Soil enzymes have an extremely important role in organic matter formation and decomposition, stabilization of soil structure and nutrient cycling and transformation. Soil enzymes are also involved in the remediation of contaminated soils. Soil enzyme activities can serve as indicators of microbial growth, activity and soil ecosystem health. Many soil enzymes can be powerful biological indicators for assessing heavy metal toxicity. Large amounts of enzymes are present in soils, including amylases, arylsulfatases, glucosidases, cellulases, chitinases, dehydrogenases, phosphatases, proteases, ureases and peroxidases of plant, animal and microbial origin (Bakshi & Varma, Reference Bakshi, Varma, Shukla and Varma2011; Dotaniya et al., Reference Dotaniya, Aparna, Dotaniya, Singh, Regar and Kuddus2019; Cui et al., Reference Cui, Wang, Wang, Zhang and Fang2021; Fanin et al., Reference Fanin, Mooshammer, Sauvadet, Meng, Alvarez and Bernard2022).

Soil peroxidases represent important enzymes that use hydrogen peroxide (H₂O₂) as an electron acceptor. This group of enzymes catalyses oxidation reactions via the reduction of H₂O₂; they are considered to be used by soil microorganisms as lignolytic enzymes because they can degrade molecules that lack a precisely repeating structure. Lignin breakdown results in significant contributions to soil N and C pools and makes available nutrients to soil microbes (Sinsabaugh, Reference Sinsabaugh2010; Plante et al., Reference Plante, Stone, McGill and Paul2015; Hassan et al., Reference Hassan, Hamid, Auta, Pariatamby, Ossai, Barasarathi, Ahmed, Maddela, Abiodun and Prasad2022).

Recently, it was observed that soil magnetic iron oxide particles exhibiting peroxidase-like (P-L) activity can substantially influence peroxidase activity measured in soil suspensions (Safarik & Prochazkova, Reference Safarik and Prochazkova2022). It has to be taken into account that this activity can be caused both by enzymes (peroxidases) of microbial, plant and animal origin and by inorganic peroxidase-mimetic minerals. A new research area called ‘soil nanozymology’, devoted to the study of soil-related nanozymes and other enzyme-mimetic materials, has been defined (Safarik & Prochazkova, Reference Safarik and Prochazkova2022).

In further experiments, soil autoclaved twice at 121°C for 20 min and, after drying, heated in a hot air dryer (18 h, 200°C) to degrade natural peroxidases, with completely removed magnetic iron oxide particles, still exhibited P-L activity using N,N-diethyl-p-phenylenediamine sulfate salt (DPD) as the substrate. In fact, several soil constituents might be responsible for this behaviour, including antiferromagnetic hematite (Chaudhari et al., Reference Chaudhari, Chaudhari and Yu2012) or specific clays (Feng et al., Reference Feng, Wang, Zhang, An, Lin and Tong2021). To test the behaviour of three commercially available soil-related clays, namely bentonite, halloysite and montmorillonite (Mnt; all from Sigma-Aldrich, USA), their P-L activity was tested with DPD as the substrate. It was observed that Mnt exhibited strong P-L activity (see Fig. 1), whereas bentonite and halloysite exhibited no measurable P-L activity, although bentonite consists mainly of Mnt.

Figure 1. P-L activity of Mnt (Sigma-Aldrich). Upper figures – non-swollen Mnts: A = Mnt suspension without DPD; B = Mnt suspension with DPD + H₂O₂ before centrifugation; C = Mnt with DPD + H₂O₂ after centrifugation. Bottom figures – swollen Mnts; D = Mnt suspension without DPD; E = Mnt suspension with DPD + H₂O₂ before centrifugation; F = Mnt with DPD + H₂O₂ after centrifugation.

Mnt is a common clay component of soils belonging to the smectite group and representing a 2:1-type clay mineral with good cation-exchange capacity (CEC) and swelling properties. Mnt is also an efficient, low-cost adsorbent, especially for various cationic contaminants (Zhu et al., Reference Zhu, Chen, Zhou, Xi, Zhu and He2016). Typical Mnt-rich soils are Vertisols exhibiting deep black colour and seasonal severe cracking after drying and swelling upon being water-logged. Vertisols form both in tropical and subtropical regions with high precipitation and in subarid areas (McGarry, Reference McGarry, Ahmad and Mermut1996; Pal et al., Reference Pal, Wani and Sahrawat2012; Jordanova, Reference Jordanova and Jordanova2017). Soils containing high Mnt contents have been tested as additives to improve the quality of sandy soils and to increase crop yields (Jia et al., Reference Jia, Zhang, Yang and Zhang2018; Yu et al., Reference Yu, Tariq and Yang2022).

To characterize the P-L activity of Mnt in more detail, four different Mnt preparations were tested. Mnt K-10 powder was from Sigma-Aldrich (catalogue no. 69866; produced by the calcination of native Mnt). Dellite® HPS (Na-Mnt, CEC = 128 meq 100 g^–1) and Dellite® LVF (Na-Mnt, CEC = 105 meq 100 g^–1) were supplied by Laviosa Advanced Mineral Solutions (Italy), whereas Shrimp Nature Mnt powder (no detailed information available) was obtained from Shrimpworld.cz (Czech Republic).

