One-step degradation of 4 classes of β-lactam using β-lactamase enzyme cocktail

Antibiotics, particularly β-lactams, are emerging environmental contaminants that induce antibiotic-resistant bacteria or genes, which threaten public health. Enzymatic degradation is an environmentally friendly approach to reducing residual antibiotics; however, its application is limited by enzyme's narrow substrate spectrum, which cannot deal with the co-occurrence of multiple β-lactam classes in the environment. To address this issue, four representative β-lactamases were biochemically characterized and two of them (CTX-33 to VIM-1) were selected because of their broader substrate spectrum and better stability. Compared to a single enzyme that can only degrade the ring structure of three classes of β-lactam antibiotics at most, the enzyme cocktail (with a ratio of CTX-33 to VIM-1 of 4:1) was able to hydrolyze nineteen antibiotics from four different β-lactam families. In laboratory experiments, the enzyme cocktail demonstrated degradation efficiencies of over 99% for the four classes of β-lactams (penicillin, cephalosporin, carbapenem, and monobactam) within the concentration range of 1 to 100 mg/L, both individually and simultaneously. Moreover, when applied to pharmaceutical industry wastewater, pig farm wastewater, and river water, over 99% simultaneous degradation of β-lactams was observed at 1 mg/L within 60 min and at 100 mg/L within 5 h. This is the first report of an enzymatic method capable of simultaneously removing antibiotics from four β-lactam classes. This study offers a novel and eco-friendly approach to β-lactam degradation.