Zymography is a widely used technique enabling visualization of in-gel peptidase/protease hydrolytic activities. This technique is used to study the activity of bacterial peptidoglycan (PG) hydrolytic enzymes named autolysins. Zymography is particularly suited for PG autolysin characterization as bulk PG is notorious to work with due to its highly insoluble nature. This recalcitrant property of PG therefore makes the set-up of PG hydrolytic activity assay very challenging. In the course of studying the catalytic activity of the CwlS protein, a D,L NlpC/P60 endopeptidase possessing multiple LysM carbohydrate-binding domains from Bacillus subtilis, we observed a potential artifact of the zymography technique. The generation of CwlS truncated mutants impaired in their PG binding capacity presented lower apparent hydrolytic activities on zymograms. Furthermore, a catalytically dead version of CwlS, or a CwlS mutant that possesses only its LysM domains and no catalytic domain, maintained similar apparent PG hydrolytic properties as wild-type CwlS on zymograms. Additionally, a mutant harboring twelve mutations in the four LysM domains, previously demonstrated to be unable to bind PG but has a similar net positive charge as the wild-type protein also presented apparent activity on zymogram. We demonstrate in this study that zymography results, which are meant to be interpreted in favor of apparent PG hydrolytic activities, are instead reflecting impairment of gel staining probably due to the very high net positive charge of the protein.

Zymography是一种广泛使用的技术，可实现凝胶内肽酶/蛋白酶水解活性的可视化。该技术用于研究称为自溶素的细菌肽聚糖（PG）水解酶的活性。Zymography特别适用于PG自溶素的表征，因为块状PG由于其高度不溶性而臭名昭著。因此，PG的这种顽强特性使PG水解活性测定的设置非常具有挑战性。在研究CwlS蛋白的催化活性的过程中，具有多个来自枯草芽孢杆菌的LysM碳水化合物结合域的D，L NlpC / P60内肽酶，我们观察到了酶谱技术的潜在伪像。CwlS截短突变体的PG结合能力受损的一代在酶谱图上表现出较低的表观水解活性。此外，CwlS的催化死亡形式，或仅具有LysM结构域而没有催化结构域的CwlS突变体，在酶谱图上具有与野生型CwlS相似的表观PG水解特性。另外，在四个LysM结构域中具有十二个突变的突变体，先前已证明无法结合PG，但具有与野生型蛋白相似的净正电荷，在酶谱图上也表现出明显的活性。在这项研究中，我们证明了酶谱学结果，可以解释为有利于表观PG水解活性，