# Designing an ecofriendly catalyst for a sustainable use of water resources

Dehui Qiu, Xiaobo Zhang, Fan Tian, Yuan Liu, Fangni He, Xinrong Yan, Shijiong Wei, Jean-Louis Mergny, David Monchaud, Shujuan Zhang, et al.

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Dehui Qiu, Xiaobo Zhang, Fan Tian, Yuan Liu, Fangni He, et al. Designing an ecofriendly catalyst for a sustainable use of water resources. National Science Review, 2025, 13 (2), pp.nwaf447. 10.1093/nsr/nwaf447. hal-05492278

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# Designing an ecofriendly catalyst for a sustainable use of water resources

Dehui Qiu<fnref n="1" />,†, Xiaobo Zhang<fnref n="1" />,†, Fan Tian²,†, Yuan Liu<fnref n="1" />, Fangni He<fnref n="1" />, Xinrong Yan<fnref n="1" />, Shijiong Wei<fnref n="1" />, Jean-Louis Mergny<fnref n="1" />,<fnref n="3" />, David Monchaud ID<fnref n="4" />, Shujuan Zhang ID⁵, Huangxian Ju<fnref n="1" /> and Jun Zhou<fnref n="1" />,*

## ABSTRACT

The printing and dyeing industry is one of the most polluting (~20% of global clean water pollution), water-consuming and energy-wasting sectors in the manufacturing field, highlighting the need to find green catalysts to improve its sustainability. Herein, a novel artificial green catalyst was developed, known as a bifunctional chimeric peptide DNAzyme (bi-CPDzyme), comprising peptide, DNA and hemin moieties. This catalyst displays both catalase (CAT) and peroxidase (POD) activities. The turnover number ($k_{cat}$) of the optimized bi-CPDzyme prototype (G-quadruplex-Hemin-HRRHKHRRH) surpasses the natural CAT/POD bifunctional enzyme KatG, and competes with individual CAT and POD enzymes. This remarkable performance is attributed to the strategic combination and incorporation of histidine (H) and arginine (R) residues, which effectively trap hydrogen peroxide ($H_2O_2$) near the catalytic center via hydrogen bond formation, thus facilitating the generation of the active intermediate compound I, as supported here by theoretical calculations. Significantly, bi-CPDzyme achieves efficient decomposition of bleaching-derived residual $H_2O_2$ in a water-/energy-saving manner, while degrading dyes from textile industry effluents even in complex real samples, in addition to being easily recyclable and implementable. These findings make bi-CPDzyme a cutting-edge and environmentally friendly catalyst, positioning it at the forefront of advancements towards creating a sustainable society.

**Keywords:** artificial enzyme, chimeric peptide DNAzyme, catalytic mechanism, saving water, wastewater treatment

## INTRODUCTION

The printing and dyeing industry is an economic sector with many ramifications in our daily lives. However, the related industrial processes are complex (multistep), expensive (being time-, water-, energy- and material-consuming, notably because of the boiling, bleaching, dyeing, printing and post-treatment steps) and not ecofriendly [1,2]. As an example, hydrogen peroxide ($H_2O_2$) used for the bleaching process that is performed at high temperatures and under strong alkaline conditions, must be completely decomposed before the dyeing step, which necessitates multiple harsh and energy-intensive washing procedures. Subsequently, the treatment of wastewater generated during the dyeing step also requires high water

consumption to remove contaminants before being released into watercourses [3–5].

A method to eliminate $H_2O_2$ relies on the use of the catalase (CAT) enzyme [6,7], the disadvantage of which being its sensitivity to high temperature and pH [8]. In addition, the dyes used for staining, along with dye intermediates, acids, bases, by-products and several organics used all along the process, are pollutants [9,10] that necessitate efficient coping strategies like physical, chemical and biological treatments [11]: some of them are efficient but also have a cost from both financial and environmental points of view [12]. In the search for more ecofriendly catalysts, there is a growing need for greener alternatives [13]. One promising candidate is the peroxidase

<footnote n="1">¹State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, China; ²Key Laboratory of Green Chemical Process of Ministry of Education, School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, China;</footnote>

<footnote n="3">³Laboratoire d'Optique et Biosciences (LOB), Ecole Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, Palaiseau 91120, France;</footnote>

<footnote n="4">⁴Institut de Chimie Moléculaire de l'Université de Bourgogne (ICMUB), CNRS UMR6302, Université Bourgogne Europe (UBE), Dijon 21078, France and ⁵State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China</footnote>

<footnote>*Corresponding author. E-mail: jun.zhou@nju.edu.cn</footnote>

<footnote>†Equally contributed to this work.</footnote>

<footnote>Received 2 July 2025; Revised 29 August 2025; Accepted 15 October 2025</footnote>
(POD) enzyme extracted from multiple species, which has been employed for the remediation of commercial dyes [14–16]. However, both CAT- and POD-based systems are vulnerable to extreme pH and temperatures, and unsuitable for use in organic solvents [17]. Therefore, massive efforts are being invested to develop new materials such as Fenton reactive nanomaterials [18,19] and enzyme-MOFs [20] for instance, aimed at being used as substitutes for natural enzymes; however, these synthetic systems are not devoid of issues, including low recyclability rates and possible generation of hazardous waste [21,22].

