物活性，已广泛应用于医药、农业、食品和化工等行业。特别是聚合度为 4 至 7 的较高 N-乙酰 COSs ((GlcNAc)4-(GlcNAc)7) 显示出良好的抗肿瘤和抗菌活性，并对自然杀伤细胞具有强烈的刺激活性。因此，发现一种既能产生GlcNAc，又能合成N-乙酰COSs的β-N-乙酰氨基葡萄糖苷酶（NAGase）具有重要意义。编码新型β-N-乙酰氨基葡萄糖苷酶的基因，命名为CmNAGase，是从梅园几丁质分解杆菌SYBC-H1中克隆的。推导的 CmNAGase 氨基酸序列包含一个糖苷水解酶家族 20 催化模块，该模块与大多数已充分表征的 NAGase 的相应结构域显示出低同一性 (12-35%)。CmNAGase 基因在大肠杆菌BL21 (DE3) 细胞中以活性形式高度表达。纯化的 CmNAGase 对 p-硝基苯基-N-乙酰氨基葡萄糖 (pNP-GlcNAc) 的比活性为 4878.6 U/mg 蛋白质。CmNAGase 的分子量为 92 kDa，其最佳活性为 pH 5.4 和 40 °C。PNP-GlcNAc 的 CmNAGase 的 Vmax、Km、Kcat 和 Kcat/Km 分别为 16,666.67 μmol min-1 mg-1、0.50 μmol mL-1、25,555.56 s-1 和 51,111.12 mL μmol-1 s-1。对 N-乙酰 COSs 和胶体几丁质的水解产物的分析表明，CmNAGase 是一种典型的外作用 NAGase。特别，CmNAGase可以分别从(GlcNAc)2-(GlcNAc)6合成更高的N-乙酰COSs((GlcNAc)3-(GlcNAc)7)，表明它具有转糖基化活性。此外，CmNAGase 还具有对 GlcNAc 的反向水解活性，可合成各种连接的 GlcNAc 二聚体。本研究中记录的观察结果表明，CmNAGase 是一种具有外作用、转糖基化和反向水解活性的新型 NAGase，表明其可能应用于生产 GlcNAc 或更高的 N-乙酰 COS。