Au Ho-Wah, Tsang Man-Wah, Chen Yu Wai, So Pui-Kin, Wong Kwok-Yin, Leung Yun-Chung
Department of Applied Biology and Chemical Technology, State Key Laboratory of Chemical Biology and Drug Discovery, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
Lo Ka Chung Research Centre for Natural Anti-Cancer Drug Development, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
PLoS One. 2020 Oct 30;15(10):e0241594. doi: 10.1371/journal.pone.0241594. eCollection 2020.
β-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated β-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for β-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP β-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to β-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four β-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 μM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated β-lactamases as biosensing devices for β-lactam antibiotics.
β-内酰胺抗生素检测在食品安全控制、环境监测和药代动力学研究中具有重要意义。在此，我们报告了两种BADAN偶联的β-内酰胺酶E166Cb和E166Cb/N170Q的开发，作为用于β-内酰胺抗生素检测的灵敏生物传感器。这些生物传感器是通过将环境敏感的BADAN探针偶联到PenPβ-内酰胺酶E166C和E166C/N170Q突变体活性位点的166位构建而成。它们在β-内酰胺抗生素作用下产生荧光开启信号。E166Cb的分子动力学模拟表明，开启信号可能归因于BADAN微环境的极性变化以及抗生素结合后附近Tyr-105对BADAN荧光猝灭作用的消除。在检测四种β-内酰胺类药物（青霉素G、青霉素V、头孢噻肟和莫西沙星）时，E166Cb和E166Cb/N170Q均以抗生素浓度依赖性方式产生信号输出，动态范围为10 nM至1 μM。与E166Cb相比，E166Cb/N170Q由于其水解抗生素分析物的能力较低，通常表现出更稳定的信号。E166Cb和E166Cb/N170Q的整体生物传感器性能与其各自的荧光素修饰对应物E166Cf和E166Cf/N170Q相当。但相比之下，BADAN偶联酶显示出更高的灵敏度，在检测莫西沙星时响应更快，对青霉素G的荧光信号更稳定。本研究说明了BADAN偶联的β-内酰胺酶作为β-内酰胺抗生素生物传感装置的潜力。
