The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62-75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6-7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

中文翻译：

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粪肠球菌的新型N-乙酰氨基葡萄糖苷酶（AtlD）的克隆和鉴定。

为了进行生化鉴定，从大肠杆菌个体干酪分离的粪肠球菌菌株的atlD基因被克隆，测序并在大肠杆菌中表达。通过LC-MS / MS获得了异源蛋白的部分氨基酸序列，其对应于称为AtlD的新型肽聚糖水解酶。通过SDS-PAGE，酶谱，Western印迹和排阻色谱法测定，其分子量为62-75kDa。电聚焦的等电点（pI）为4.8。它具有N-乙酰氨基葡萄糖苷酶活性，最佳pH和温度分别在6-7和50°C之间。它在1,000 mM的NaCl溶液中保留了85％的活性，但是对二价离子特别是Zn2 +敏感。对单核细胞增生李斯特菌，金黄色葡萄球菌，和临床来源的肠球菌菌株。由于它具有对抗病原菌的活性，并且由于它在食品基质中存在的离子强度，温度和pH值下具有一定的能力，因此可以在食品工业中用作添加剂。这项研究将有助于设计新的天然抗菌剂来抵抗食源性疾病，并且也可以用作工业或医院卫生剂。