Two parameters must be fine-tuned and combined to pave the way for next-generation green catalysts: first, the enzymatic system must be usable in harsh experimental conditions found in the printing and dyeing industry (extreme pH and temperatures, practically convenient solvents); second, the CAT and POD activities must be combined to optimize the processing of high concentrations of H₂O₂ and wastewater treatment. Such bifunctional enzymes exist in nature, e.g. the catalase-peroxidase KatG [23,24], but they suffer from an intrinsic fragility limitation along with a rather low POD activity despite a high CAT activity [25]. More practically convenient, so-called the artificial enzymes have been reported over the past years including engineered metalloenzymes, de novo designed enzymes [26–28], G-quadruplex (G4)-based DNAzymes [29], along with several nanozymes (Prussian blue, Mn@Bi₂Se₃ among others) [30–32]. These systems usually exhibit good POD activity but relatively poor CAT activity. Recently, we proposed the concept of chimeric peptide-DNAzyme (CPDzyme): this system combines the advantages of both natural enzymes (high catalytic activity) and DNAzymes (high chemical robustness) [33]. Our first CPDzyme prototype was found to be both efficient, with a POD activity surpassing (kcat) that of the parent horseradish peroxidase (HRP), and robust, being usable in a wide range of non-physiological environments. However, the CAT activity of CPDzymes remains to be established and/or optimized.

In this context, we report here on the design and synthesis of the first CAT/POD bifunctional CPDzyme (bi-CPDzyme) system along with its use for tackling the environmental issues associated with the printing and dyeing industry. To this end, we took the CPDzyme concept a step further: we assembled a G4 DNA and a short active peptide around a hemin (chloropropoporphyrin IX iron(III)) core (Fig. 1a), thereby benefitting from its two chemically addressable carboxylic arms which are easily converted into two amide bonds

(red circles, Fig. 1b). The resulting bi-CPDzyme was found to exceed the activity of KatG, which results from the cumulative effect of multiple histidine-arginine (His-Arg) repetitions inside the active site of the artificial enzyme, as compared to a single repetition for the natural enzyme (Fig. 1c). More importantly, this system is catalytically competent under non-physiological conditions and can be recycled quite efficiently. Its potential relevance for strategic applications is demonstrated herein for both one-step bleaching (in which bi-CPDzyme decomposes H₂O₂ without the need for multiple washings at high temperatures or pH adjustment, thus reducing water and energy usage) and wastewater disposal (degrading dyes in complex solvent mixtures) (Fig. 1d). As further detailed hereafter, bi-CPDzyme satisfies the key performance criteria expected of a promising green catalyst, as this artificial enzymatic system is highly efficient, made of non-toxic and harmless raw materials on the basis of environment-friendly processes.

# RESULTS AND DISCUSSION

## Synthesis and characterization of bi-CPDzymes

Our artificial enzyme systems, bi-CPDzymes, were constructed (see details in the Methods section) and optimized for peptide linking (described further below). Briefly, the central hemin core (NHS-hemin-NHS) was connected to the G4 unit on one side (biotin-T₉/iPCLink/TG₃TG₃TG₃TG₃-NH₂, itself linked to streptavidin (STV)-coated magnetic beads (MBs), with iPCLink: internal photocleavage site), and to the peptide arms on the other side (Table S1). The iPCLink was used to free the complex from the MBs (UV irradiation) and G4 folding was ensured by the presence of potassium cations (Fig. S1). The structural integrity of bi-CPDzyme (Figs S1–S4) was demonstrated by UV and fluorescence measurement, to testify to the presence of both G4 and hemin in the bi-CPDzyme structure, along with electrospray ionization (ESI-MS) and inductively coupled plasma (ICP-MS) mass spectrometry, to characterize the whole assembly through mass/charge ratio and metallic content.

## Lessons from natural enzymes to design bi-CPDzymes

Both peroxidases HRP (PDB ID: 1W4Y, Fig. 2a) and cytochrome c peroxidase (CcP, PDB ID: 4CVJ, Fig. S5a) share a remarkably conserved histidine
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Bi-CPDzyme assembly and applications. (a) Bi-covalent assembly schematic (details in Fig. S1). (b) Hemin catalytic center with dual amide bonds (red ovals) linking DNA/peptides (aa: amino acid). (c) Schematic illustrations of the G4-Hemin-peptide, with G4 (gray), hemin (green) and active peptide (blue) highlighted, mimicking peroxidase (POD) and catalase (CAT). (d) Flowchart of the application of bi-CPDzyme in the dyeing and printing process using its CAT and POD activities.</figcaption></figure>

