A Preliminary Study On Penicillin Binding Protein3and Its Application In Determination Of β-lactam Antibiotics

The (3-lactam antibiotics are the oldest group of antibiotics that widely used in the prevention and treatment of bacterial infections. Illegal use and abuse of these antibiotics may result in the presence of residues in animal edible products which can lead to food safe problems and have arised widespread concern at home and abroad. There is an urgent need to develop a fast, reliable and efficient detection method for montoring these antibiotics residues in food samples to ensure food safety and human health. Nowadays, most immunoassays focus on detecting one analyte or several structural-related analytes. Penicillin binding proteins (PBPs) which could specifically recognize intact (3-lactam antibiotics, have become a hotspot in the detection of β-lactams. In this study, a fast assay was developed based on PBP3from Streptococcus pneumoniae R6for the detection of β-lactams in milk, and a preliminary study on the structure of PBP3was conducted by molecular docking and site-directed mutagenesis.The gene pbp3was amplified by PCR and cloned in expression vector pET28b, and a six-histidine fusion protein was overexpressed in Eschericha coli BL21(DE3). Efficient expression was achieved at20℃for16h with0.2mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG) by optimization. PBP3was purified by Ni-NTA affinity chromatography with the soluble expression efficiency of3mg/100mL. The specific recognitions between PBP3and β-lactams could be affected greatly by pH, ionic strength and the presence of organic solvent.Five β-lactam antibiotics were coupled to horseradish peroxidase (HRP) by1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) method, and ampicillin conjugate, labelled as AMP-HRP, was selected due to the high affnity with PBP3. A microplate assay based on PBP3and AMP-HRP was developed and optimized for determination of27β-lactams in milk, with limits of detection (LODs) ranging from0.26ng mL-1to109.46ng mL-1, IC5o values ranging from1.83ng mL-1to426.02ng mL-1. Except cefoperazone, most of the β-lactams could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from63.97%to107.26%in whole milk and from74.06%to106.31%in skimmed milk, with coefficients of variation (CVs) less than16%.In this study, the interactions between PBP3and12β-lactams were investigated by molecular docking using Surflex X-2.0. Hydrogen-bonding and hydrophobic effects were the dominant force during the docking. Besides the important amino acids reported before, five amino acids with high occurrences, Ser97, Asnl63, Thr166, Thr243and Asp244, were also participated in the formation of hydrogen bond and binding pocket, and played an important role in the interaction model. The comparative analysis of docking models indicated that the size and structure of the side chain on β-lactams directly impact on the geometric matching and energy change between β-lactams and PBP3, resulting in the differences on affnity. Five disulfide mutants were obtained by site-directed mutagenesis, and displayed some differences on affinity and thermostability compared with PBP3wildtype.Among them, the thermostability of A353C/E393C mutant improved to some extent, while the affinity was not be affected. The investigation on affnity and thermostability suggested that the flexible regions in the C-terminal, which was beyond the active sites of PBP3, might be important for thermostability. Our preliminary results provided a theoretical basis for the subsequent evolutions of PBP3in vitro.