Grishin Alexander V, Konstantinova Svetlana V, Vasina Irina V, Shestak Nikita V, Karyagina Anna S, Lunin Vladimir G
N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia.
All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, 127550 Moscow, Russia.
Antibiotics (Basel). 2020 Dec 17;9(12):917. doi: 10.3390/antibiotics9120917.
Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen . Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.
抗菌溶素是一类能够水解细菌肽聚糖的酶，由于渗透裂解作用，可导致细菌细胞迅速死亡。溶葡萄球菌酶是对重要医院病原体具有活性的最有效且研究充分的溶素之一。与大多数其他溶素类似，溶葡萄球菌酶由酶结构域和肽聚糖结合结构域组成，这两个结构域均影响其抗菌活性。因此，能够独立研究这两个结构域的活性是很有必要的。溶葡萄球菌酶可裂解葡萄球菌肽聚糖中的五肽甘氨酸交联桥。在此，我们报告一种基于肽氨基与茚三酮的显色反应来研究溶葡萄球菌酶对分离出的五肽甘氨酸肽的催化活性的方法。与先前报道的测定方法不同，该方法不需要内部化学合成或专门设备，并且大多数实验室都能轻松进行。我们展示了使用该方法来研究乙二胺四乙酸（EDTA）处理对溶葡萄球菌酶活性的影响。我们进一步使用该方法来测定溶葡萄球菌酶对分离出的五肽甘氨酸的催化效率，并将其与对整个葡萄球菌细胞的表观催化效率进行比较。这些结果突出了溶葡萄球菌酶的酶结构域和肽聚糖结合结构域对其溶菌活性的相对影响。