(His42 and His52 for HRP and CcP, respectively) at the core of their active site. This histidine, along with arginine (Arg38 and Arg48 for HRP and CcP, respectively) plays a crucial role in the formation

and stabilization of an intermediate compound referred to as compound I [34,35] during the catalytic reaction. In addition, an asparagine (Asn70 or Asn82 for HRP and CcP, respectively) facili-
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Bi-CPDzyme design and activity analysis. (a–f) Key heme-binding residues in HRP (a) and CAT (d) and their effects on POD (b, e) and CAT (c, f) activities in bi-CPDzyme. (g, h) Performance of engineered peptides (vs G4-Hemin control). (i, j) Top-view structures of G4-Hemin-KHRRH (i) and -KHRRHRH (j) systems (red box: hemin occupancy; blue oval: tertiary H + R).</figcaption></figure>
tates the process by forming hydrogen bonds with this His to increase its basicity [36,37]. This amino acid network is reminiscent of both the active site of KatG and the cofactor binding region of CcP [38]. Also, each subunit of tetrameric CAT (PDB ID: 1DGG, Fig. 2d) contains its own heme and a combination of His75 + Asn148 as well as a serine (Ser114) that plays the same role as Asn in PODs (Fig. S5a) [39,40].

We exploited these amino acids' roles in the bi-CPDzyme prototypes (Fig. S5b): the effect of single/multiple amino acid(s) on the POD/CAT bifunctional activity was evaluated by the model oxidation reaction between 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and H₂O₂ for the POD activity, and monitoring the concentration of produced O₂ for the CAT activity. Among the core HRP amino acids, Phe was discarded as it was shown not to affect CPDzyme activity [33]. The effects of three amino acids His, Arg and Asn (using a Lys (K) as a linker) on CPDzyme activity are summarized in Fig. 2b and c. Briefly, (i) for a single amino acid addition, G4-Hemin-KH showed the highest bifunctional activities, with an initial velocity of ABTS oxidation (POD-like activity) V₀-POD = 258 nM/s and an initial O₂ production (CAT-like activity) V₀-CAT = 5.8 µM/s. This result suggests that His plays an essential catalytic role [41,42]; (ii) for two amino acids, the best combination was His + Arg (H + R), which was found to increase both POD- and CAT-like activities, with V₀-POD = 432 nM/s and V₀-CAT = 11.3 µM/s, respectively. This enhancement may originate in the basicity of the Arg residue (pKa = 12.5), which could activate H₂O₂ in coordination with His and accelerate the formation of compound I [43,44], thus making the H + R combination central to the catalysis; (iii) for three amino acids, no significant improvements were noticed using His, Asn and Ser combinations (Fig. 2e, f). As this first series of results demonstrated that the H + R combination provides the strongest activation, we decided to increase the number of H + R combinations (Fig. 2g, h): (i) both POD- and CAT-like activities benefitted from the extension of H + R tails with V₀-POD = 982 nM/s and V₀-CAT = 23.2 µM/s for G4-Hemin-KHRRH, thereby being 2.3- and 1.9-fold higher than a single H + R combination (G4-Hemin-KHR); (ii) the H + R tail was further extended to G4-Hemin-KHRRHRH and -KHRHRHR, but without any increase in efficiency, likely due to the steric hindrance within the active site of CPDzyme. This is illustrated in Fig. 2i and j, where the red square represents the space

occupancy of hemin and the dark blue oval the third H + R repeat within peptide-KHRRHRH, which is outside of the active site.

## Design of optimized bi-CPDzymes

To further benefit from the available space above the hemin binding site, the position of the Lys residue was shifted (towards the N- or C-terminal, or in the middle of the oligopeptide) and the consequence on the catalytic activity of the resulting bi-CPDzyme was monitored (Fig. 3a-c and Fig. S6): (i) no position-dependent effect was assessed within the HRRH tail (neither in the L- nor D-series); (ii) in the (H + R)₂ tail, this effect was also weak, with V₀-POD between 982 and 1006 nM/s and V₀-CAT between 21.9 and 23.2 µM/s; (iii) in the (H + R)₃ tail, a noticeable effect was obtained with HRRHKHR (or RHKHRRH) when the Lys was located at intermediate positions, with V₀-POD up to 1331 nM/s and V₀-CAT up to 38.9 µM/s, which represents a 3.1- and 3.3-fold improvement as compared to a bi-CPDzyme displaying a single H + R (V₀-POD = 432 nM/s and V₀-CAT = 11.9 µM/s); (iv) in the (H + R)₄ tail, a similar improvement was obtained with HRRHKHRRH, with (V)₀-POD = 1640 nM/s and V₀-CAT = 49.3 µM/s, which represents a 3.8- and 4.1-fold improvement as compared to a bi-CPDzyme displaying a single H + R; (v) finally, no positive effects were seen when further extending the H + R tail to five units. This second series of results provides interesting insights into the available space within the active site and how the H + R tail length can be fine-tuned to maximize the catalytic activity of the resulting bi-CPDzymes.

