Zirconium MOF Cage Turns Horseradish Enzyme Into a Reusable Water-Cleaning Catalyst

Water pollution by trace organic chemicals has become one of the most stubborn environmental problems of the modern era. Pharmaceuticals, pesticides, personal-care products, and industrial compounds are now routinely detected in drinking water, groundwater, seawater, and wastewater around the world, often at concentrations measured in nanograms to a few hundred micrograms per liter. Even at these vanishingly small levels, many of these so-called emerging pollutants have been linked to oxidative stress, growth inhibition, and developmental, behavioral, and reproductive harm in aquatic organisms. Conventional treatment trains were never designed to strip out such molecules, which has pushed researchers toward biocatalytic approaches that exploit the exquisite selectivity of enzymes. A new study published in Environmental Science and Pollution Research reports a promising step forward: horseradish peroxidase anchored onto an amino-functionalized zirconium metal–organic framework, known as HRP@UiO-66-NH2, that degraded nearly all of two stubborn pollutants within half an hour and could be reused repeatedly with almost no loss of activity.

The research team, led by Khadega A. Al-Maqdi of UAE University together with colleagues at Khalifa University, the University of Arkansas at Little Rock, and collaborators, chose horseradish peroxidase (HRP) for good reason. Peroxidases are oxidoreductase enzymes that use hydrogen peroxide to oxidize a broad range of substrates, and HRP in particular has long attracted attention for water remediation because of its high catalytic efficiency and selectivity. The catch is operational: free enzymes in solution are fragile, difficult to recover, and quickly lose activity under the temperature swings, pH extremes, and oxidative conditions of real treatment scenarios. Poor reusability drives up cost and has largely confined enzyme-based remediation to the laboratory. Immobilization on a solid support is the standard remedy, and the nature of that support can make or break the technology.

The support selected here was UiO-66-NH2, a zirconium-based metal–organic framework (MOF) built from zirconium chloride and 2-aminoterephthalic acid. MOFs are crystalline, highly porous materials in which metal ions or clusters are coordinated to organic linkers, producing enormous internal surface areas and tunable pore architectures. UiO-66-NH2 brings additional advantages: the strong zirconium–oxygen coordination bonds confer exceptional structural and aqueous stability, while pendant amino groups on the linkers promote hydrogen bonding and electrostatic interactions at the enzyme–MOF interface, helping the protein adhere and remain stable. Immobilization was deliberately simple and gentle. Five milligrams of the MOF were dispersed in solutions of HRP at concentrations ranging from 0.5 to 4 milligrams per milliliter, shaken for four hours, and then collected by centrifugation and washed to remove unbound enzyme. This physical adsorption approach preserves the enzyme’s native structure better than covalent coupling or crosslinking, at the cost of a need to verify that the protein does not leach away.

Optimization experiments showed that relative activity rose as the HRP loading concentration increased from 0.5 to 1 milligram per milliliter but plateaued beyond that point, indicating that the MOF had reached its maximum uptake capacity at 1 milligram per milliliter, which was used for all subsequent work. Leaching tests addressed the Achilles heel of physical immobilization: after the first wash, minimal enzyme was detected in the wash solution, and after the second wash no unbound HRP appeared in the supernatant at all, confirming that the enzyme was effectively anchored. The composite was then subjected to a battery of characterization techniques. Scanning electron microscopy revealed that the MOF crystallized as ball-shaped particles whose morphology was unchanged by enzyme loading, while energy-dispersive X-ray spectroscopy detected increases in the carbon, nitrogen, and oxygen signals after immobilization, consistent with protein incorporation. Powder X-ray diffraction showed that the characteristic diffraction peaks of the framework survived intact, and thermogravimetric analysis revealed an additional low-temperature weight loss in the composite attributable to protein decomposition, further evidence of successful binding.

Brunauer–Emmett–Teller surface area measurements added a telling detail. The bare MOF had a surface area of 256.27 square meters per gram, which dropped to 207.33 square meters per gram after enzyme loading, with pore volume falling from 0.17 to 0.14 cubic centimeters per gram. Both materials displayed Type I nitrogen adsorption isotherms, indicating predominantly microporous character. The reduction suggests that HRP molecules partially blocked the framework’s pores, a pattern seen repeatedly in other enzyme–MOF systems such as laccase on MIL-53(Al) and beta-lactamase on ZIF-8. Fourier-transform infrared spectroscopy told a complementary story: the amide I and amide II bands characteristic of the protein’s peptide bonds overlapped with the framework’s own absorptions in the 1500 to 1700 per centimeter region, producing broadened signals without shifts or signs of framework degradation. Together the data confirmed that immobilization neither destroyed the MOF’s crystallinity nor its thermal stability, which persisted up to roughly 350 to 400 degrees Celsius.

Functional testing revealed that immobilization did more than just fix the enzyme in place; it changed its operating envelope. Both free and immobilized HRP performed best at pH 4, with a narrow operational window from pH 3 to 5, but the immobilized enzyme showed roughly 30 percent higher activity than the free enzyme at pH 3, hinting at improved stability or substrate accessibility under acidic conditions. More striking was the temperature shift: free HRP peaked at 30 degrees Celsius, while HRP@UiO-66-NH2 reached maximum activity at 40 degrees and outperformed the free enzyme by around 10 percent or more at 50 and 70 degrees. The peroxide story was equally significant. Free HRP achieved maximum activity at about 0.72 millimolar hydrogen peroxide and then declined sharply, because excess peroxide drives the enzyme’s heme into irreversibly inactive species. The immobilized enzyme peaked at 1.08 millimolar and retained substantial activity at 1.62 and 2.43 millimolar, indicating that the framework shields the protein from oxidative damage and restricts the conformational changes that lead to heme destruction.

