State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic of China.
Appl Microbiol Biotechnol. 2021 Apr;105(7):2901-2909. doi: 10.1007/s00253-021-11229-7. Epub 2021 Mar 22.
In this study, we report a chromogenic reaction between magnesium ascorbyl phosphate (MAP) and ferric chloride to generate a Brown-Red clathrate, while the Treated MAP by phosphatases forms Colorless (BRTC) product with ferric chloride. The BRTC was indicative of phosphatase activity-mediated excision of phosphorous group from MAP and utilized to screen phosphatases from bacterial cell lysates. From ten tested strains, BRTC was observed in the cell lysate of Salmonella enterica subsp. enterica serovar Cerro 87. BRTC was again employed to track phosphatase activity of the resuspensions of the ammonium sulfate graded precipitations of the cell lysate. Two phosphatases, PhoN and YcdX, were identified by LC-MS/MS analysis in the protein fraction giving most obvious BRTC phenotype and validated by examination of in vitro activity of the purified proteins. KEY POINTS: • BRTC is labelling-free, naked-eye visible, and independent of any facilities. • BRTC can directly screen phosphatases from microbial cell lysates. • Using BRTC system, two phosphatases were identified in Salmonella enterica subsp. enterica serovar Cerro 87.
在这项研究中，我们报告了镁抗坏血酸磷酸酯（MAP）和氯化铁之间的显色反应，生成棕红色包合物，而经磷酸酶处理的 MAP 与氯化铁形成无色（BRTC）产物。BRTC 指示了磷酸酶活性介导的 MAP 中磷基团的切除，并用于从细菌细胞裂解物中筛选磷酸酶。在十种测试菌株中，沙门氏菌亚种肠亚种塞罗 87 的细胞裂解物中观察到 BRTC。BRTC 再次用于跟踪细胞裂解物的硫酸铵分级沉淀的悬浮液中的磷酸酶活性。通过 LC-MS/MS 分析在产生最明显 BRTC 表型的蛋白质部分中鉴定出两种磷酸酶 PhoN 和 YcdX，并通过检查纯化蛋白质的体外活性进行验证。 要点： • BRTC 是无标记的、肉眼可见的，且不依赖于任何设备。 • BRTC 可直接从微生物细胞裂解物中筛选磷酸酶。 • 使用 BRTC 系统，在沙门氏菌亚种肠亚种塞罗 87 中鉴定出两种磷酸酶。