## Proposed mechanism of bi-CPDzyme catalysis

A schematic representation of the bifunctional POD/CAT catalytic cycle can be seen in Fig. 3d. The generation of Por⁺-Fe⁴⁺=O (compound I, Cpd I) [45], an important active intermediate in the POD/CAT catalytic cycle, is shown schematically in Fig. 3e. His-N and Arg-NH form intermolecular hydrogen bonds with H₂O₂ and then the H + R coordinates H₂O₂ delivery to the Por-Fe¹¹¹ center to form compound 0 (Cpd 0). Subsequent O-O bond cleavage generates Cpd 0* and the free OH⁻ forms a hydrogen bond with Arg-NH. Through the mediation of a single H₂O molecule, the proton is transferred from Fe-O-H to His-N to regenerate Cpd I. The EPR spectrum
<figure><img src="image_4.png" /><figcaption>**Figure 3.** H + R pair evolution in bi-CPDzyme and catalytic mechanism. (a) Top-view structural progression from single to quadruple H + R pairs. (red box: four pairs maximize hemin's space occupancy). (b, c) Activity assays: POD (b) and CAT (c) performance. (d) Catalytic mechanism of bi-CPDzyme (CAT: light blue; POD: orange). (e) Intermediate compound I formation (Fe(III)/Fe(IV) transition: green/red spheres).</figcaption></figure>
of Cpd I harbors an asymmetrical signal with a g-value of 1.997 at 77 K (Fig. S7), which is similar to that of HRP (g = 1.995, 4.2 K) [46]. Meanwhile, OH⁻ attracts a proton from Arg to form stable H₂O, which is then exchanged with the protonated His to regenerate the initial Arg-NH/His system. In the absence of a substrate, Cpd I reacts with a second molecule of H₂O₂ to produce O₂ and H₂O, the bi-CPDzyme thus exhibiting a CAT activity [41,47]. In the presence of a substrate (AH), Cpd I is reduced to Por-FeIV=OH (Cpd II) and produces the radical A'; a second AH molecule reduces Cpd II to the native bi-CPDzyme FeIII and produces a second radical A', the bi-CPDzyme thus exhibiting a POD activity. This mechanism highlights the positive and coordinated contributions of the H + R pairs, found to improve both the speed and efficiency of the catalysis. The proposed mechanism here was supported by the cumulative effect of H + R pairs: the catalytic activity of the bi-CPDzyme system does indeed positively correlate with the number of H + R pairs (for (H + R)≤4, Fig. 3b, c). This observation indicates that the H + R pairs help capture H₂O₂ and accelerate the formation of Cpd I, which are the two important steps in the POD/CAT-catalyzed reaction. With (H + R)≥4, such a cumulative effect is lost, presumably due to the introduction of steric hindrance within the active site (see Fig. 2j).

## The roles of H + R pair in catalysis

Density functional theory (DFT) calculations were performed to map the formation of Cpd 0 and Cpd I. In the 'simple' G4-Hemin system, H₂O₂ first binds to Por-FeIII to give Cpd 0; subsequent O-O cleavage furnishes Cpd 0*, whose proton transfer to Cpd I plus two H₂O molecules proceeds via TS1 (ΔG‡ = 0.80 kcal/mol, red, Fig. 4a, Fig. S8a and Movie S1). Despite this low barrier, kinetic trapping of H₂O₂ is hindered because surrounding bulk water can hydrogen-bond with the peroxide and delocalize it away from the iron center. In the G4-Hemin-KHR system, this issue is alleviated as His-N and Arg-NH together with the iron center pre-organize H₂O₂ and its fragments. Their concerted action lowers kinetic dispersion: cleavage proceeds through TS1 (ΔG‡ = 6.39 kcal/mol, blue, Fig. 4a and Fig. S8b) that simultaneously involves Fe and Arg-NH, after which the nascent OH⁻ is captured by Arg-NH and protonated to yield H₂O. Accordingly, G4-Hemin relies on a single TS1 in which a water molecule shuttles one proton. In G4-Hemin-KHR the process is split: TS2 (ΔG‡ = 1.11 kcal/mol) moves a proton from Fe-OH to His-N via water, and TS3 (ΔG‡ = 1.76 kcal/mol) relays the proton

from His-NH⁺ to Arg-N, again through a bridging water, to complete the cycle (Movie S2). Although the overall barrier for Cpd I formation is higher in G4-Hemin-KHR than in G4-Hemin (6.39 vs 0.8 kcal/mol), the amino acids restrict H₂O₂ and its fragments to the iron center, kinetically promoting the Por-FeIII/H₂O₂ encounter.