Reusability, the economic linchpin of any immobilized enzyme, proved robust. Over four consecutive reaction cycles, HRP@UiO-66-NH2 retained approximately 95 percent of its initial activity, with a decline to around 60 percent only in the fifth cycle, likely from leaching or denaturation. The authors are careful to note, however, that post-use characterization was not performed, so retained catalytic activity demonstrates functional reusability but does not directly prove that the framework’s crystallinity and morphology survived the cycles; future work combining post-cycle diffraction, spectroscopy, microscopy, and leaching measurements is needed to distinguish enzyme deactivation, mass-transfer limitations, and possible structural changes in the MOF.

The real test came with pollutants. The team challenged the biocatalyst with a mixture of nine structurally diverse emerging pollutants detected in water sources across China, the United Arab Emirates, Poland, and Brazil: 2-mercaptobenzothiazole (a rubber-industry chemical), paracetamol, the antibiotic sulfamethoxazole, the preservative methylparaben, salicylic acid, the antibiotic lincomycin hydrochloride, the beta-blocker atenolol, the fungicide thiabendazole, and the insect repellent DEET. Degradation was quantified by liquid chromatography–tandem mass spectrometry in multiple-reaction-monitoring mode, a sensitive approach that tracks the disappearance of each compound’s characteristic molecular transitions. Within 30 minutes, HRP@UiO-66-NH2 degraded 99.7 percent of 2-mercaptobenzothiazole and 97.4 percent of paracetamol, while the remaining pollutants were removed far less efficiently, a pattern consistent with earlier reports that certain peroxidases struggle with specific substrates, possibly because of competition or inhibition within the mixture rather than inherent inability.

That gap is where redox mediators enter the picture. The team added 1-hydroxybenzotriazole (HOBT), a small molecule that HRP oxidizes to a reactive species capable of transferring oxidative equivalents to pollutants that interact poorly with the enzyme’s active site. The effect was dramatic and pollutant-dependent. Degradation of sulfamethoxazole rose from 23.3 to 53.1 percent, methylparaben from 14.5 to 57.6 percent, and salicylic acid from 12.4 to 54.3 percent, while MBT and paracetamol, already nearly eliminated, showed no further change, and thiabendazole, lincomycin, and atenolol remained largely untouched. Comparisons with free HRP added nuance: free enzyme plus HOBT pushed sulfamethoxazole degradation to 80.2 percent, roughly 30 percentage points higher than the immobilized version, possibly because minor conformational changes imposed by the support alter substrate selectivity. Yet the immobilized enzyme outperformed free HRP on DEET by more than 10 percent, encouraging for a notoriously recalcitrant compound. Unlike MOF-based adsorption, which merely accumulates pollutants on finite surface sites and requires regeneration, the enzyme–MOF composite catalytically transforms susceptible molecules through repeated oxidation cycles powered by hydrogen peroxide.

The authors are candid about limitations. The experiments were conducted in a controlled buffered model-water system, so performance may differ in real wastewater laden with organic matter, dissolved ions, and competing contaminants. Removal of a parent compound does not confirm complete mineralization or reduced toxicity, and transformation products were not identified, meaning the proposed mediator-assisted mechanisms remain plausible rather than proven pathways. Still, the study demonstrates that a zirconium MOF can simultaneously protect a fragile enzyme, extend its thermal and peroxide tolerance, enable four-cycle reuse, and, with a selectively applied redox mediator, broaden its reach across chemically diverse micropollutants. The researchers propose evaluating HRP@UiO-66-NH2 as a targeted polishing treatment for emerging pollutants, with mediators deployed according to pollutant-specific effects, and call for follow-up work on transformation products, toxicity before and after treatment, real-matrix performance, and scalability. If those hurdles are cleared, enzyme–MOF composites of this kind could move from the laboratory bench toward a genuine role in safeguarding drinking water.

Subject of Research: Immobilization of horseradish peroxidase on the metal–organic framework UiO-66-NH2 for enzymatic degradation of emerging water pollutants

Article Title: Efficient enzyme-mediated degradation of various emerging pollutants, including 2-mercaptobenzothiazole, paracetamol, and sulfamethoxazole, using HRP@UiO-66-NH2

Article References: Al-Maqdi, K. A., Alneyadi, S., Elmerhi, N., Siddig, L. A., Alzamly, A., Shah, I., & Ashraf, S. S. (2026). Efficient enzyme-mediated degradation of various emerging pollutants, including 2-mercaptobenzothiazole, paracetamol, and sulfamethoxazole, using HRP@UiO-66-NH2. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-026-38269-8

Image Credits: AI Generated

DOI: 10.1007/s11356-026-38269-8

Keywords: horseradish peroxidase, UiO-66-NH2, metal–organic framework, enzyme immobilization, emerging pollutants, water remediation, bioremediation, redox mediator, HOBT, sulfamethoxazole, paracetamol, LC–MS/MS