Mechanistic insights into extracellular ampicillin degradation by Antarctic Pseudomonas sp. WH-5 and engineering of a robust β-lactamase

β-Lactam antibiotic contamination is a major environmental driver of antimicrobial resistance, yet whole-cell bioremediation is often limited by poor environmental adaptability and catalyst instability. In this study, we report an Antarctic Pseudomonas sp. WH-5 whose cell-free supernatant removed 100 μg/mL ampicillin (AMP) within 3 h and retained strong activity over 10-50°C and pH 5-10, demonstrating greater robustness than cell-based fractions. Genomic analysis identified a class C β-lactamase as the key driver of this activity. Semi-rational engineering further improved enzyme performance, and mutant N233R displayed a 75.7% increase in activity at 60°C together with enhanced pH tolerance. LC-MS revealed a synergistic extracellular degradation pathway in which multiple hydrolases promoted further transformation of ring-opened intermediates. Antibacterial assays and toxicity prediction demonstrated effective detoxification, generating low-toxic and non-mutagenic products. This study provides a robust and safe Antarctic resource for future enzymatic treatment of β-lactam antibiotics and offers a promising route for integrating extremophile-derived biocatalysts with enzyme engineering for environmental applications.