Next, we calculated the fate of Cpd 0* in both G4-Hemin and G4-Hemin-KHR systems with ab initio molecular dynamics (AIMD). The monitored O-H distance of Fe-OH in the G4-Hemin system fluctuates around 1 Å (black line, Fig. 4b), indicating that the proton is hardly removed to generate Cpd I. The cleaved OH⁻ drifts among bulk water molecules and away from Fe (Fig. S9, Movie S3), underscoring the sluggish Cpd 0* → Cpd I step. In G4-Hemin-KHR, this O-H distance rises and fluctuates around 2 Å (after 281 fs) while the N-H distance of His-N decreases to 1 Å (red and blue lines, Fig. 4b). The derived pair distribution functions (PDFs) for His-N show a distinct peak centered at 1 Å (solid lines, Fig. 4c) with an integral of 1 each (dashed lines, Fig. 4c), confirming N-H formation. The Fe-O PDF, however, has only a weak 1 Å feature integrating to ~0, evidencing successful Cpd I generation. In G4-Hemin, the Fe-O PDF shows a single 1 Å peak integrating to 1, confirming that no OH⁻ remains near the iron and the proton is never abstracted. Taken together, AIMD snapshots (Fig. S10) reveal that only in G4-Hemin-KHR can the proton shuttle smoothly from Fe-O to His-N. Moreover, the free OH⁻ exhibits an oxygen coordination number of 2, while Arg-N1 shows 1, indicating a hydrogen bond between OH⁻ and Arg-N1···H (Fig. S11). These results corroborate the DFT pathway for Cpd 0* → Cpd I in G4-Hemin-KHR (Fig. S8b, Movie S4). The results of these calculations reveal the catalytic importance of the H + R pair: the hydrogen-bonds form between guanidinium group of Arg and H₂O₂, which can stabilize Cpd 0 (Por-FeIII–H₂O₂, TS1) to facilitate O-O bond cleavage. The guanidinium group subsequently captures the nascent OH⁻ via hydrogen bonding, spatially constraining this proton-accepting species near the hemin center. Through a bridging water molecule, His mediates proton abstraction from Fe-OH, driving Cpd I (Por⁺-FeIV=O) formation. Then, the confined OH⁻ extracts a proton from the guanidinium group to form H₂O, which ultimately participates in proton transfer with the protonated His, regenerating the catalytically competent H + R pair.

We then further characterized the advantages of multiple H + R pairs in bi-CPDzyme by calculating the electrostatic potential (ESP): this re-
<figure><img src="image_5.png" /><figcaption>**Figure 4.** The proposed catalytic processes mediated by amino acids. (a) Energetic diagram of possible reaction intermediates and transition states (TS) during the generation of compound I by G4-Hemin and G4-Hemin-KHR. (b) Evolutions of the distance between the Fe–O/His-N and the H and (c) the pair distribution functions of O/N and H atoms. (d) Binding energies of G4-Hemin and bi-CPDzymes with one, two, four and eight H₂O₂, and K_m values (POD/CAT) from Table 1. (e) Adsorption energy (Eads) of H₂O₂ to binding sites in the cavity of bi-CPDzyme.</figcaption></figure>
<table><caption>**Table 1.** Kinetic parameters of POD and CAT reactions catalyzed by bi-CPDzyme.</caption><thead><tr><th></th><th></th><th>K<sub>m</sub><sup>a</sup> (mM)</th><th>V<sub>max</sub><sup>b</sup> (μM·s<sup>-1</sup>)</th><th>k<sub>cat</sub><sup>c</sup> (s<sup>-1</sup>)</th><th>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup>·mM<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="2">G4-Hemin</td><td>POD</td><td>147 ± 26</td><td>0.167 ± 0.018</td><td>33.4 ± 3.6</td><td>0.20 ± 0.05</td></tr><tr><td>CAT</td><td>118 ± 5</td><td>4.81 ± 0.09</td><td>482 ± 9</td><td>4.1 ± 0.26</td></tr><tr><td rowspan="2">G4-Hemin-KHR</td><td>POD</td><td>170 ± 20</td><td>2.149 ± 0.153</td><td>430 ± 31</td><td>2.53 ± 0.54</td></tr><tr><td>CAT</td><td>172 ± 27</td><td>18.2 ± 1.27</td><td>1820 ± 127</td><td>10.6 ± 2.85</td></tr><tr><td rowspan="2">G4-Hemin-KHRRH</td><td>POD</td><td>121 ± 18</td><td>4.191 ± 0.355</td><td>838 ± 71</td><td>6.92 ± 1.90</td></tr><tr><td>CAT</td><td>149 ± 15</td><td>41.03 ± 1.71</td><td>4103 ± 171</td><td>27.5 ± 4.36</td></tr><tr><td rowspan="2">G4-Hemin-RHKHR</td><td>POD</td><td>120 ± 17</td><td>4.003 ± 0.318</td><td>801 ± 64</td><td>6.68 ± 1.72</td></tr><tr><td>CAT</td><td>153 ± 17</td><td>36.67 ± 1.81</td><td>3667 ± 181</td><td>24.0 ± 4.33</td></tr><tr><td rowspan="2">G4-Hemin-HRRHKHRRH</td><td>POD</td><td>101 ± 11</td><td>6.226 ± 0.376</td><td>1245 ± 75</td><td>12.3 ± 2.37</td></tr><tr><td>CAT</td><td>104 ± 5</td><td>63.41 ± 1.15</td><td>6341 ± 115</td><td>61.0 ± 4.24</td></tr></tbody></table>

revealed that the positive charge increased with the number of H + R pairs, which is favorable to electrostatically attract the O atom of H₂O₂ (Fig. S12). Furthermore, the binding energies (docking) of bi-CPDzymes to one, two, four and eight H₂O₂ molecules progressively decrease as the number of H + R pairs increase from 0 to 4 (Figs S13 and S14), implying an increase in affinity of bi-CPDzyme for H₂O₂, as supported by the Kₘ value measured for the POD/CAT activity (Fig. 4d). Finally, the adsorption energies (Eads) of H₂O₂ to the binding sites of bi-CPDzyme were evaluated by DFT and we found that one, two and four H + R pairs could adsorb one, two and four H₂O₂ molecules with Eads of -23.39, -33.62 and -48.16 kcal/mol, respectively (Fig. 4e). Therefore, the G4-Hemin-HRRHKHRRH system seems to be optimized for binding to multiple H₂O₂ molecules, thus increasing the probability of the enzyme binding to H₂O₂ and the reaction between hemin and H₂O₂.

