Peroxidase-Mediated Bioremediation of Lead and Zinc by Indigenous Bacterial Isolates: Enzymatic Characterization, Biosorption Kinetics, and Mechanistic Gene Expression Analysis

Abstract

Background: Heavy metal contamination by lead (Pb) and zinc (Zn) poses a critical threat to environmental and public health. This study aimed to isolate and characterize indigenous metal-resistant bacteria from contaminated agricultural soil in Wasit Governorate, Iraq, and to establish a quantitative link between peroxidase enzyme induction and metal removal capacity. Methods: Eighteen bacterial isolates were recovered from Pb- and Zn-contaminated soil and screened for metal tolerance, peroxidase induction, and metal removal. The two superior isolates were identified by 16S rRNA gene sequencing. Peroxidase specific activity and Michaelis–Menten kinetics were determined spectrophotometrically under graded metal stress. Resting-cell biosorption was modelled using pseudo-first-order, pseudo-second-order, and Langmuir/Freundlich isotherm models. FTIR and SEM-EDX characterized surface interactions, and transcriptional responses of peroxidase-related genes (katE, ahpC, bcp) were screened by RT-qPCR. Results: Two isolates – Bacillus sp. PB-07 and Pseudomonas sp. ZN-12 – exhibited minimum inhibitory concentrations (MICs) of 18 mM Pb2+ and 8 mM Zn2+, respectively. Peroxidase-specific activity increased dose-dependently, reaching 38.7 ± 2.8 U/mg protein (212% induction; p < 0.001) in PB-07 and 35.2 ± 2.1 U/mg protein (133% induction) in ZN-12. Resting cells removed 78.4 ± 3.2% Pb2+ and 65.2 ± 2.9% Zn2+ within 48 h, best described by pseudo-second-order kinetics (R2 > 0.994) and Langmuir isotherms (qmax = 89.3 and 71.6 mg/g). A strong positive correlation (r ≥ 0.94; p < 0.001) was observed between peroxidase activity and metal removal efficiency. RT-qPCR revealed significant upregulation of ahpC (up to 6.2-fold), katE (up to 4.8-fold), and bcp (up to 3.4-fold) under metal stress. Conclusion: These findings establish a coherent enzymatic framework linking peroxidase induction to metal detoxification and position these indigenous isolates as strong candidates for field bioremediation applications.