Production, purification and biochemical characterization of a novel DyP-type peroxidase from an indigenous metal-resistant Pseudomonas sp. ZN-12: statistical optimization, comparison with commercial horseradish peroxidase, and molecular docking for enzym

Abstract

Enzymatic bioremediation offers a selective, low-sludge alternative to physicochemical heavy-metal treatment, but its economics are constrained by the cost of commercial enzymes such as horseradish peroxidase (HRP). Here we report the production, purification and full biochemical characterization of a novel dye-decolorizing (DyP-type) peroxidase from an indigenous, metal-resistant Pseudomonas sp. ZN-12 isolated from contaminated soil in Wasit, Iraq, and benchmark it directly against commercial HRP (Sigma-Aldrich Type VI-A) for the enzymatic detoxification of Pb(II) and Zn(II). A six-factor Box–Behnken design raised peroxidase yield 3.7-fold over unoptimized medium (validated 31.8 ± 1.2 U mL⁻¹; model R² = 0.962), with pH, glucose and H₂O₂ induction the dominant factors. A four-step purification (ammonium-sulfate fractionation, DEAE ion exchange, phenyl-Sepharose hydrophobic interaction and Sephadex G-100 gel filtration) delivered an 84.5-fold-enriched, homogeneous enzyme of 412.5 U mg⁻¹ specific activity. The purified protein was a 38 ± 1 kDa monomer with a Soret band at 405 nm (Reinheitszahl 2.6) and a substrate preference for ABTS over guaiacol — the diagnostic signature of a DyP-B peroxidase. Relative to HRP, the indigenous enzyme was less thermostable (t½ 240 vs >480 min at 40 °C) but markedly more tolerant of the target metals (38%/24% activity inhibition by 5 mM Pb²⁺/Zn²⁺ versus 61%/45% for HRP). Under separately optimized conditions (RSM desirability 0.94), the indigenous enzyme removed 91.6 ± 2.4% Pb(II) — statistically equal to HRP (93.4%; p = 0.18) — and 82.4 ± 2.7% Zn(II), significantly exceeding HRP (71.6%; p = 0.003). Molecular docking rationalised this pattern: both metals bound the open DyP-B distal pocket (His₅₆/Arg₃₈/Glu₈₄) more tightly than the sterically crowded HRP pocket, with the largest advantage for Zn²⁺ (ΔG = −8.1 vs −5.8 kcal mol⁻¹). The indigenous DyP-type peroxidase is thus a locally producible, metal-tolerant biocatalyst that matches HRP on lead and surpasses it on zinc, offering a cost-effective route to enzymatic heavy-metal remediation.