Visualized characterization of bacterial penicillin G acylase for the hydrolysis of β-lactams using an activatable NIR fluorescent probe

Publication date: Available online 14 February 2020Source: Sensors and Actuators B: ChemicalAuthor(s): Zhenhao Tian, Lei Feng, Lu Li, Xiangge Tian, Jingnan Cui, Houli Zhang, Chao Wang, Huilian Huang, Baojing Zhang, Xiaochi MaAbstractPenicillin G acylase (PGA) identified from bacteria, is used as industrial biocatalyst for the hydrolysis of penicillins along with the production of 6-aminopenicillanic acid, a key intermediate of the semi-synthetic β-lactam antibiotics. Due to wide usage of PGA, some novel homology bioactive enzymes are investigated in various bacteria, and visualized analysis technique would be necessary and helpful for the development and utilization of PGA function in living systems. In the present work, an activated off-on near-infrared (NIR) fluorescent probe (DDAP) deduced from a novel fluorophore has been designed and developed for sensitively and selectively detecting endogenous PGA in real-time. The hydrolysis mechanism has been elucidated using in silico docking analysis, which confirmed DDAP as the specific substrate of PGA. Using DDAP as the fluorescent probe, the endogenous bacterial PGA was imaged in living systems successfully, which could be applied to identify bacteria with PGA expression from complex microorganism samples. Furthermore, using DDAP as the staining agent, the endogenous PGA in various bacteria has been profiled visually on native-PAGE gel, and the PGA activity in bacterial lysates could be effectively determined. The PAGE-gel for...

Source: Sensors and Actuators B: Chemical - February 14, 2020 Category: Chemistry Source Type: research