Single amino acid bionanozyme for environmental remediation

Enzymes efficiently catalyze challenging biochemical reactions but face practical limitations for environmental applications, including high production costs, limited stability under varying conditions, and difficulties in recycling. Laccases, multicopper oxidases capable of oxidizing diverse phenolic pollutants using molecular oxygen (forming water), are attractive yet constrained by the same issues. Nanozymes offer a cost-effective, robust alternative, but most reported systems require complex synthesis and do not structurally resemble native enzymes. Minimalistic, peptide-based bionanozymes have emerged, yet designing laccase mimics remains challenging due to the enzyme’s complex multicopper active site and mechanism. The research question addressed here is whether the self-assembly of a single amino acid, phenylalanine, coordinated with redox-active Cu2+ into a 2D layered structure can mimic laccase’s oxidative function for environmental remediation and biosensing, while overcoming stability, cost, and reusability limitations.

The paper situates the work within enzyme catalysis and environmental remediation using oxidative enzymes, emphasizing laccases for phenol oxidation and their eco-friendly use of O2. It reviews the rise of nanozymes (metal oxides, metals, chalcogenides, carbons, MOFs, etc.) that often suffer from harsh synthesis and lack biomimetic features. Peptide-based minimalistic systems are highlighted for ease of synthesis, biodegradability, and functional supramolecular assembly, with prior examples of catalytic peptide assemblies and laccase-mimicking nanozymes using non-peptidic scaffolds. Despite progress, de novo laccase-mimicking bionanozymes remain scarce due to the complexity of laccase’s active site, motivating a minimalist single–amino acid approach.

• Design and synthesis: Phenylalanine (F) was used as a multidentate ligand to coordinate Cu2+ via amine and carboxylate functionalities in alkaline conditions. An aqueous CuCl2 (5 mM) solution (1 eq) was added slowly to an alkaline F solution (10 mM F, 10 mM NaOH; 2 eq) at 60 °C, yielding blue, thin plate-like F–Cu crystals at the liquid–air interface. Crystals were collected by filtration and washed.
• Structural characterization: Single-crystal X-ray diffraction revealed an octahedral Cu2+ coordination with tetragonal distortion: two F ligands chelate one Cu2+ via O(carboxylate)/N(amine), forming five-membered chelate rings. These units form infinite, covalently linked 2D sheets along a–b, which stack via van der Waals forces into layered crystals. AFM and HRSEM showed micrometer-scale 2D sheets; AFM indicated ~260 nm thick stacks consisting of 10–20 nm layers. Mild ultrasonication exfoliated crystals to nanosheets ~1.8 nm thick (consistent with ~1.6 nm monolayer from SCXRD). PXRD of nanosheets matched the crystal structure. UV–vis showed a broad 615 nm d–d band characteristic of Jahn–Teller-distorted Cu2+. FTIR exhibited a metal–oxygen stretch at 555 cm−1 and red-shifted amine band, consistent with carboxylate/amine–Cu2+ coordination. EDX confirmed Cu, C, O; TGA showed high thermal stability with decomposition at 273 °C and 332 °C.
• Catalytic assays (laccase-mimicking): The benchmark oxidation of 2,4-dichlorophenol (2,4-DP) in the presence of 4-aminoantipyrine (4-AP) was used. Reaction mixtures (PBS 1X, pH ~7.25, 25 °C) contained 0.6 mM 2,4-DP, 0.5 mM 4-AP, and either F–Cu (0.1 mg/mL) or laccase (0.1 mg/mL). Formation of the antipyrilquinoneimine dye was monitored at 510 nm. Initial rates were measured across [2,4-DP] = 0–0.7 mM (with 0.5 mM 4-AP) and fit to Michaelis–Menten to obtain Vmax and KM; kcat and kcat/KM were calculated.
• Controls and mechanism probes: Activity controls included free Cu2+, F alone, mixtures without one component, and Cu complexes of other amino acids (Gly, Trp, His, Cys) and F–Zn. Oxygen dependence was tested by bubbling N2 vs ambient air. Generation of H2O2 was tested using ABTS/HRP after removing F–Cu by centrifugation; addition of exogenous H2O2 served as positive control. EPR probed Cu2+/Cu+ redox states during reaction; in situ optical microscopy visualized reaction progress on crystal surfaces; filtration experiments assessed leaching contributions.
• Stability and recyclability: Relative activity was measured under varying pH (3–9), ionic strengths (NaCl 0–600 mM), real waters (tap, river, seawater), storage (up to 30 days in buffer; up to 210 days in air), and thermal pre-incubation (0–100 °C, 1 h). Recyclability was tested over 15 cycles by centrifugation, washing, and reuse each cycle.
• Substrate scope: Oxidation of various phenolic contaminants (phenol, catechol, hydroquinone, 2-aminophenol, 2,6-dimethoxyphenol, 2-naphthol, 2-nitrophenol, 2,4-DP, 2,4,6-trichlorophenol) was compared between F–Cu and laccase at equal mass loading.
• Computational studies: Spin-polarized DFT (FHI-aims, PBE with TS dispersion) on a tetrameric F–Cu cluster resembling an edge (100) surface, including O2, H2O, and phenol molecules. NEB climbing image calculations identified transition states. Spin configurations were examined; spin densities were analyzed to track radical formation.
• Neurotransmitter detection: Oxidation of catecholamines (epinephrine, dopamine, norepinephrine, L-DOPA) was monitored colorimetrically. For epinephrine (EP), absorbance at 485 nm (adrenochrome) was tracked over time. Calibration curves (20–100 µM EP) were used to determine LOD (3σ/b) for F–Cu vs laccase at 0.1 mg/mL, PBS 1X, 22–25 °C.

