Characterization and Statistical Optimization of Enterobatin Synthesized by Escherichia coli OQ866153

Microorganisms produce siderophores, which are secondary metabolites with a high affinity for iron. Siderophores have received significant attention due to their diverse applications in ecological and clinical research. In this study, siderophores production by Escherichia coli OQ866153 was optimized using two-stage statistical approach involving Plackett-Burman design (PBD) and response surface methodology (RSM) using central composite design (CCD). Out of 23 variables, succinate, tryptophan, Na₂HPO₄, CaCl₂, agitation, and KH₂PO₄ were found to have the most significant effect on siderophores production in the first optimization stage with the highest SU% of 43.67%. In the second stage, RSM using CCD was utilized, and the optimal conditions were determined to be 0.3 g/l succinate, 0 g/l tryptophan, 6 g/l Na₂HPO₄, 0.1 g/l CaCl₂, 150 RPM agitation, and 0.6 g/l KH₂PO₄, resulting in a maximum siderophore units (SU%) of 89.13%. The model was significant, as indicated by the model f-value of 314.14 (p-value = 0.0004) and coefficient of determination R² of 0.9950. During validation experiments, the obtained maximum SU% was increased up to 87.1472%, which was two times as the value obtained under ordinary conditions (46.62%). The produced siderophores were purified and characterized using ¹H, ¹³C NMR, IR spectroscopy. The obtained results indicated that the compound was enterobactin and entABCDEF genes were further detected in Escherichia coli OQ866153 extracted DNA. To our knowledge, this is the first report of statistical optimization for enterobactin synthesis by an E. coli strain isolated from a clinical source in Egypt.