## Kinetic analysis of bi-CPDzymes catalysis

To gain more accurate insights into the catalytic performance of bi-CPDzymes, their steady-state kinetics were analyzed (Table 1 and Figs S15-S17). The POD and CAT turnover numbers (kcat) of G4-Hemin were used as references, with kcat = 33.4 s⁻¹ and 482 s⁻¹, respectively. The kcat of G4-Hemin-KHR reached 430 s⁻¹ (POD) and 1820 s⁻¹ (CAT). Significant improvements were observed with two H + R pairs (G4-Hemin-KHRRH and G4-Hemin-RHKHR showed roughly the same kinetic parameters) while G4-Hemin-HRRHKHRRH provided the best results, with kcat = 1245 s⁻¹ (POD), which is ~2.4-fold of the natural enzyme HRP (kcat = 521 s⁻¹) [33], and kcat = 6341 s⁻¹ (CAT), which is ~0.77-fold

of natural CAT (the kcat of the natural CAT is 32.891 s⁻¹, and reported kcat in the literature ranges from 9628 to 74 800 s⁻¹ [48,49], but it should be noted that natural CAT is a tetramer, the kcat value for a single active center is thus 8223 s⁻¹) (Fig. S18). Interestingly, G4-Hemin-HRRHKHRRH outcompetes the bifunctional KatG enzyme (with kcat = 7.7-25 s⁻¹ (POD) and 2950-7770 s⁻¹ (CAT)) [25], thus being 50- to 162-fold (POD) and 0.8- to 2.1-fold more active (CAT) than KatG, and also offers significant advantages over other reported bifunctional artificial enzymes (Table S2). The Michaelis constant (Kₘ) of bi-CPDzymes decreases with the increase in the number of H + R pairs (from 170 to 101 mM (POD) and 172 to 104 mM (CAT)), which corresponds to an increase kcat/Kₘ value, from 2.53 to 12.3 s⁻¹ mM⁻¹ (POD) and 10.6 to 61.0 s⁻¹ mM⁻¹ (CAT). The Kₘ value being higher than that of natural enzymes, in which the unique substrate channel actively participates in catalysis. Of note, the high H₂O₂ concentrations used in routine experimental setups make the kcat value more important than Kₘ; therefore, the excellent kcat of bi-CPDzymes make them very promising for practical applications.

## Chemical stability of bi-CPDzymes

The printing industry makes most frequent use of redox reactions under conditions not suited to the use of natural enzymes (high temperatures, strongly acidic and basic conditions, organic solvents, etc.). As CPDzymes were previously shown to be highly robust in harsh conditions [33], we decided to assess the activity of bi-CPDzymes under these conditions: the results seen in Figs S19 and S20 demonstrate the operability of G4-Hemin-HRRHKHRRH: (i) it

<footnote><sup>a</sup>[H₂O₂] mM; <sup>b</sup>POD kinetic parameters measured by 5 mM ABTS oxidation; <sup>c</sup>POD and CAT reactions catalyzed by 5 and 10 nM bi-CPDzyme.</footnote>
works within a wide pH range of 2 to 12; for in-
stance, neutral and weakly acidic conditions fa-
vor POD-like activity, and CAT-like activity ex-
hibits higher tolerance between pH 4 and 10
(Fig. S19a). (ii) It shows the robustness in so-
lutions containing 50% (v/v) of various organic
solvents, retaining 25%-57% of its POD-like ac-
tivity and 58%-92% of its CAT-like activity rel-
ative to buffer solutions (Fig. S19b). (iii) It dis-
plays excellent activity under thermal stress: af-
ter a 1-h treatment at 95°C, the POD-like and
CAT-like activities are 86% and 57% maintained,
respectively (Fig. S19c). (iv) It preserves >95%
of its initial activity after 21 days of storage at
room temperature (Fig. S20a). (v) It exhibits high
reusability, evidenced by maintaining >90% activ-
ity after 10 catalytic cycles when immobilized on
MBs (Fig. S20b). Collectively, these outstanding
performances highlight the usability potential of
bi-CPDzymes under actual complex conditions.

Applications of bi-CPDzymes

The ultimate application of bi-CPDzymes lies
in their ability to decompose H₂O₂ and decon-
taminate dye wastewater originating from the
printing industry. As schematically represented
in Fig. 1d, the industrial boiling and bleaching
steps require the use of both an excess of H₂O₂ at
high temperatures and strong alkaline conditions.
The CAT-like activity of bi-CPDzyme is intended
to decompose H₂O₂ into H₂O and O₂ while its
POD-like activity is intended to degrade the
contaminants found in dye wastewater.