• Structure and formation: F–Cu forms hierarchical 2D van der Waals layered crystals comprising infinite covalent 2D sheets of Cu–phenylalaninate; ultrasonication yields ~1.8 nm nanosheets (near-monolayer by SCXRD estimate ~1.6 nm). UV–vis shows a 615 nm Cu2+ d–d band; FTIR and EDX confirm coordination and composition; TGA indicates decomposition at 273 °C and 332 °C.
• Superior laccase-mimicking catalysis: In the 2,4-DP/4-AP assay at equal mass (0.1 mg/mL), F–Cu produces stronger product absorbance at 510 nm than laccase. Michaelis–Menten parameters (2,4-DP, 25 °C): F–Cu Vmax = 6×10−5 mM s−1; KM = 0.19 mM; kcat = 11.9 s−1; kcat/KM = 62.65 mM−1 s−1. Laccase Vmax = 3×10−6 mM s−1; KM = 0.06 mM; kcat = 1.9×10−3 s−1; kcat/KM = 0.03 mM−1 s−1. Per molecular weight, F–Cu achieves >6 orders higher catalytic efficiency: (kcat/KM)/M.Wt = 1.60×10−1 (g−1 l−1)−1 s−1 vs 4.0×10−7 for laccase.
• Essential assembly and metal identity: Cu2+ alone shows ~16% relative activity vs F–Cu; other amino acid–Cu complexes show lower activities (W–Cu 40%, G–Cu 25%, H–Cu 19%, C–Cu 1.5%); F–Zn shows ~1.4%, underscoring the necessity of Cu2+ and the crystalline F–Cu architecture.
• Laccase-like mechanism: No H2O2 detected in supernatants by ABTS/HRP unless exogenous H2O2 added; reaction is O2-dependent (rate 2.5× higher with air vs N2). EPR signal of Cu2+ at 3291 G diminishes upon substrate addition, indicating Cu2+→Cu+ reduction during catalysis. In situ microscopy shows red product forming on crystal surfaces without morphology change; filtration rules out leached species.
• Robust stability and recyclability: F–Cu retains ≥60% activity at pH 3.0 and increases to 65–120% across pH 4–9; laccase drops to 5% (pH 3) and 48% (pH 9). With increasing NaCl (0→600 mM), laccase activity falls from 100% to 1.4%, whereas F–Cu rises from 100% to 360%. In real waters, both active in tap/river; in seawater, laccase inactive, F–Cu retains 76%. Storage: laccase loses all activity by day 10; F–Cu retains ~90% after 30 days in buffer and ~95% after 210 days in air at room temperature. Thermal: laccase loses activity by 60 °C; F–Cu retains 92% after 100 °C (1 h). Recyclability: F–Cu maintains >80% activity after 15 cycles; laccase not recyclable.
• Broad substrate scope: F–Cu oxidizes all tested phenolic pollutants with higher conversions than laccase, notably chlorophenols (2,4-DP; 2,4,6-trichlorophenol), EPA-listed priority pollutants.
• Computational mechanism: DFT shows phenol approaches active Cu site; key H-atom transfer from phenol to –COO of F (barrier ~29 kcal/mol) generates a phenoxyl radical; subsequent H transfer to O2 has 9.5 kcal/mol barrier. Dimerization/polymerization of phenoxyl radicals is highly exothermic (−55 kcal/mol), rendering the overall process thermodynamically favorable. Raman changes in carboxylate vibrations support H transfer and altered Cu–carboxylate binding.
• Neurotransmitter sensing: F–Cu catalyzes epinephrine oxidation to adrenochrome with much faster kinetics (20× faster within first 10 min; Vmax ~43× laccase). Linear response 20–100 µM EP; LOD for EP is 150 nM with F–Cu vs 5 µM for laccase (~36× more sensitive). Similar superior activity observed for DA, NE, and L-DOPA. The method is simpler and ~5400× more cost-effective than laccase for comparable conversion.

The study demonstrates that a minimalistic supramolecular assembly of a single amino acid (phenylalanine) coordinated to Cu2+ forms a 2D layered bionanozyme that effectively mimics laccase’s oxidation of phenolic substrates using dissolved O2 without H2O2 generation. The redox-active, regularly spaced Cu2+/Cu+ sites in an ultrathin, high–surface area architecture enable higher catalytic efficiency than the native enzyme, while the robust inorganic–organic framework confers exceptional stability across pH, ionic strength, temperature, and storage, and permits multiple reuse cycles. Control experiments and spectroscopy support a laccase-like mechanism involving Cu redox cycling and phenoxyl radical formation; DFT clarifies the pivotal H transfer to the carboxylate and the exothermicity of radical coupling. The platform’s broad substrate scope and ultrasensitive catecholamine detection highlight its relevance for environmental remediation (e.g., chlorophenols in wastewater), analytical chemistry, and healthcare diagnostics, addressing the cost, stability, and reusability shortcomings of natural enzymes.

This work introduces a de novo, single–amino acid (phenylalanine)–Cu2+ bionanozyme that self-assembles into hierarchical 2D layered crystals/nanosheets and mimics laccase with markedly superior catalytic efficiency, robustness, reusability, and substrate universality. It enables rapid oxidation of phenolic pollutants and ultrasensitive colorimetric detection of catecholamine neurotransmitters, while being vastly more cost-effective than the natural enzyme. The simplicity and performance of this minimalist system suggest a possible link to prebiotic catalytic motifs and provide a rational design path for next-generation bionanozymes. Potential future directions include exploring other amino acid–metal combinations to tailor activity/selectivity, integrating the bionanozyme into membranes or devices for scalable water treatment, and developing multiplexed biosensing platforms.