We first implemented the bleaching post-
treatment process seen in Fig. 5, Figs S21 and S22:
to this end, a piece of textile was bleached by treat-
ment with H₂O₂ auxiliaries, the excess H₂O₂ was
removed by either washing steps or enzyme/bi-
CPDzyme treatment, and the residual H₂O₂ was
quantified by H₂O₂ test strips. The conventional
high-temperature washing process involves the
addition of H₂O₂ at 25°C followed by a heating step
(30 min at 98°C), a drainage step (the pH measured
after the drainage is between 11 and 12; the con-
centration of H₂O₂ is 14.79 mg/L) and three wash-
ings, one with cold water (25°C for 5 min), and
two with hot water (98°C for 10 min) (Fig. 5a). At
the end of this tedious process, the final concentra-
tion of H₂O₂ was 0.35 mg/L, and the decomposi-
tion rate was 97.6%. The enzymatic washing pro-
cess using CAT was adapted from the previous ap-
proach; however, as CAT is inactivated under al-
kaline conditions, glacial acetic acid was added to
adjust the pH of the solution to pH 7 during the
process (Fig. 5b), which led to an H₂O₂ decom-
position rate of 99.7%. When using bi-CPDzyme

G4-Hemin-KHRRHKHRRH at the same concen-
tration as CAT (for 30 min at pH 11–12) directly
without washings, the H₂O₂ decomposition rate
was 98.5% (Fig. 5c). The results, summarized in
Fig. 5d, indicate that using bi-CPDzyme allows
for removing the steps of hot/cold water wash-
ings and pH adjustment, while being equally ef-
ficient. Therefore, the one-step treatment with
bi-CPDzyme improves the bleaching process in a
time-, water- and energy-saving manner.

Next, we focused on wastewater decontami-
nation: the ability of G4-Hemin-KHRRHKHRRH
to degrade Basic blue 9 (BB9), a popular dye
used in industry (textiles, rubber, plastics, cos-
metics, etc.), was chosen as a model reaction. Of
note, as we aimed at working in different wa-
ter/organic mixtures, we also selected different
oxidants, soluble in the experimental conditions
(Fig. 6a). The performance of bi-CPDzyme was di-
rectly compared to that of HRP: as seen in Fig. 6b
and Fig. S23, when using H₂O₂ at pH 7, the
catalytic rate of HRP was 4.1 nM/min and that
of bi-CPDzyme 187.4 nM/min, thus being ~46-
fold higher than HRP. The catalytic rate of bi-
CPDzyme reached 141.5 nM/min in 50% ethanol
(v/v) using cumyl hydroperoxide (CHP) as an ox-
idant, 103.4 nM/min in 50% acetone (v/v) using
tert-butyl hydroperoxide (TBHP) as an oxidant,
and 108.8 nM/min in 50% acetonitrile (v/v) us-
ing H₂O₂ as an oxidant, all these conditions being
unsuited to the use of HRP.

We then compared the degradation rate of
BB9 by bi-CPDzyme with two artificial catalysts,
i.e. Fe₃O₄ nanoparticles (NPs) and G4TC/Hemin
DNAzyme: as seen in Fig. 6c, Figs S23 and S24,
under the same molar concentration of catalysts,
the degradation rate of Fe₃O₄ NPs was 1.4%, that
of G4TC/Hemin 12.9%, far behind that of bi-
CPDzyme (54.9%) in 30 min, demonstrating the
superiority of bi-CPDzyme among these artifi-
cial catalysts. We also investigated the byproducts
generated during BB9 degradation in order to as-
sess whether some of them could be harmful. As
shown in Fig. S25a, the BB9 solutions changed
from blue to colorless when they were treated by
bi-CPDzyme with 0.3 to 300 mM H₂O₂ concen-
tration for 12 h, indicating the total disappearance
of the chromogenic groups, which was confirmed
by both UV measurements (Fig. S25b) and high-
performance liquid chromatography (HPLC) (Fig.
S26). Then, the degradation products were iden-
tified by liquid chromatography-mass spectrome-
try (LC-MS): several peaks were seen correspond-
ing to several degradation intermediates (details
in Fig. S27), with the terminal degradation prod-
ucts found to be NO₃⁻ and SO₄²⁻, as detected
by ion chromatography (IC) (Fig. S28). We thus
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Water-saving application of bi-CPDzyme. (a) The conventional washing and (b) natural catalase bleaching process. (c) Direct bleaching process curve of bi-CPDzyme. (d) Essential steps and H₂O₂ decomposition rates (%) for three bleaching processes.</figcaption></figure>

showed that the degradation products of BB9 by bi-CPDzyme (Fig. 6d) include nontoxic inorganic ions, carbon dioxide and water. To go a step further, we assessed the toxicity of BB9

before and after degradation: after 1 h of treatment by bi-CPDzyme, the BB9 sample changed from blue to colorless, the absorption peak at 665 nm decreased from 0.85 to 0.002 (Fig. 6e),
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Comprehensive dye wastewater treatment by bi-CPDzyme. (a) BB9 degradation schematic with oxidants/organic solvents. (b) Comparative degradation (1 mM oxidants) in solvents (HRP vs bi-CPDzyme). (c) UV absorption kinetics (1 mM H₂O₂) with artificial catalysts. (d) Possible degradation pathway of BB9 catalyzed by bi-CPDzyme. (e) Visual/spectral changes after 1 h treatment. (f) COD removal efficiency over time. (g) HEK293T cell viability assessment.</figcaption></figure>
and the corresponding chemical oxygen demand (COD) value decreased from 200–250 mg/L to <30 mg/L, indicating the reduction of water pollution (Fig. 6f). In addition, the HEK293T cell viability was measured to evaluate the cytotoxicity of the degradation products. The cell viability increased from 47% (control) to 66%, 83% and 91% after 0.5, 1 and 12 h of degradation, respectively (Fig. 6g). Moreover, the cell viabilities range from 62% to 73% in six degradation intermediates (Fig. S29), suggesting that toxicity was reduced after treatment with bi-CPDzyme.

Finally, we investigated the functions of bi-CPDzyme in real-life examples, that is, in textile dyeing wastewater samples: two samples were taken in a textile dyeing wastewater treatment plant—one from the primary clarifier and one from a sequencing batch reactor (SBR) (Fig. S30a, b). The maximum absorption peak at 228 nm decreased for both samples after treatment with bi-CPDzymes, indicating that pollutants with conjugate absorption were degraded (Figs S30c and S31). In addition, the COD values of the two samples decreased to 60 and 30 mg/L (76% and 75% reduction, respectively) after treatment with bi-CPDzyme, and the amount of residual H₂O₂ was ~2 mg/L, indicating that the decomposition rate of H₂O₂ was >92% (Fig. S32a), and increased cell viability by 28.5% and 25% (Fig. S32b). Moreover, bi-CPDzyme retained 87% CAT activity and 88% POD activity after 7 consecutive catalytic cycles (Fig. S32c). These results thus demonstrate the high efficiency, stability, and reusability of bi-CPDzyme in real-life conditions, being able to catalytically degrade dyes and H₂O₂ in a wide range of experimental conditions. Specifically, bi-CPDzyme exhibits outstanding cost-effectiveness (21.8% of commercial enzymes, Tables S3–S5), maintains >87% activity after 7 operational cycles, and reduces wastewater toxicity by 25%–28.5%. Its exceptional environmental tolerance and reusability further support its potential for industrial-scale applications, pending future techno-economic analyses (TEAs) and pilot-scale validation.

## CONCLUSION

We report here on a bifunctional chimeric peptide DNAzyme (bi-CPDzyme) system synthesized in a simple one-step process from non-toxic and non-hazardous peptides, nucleic acids and hemin, which displays both catalase (CAT) and peroxidase (POD) activities. The catalytic performance of the optimized bi-CPDzyme (G4-Hemin-KHRRHKHRRH) prototype is superior to the cor-

responding natural bifunctional enzyme KatG and competes with individual CAT and HRP enzymes. Theoretical calculations reveal that this exceptional performance is due to the strategic incorporation of histidine (H) and arginine (R) residues in the binding sites of the catalytic system, which trap and maintain hydrogen peroxide (H₂O₂) near the catalytic center, thereby favoring the catalytic process. Importantly, the chemical robustness of bi-CPDzymes make them operatable in harsh experimental conditions (organic solvent mixtures, high temperatures, high/low pH, etc.), as demonstrated here with real-life samples, in which native enzymes cannot be used. We thus demonstrate the strategic interest of bi-CPDzymes, found to be efficient catalysts for treating printing wastewaters, decomposing both residual H₂O₂ and dyes still present in wastewaters, even after several hard washing steps. Compared with the traditional process, bi-CPDzyme not only shortens but also improves the workflow, which results in a decrease in the amount of water used. We are thus convinced that bi-CPDzymes represent a new class of artificial catalysts suited to a wide range of wastewater treatment processes, which can be furthermore easily recycled, thus decreasing both the cost *per se* and the environmental cost of the printing and dyeing industry. These artificial enzymes can undoubtedly be considered as next-generation green catalysts, fully in line with the objectives defined by the United Nations, as sustainable industrial development has been incorporated as Goal 9 in the 2030 Agenda for Sustainable Development (https://sdgs.un.org/goals/goal9).

## SUPPLEMENTARY DATA

Supplementary data are available at NSR online.

## FUNDING

This work was supported by the National Natural Science Foundation of China (22374070 and 22177047), the State Key Laboratory of Analytical Chemistry for Life Science (5431ZZXM2202, SKLACLS2109 and SKLACLS2307) and the Fundamental Research Funds for the Central Universities (020514380299).

## AUTHOR CONTRIBUTIONS

All authors contributed to this work. D.Q., X.Z. and J.Z. conceived and designed the experiments and wrote the original version of the manuscript. F.T. and Y.L. performed the theoretical calculations. F.H., S.W. and Y.X. performed some experiments. D.Q. drafted the figures. D.M., J.L.M. and H.J. provided extensive suggestions for revisions of the original manuscript.

*Conflict of interest statement.* None declared.
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