The P-L activities of all four Mnt materials were studied using DPD as the substrate, as described previously (Safarik & Prochazkova, Reference Safarik and Prochazkova2022). The reaction mixture contained 3.4 mL of deionized water, 400 μL of DPD solution (12.53 mmol L⁻¹) and 200 μL of 2% H₂O₂. A total of 5–30 mg of Mnt materials was used for the assays performed at 20°C; the incubation time was 3 or 8 min, followed by centrifugation for 2 min. Corresponding blank samples containing all of the reagents except the Mnt materials were also prepared. Absorbance at a wavelength of 551 nm was measured. In addition to the P-L assay with natural (non-swelling) Mnt samples, Mnt samples swollen in 3.4 mL of deionized water for 1 week at 20°C before the assay were also analysed.

Figure 1 shows the peroxidase-mimetic activity of the most efficient Mnt sample (commercial preparation from Sigma-Aldrich); a red-coloured oxidized DPD product was formed during the assay. Due to the fact that Mnt is a well-known adsorbent, the red-coloured reaction product was bound to Mnt powder, causing its dark red colouration. A similar situation was observed using another peroxidase substrate, namely ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), forming a blue reaction product. The observed P-L activity of this Mnt material is in agreement with a previously analysed Chinese Mnt material that was assayed for its P-L activity with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate (Feng et al., Reference Feng, Wang, Zhang, An, Lin and Tong2021). Swelling of Mnt in water did not substantially influence its ability to act as a P-L catalyst (Fig. 1d–f).

The Dellite® LVF Mnt material (Fig. 2) exhibited substantially lower P-L activity, which resulted in a weak red colouration of sedimented Mnt after the assay; no free reaction product remained in the supernatant. In addition, the two remaining Mnt materials exhibited only weak P-L activity. This suggests that materials with identical chemical compositions might not all exhibit enzyme-mimetic activity; other typical examples are cerium dioxide particles of various particle sizes (Hamidat et al., Reference Hamidat, Barakat, Ortet, Chanéac, Rose and Bottero2016).

Figure 2. P-L activity of non-swollen Mnts (Dellite® LVF). A = Mnt suspension without DPD; B = Mnt suspension with DPD + H₂O₂ before centrifugation; C = Mnt with DPD + H₂O₂ after centrifugation.

To evaluate the P-L activities of the tested Mnt materials, we measured the absorbance values of the coloured DPD reaction products in the supernatant after centrifugation; however, these were also strongly adsorbed on the Mnt surface in the sediment (Figs 1 & 2). This does not allow for measuring catalytic activity accurately. This is why 5 mg of the Mnt samples was used in the assays to limit the effects of adsorption. Therefore, arbitrary values of the specific P-L activities of the Mnt samples are given as the values expressing P-L activities not considering the adsorption of the reaction products (Table 1). The Mnt from Sigma-Aldrich exhibited the highest specific P-L activity despite the strong adsorption of the reaction product. Other Mnt preparations exhibited substantially lower specific P-L activities, and in some cases the reaction product was completely bound to the Mnt surface.

Table 1. Specific P-L activities of native and modified Mnt samples (nkat mg^–1) and absorbances at 600 nm after Mnt sample reaction with phenol in the presence of H₂O₂ for 24 or 48 h (phenol polymerization).

^a Red-coloured oxidized DPD product was bound to Mnt particles.

The Mnt easily interacts with metal ions (Bhattacharyya & Sen Gupta, Reference Bhattacharyya and Sen Gupta2008). It was observed previously that copper and iron ions bound to solid surfaces can exhibit high P-L activity (Castro et al., Reference Castro, Fortuny, Stüber, Fabregat, Font and Bengoa2013; Wu et al., Reference Wu, Xu, Chen, Zhao, Cui, Shen and Zhang2014; Pospiskova & Safarik, Reference Pospiskova and Safarik2022). Thus, Mnt with bound metal ions may exhibit even higher P-L activity than native Mnt. To test this possibility, Mnt samples were immersed in excess of 5% CuSO₄.5H₂O or FeSO₄.7H₂O solutions for 24 h. After thorough washing to remove free (unbound) metal ions, the modified clays were dried and used for P-L activity measurements. The results are presented in Table 1. As expected, metal modification usually increased specific P-L activities; iron modification led to higher values.

Materials with P-L activity have been successfully used in various biotechnology and environmental technology applications instead of natural peroxidases – for example, for the decolourization of organic dyes (Safarik et al., Reference Safarik, Prochazkova, Schroer, Garamus, Kopcansky and Timko2021) or for the removal of phenol, aniline and related compounds (Zhang et al., Reference Zhang, Zhao, Niu, Shi, Cai and Jiang2009; Jiang et al., Reference Jiang, He, Wang, Jiang, Li and Li2018). Phenol polymerization by plant peroxidases and the formation of dark reaction products have been described previously (Reihmann & Ritter, Reference Reihmann, Ritter, Kobayashi, Ritter and Kaplan2006). To test the ability of Mnt and its metal derivatives to polymerize phenol, 5 mg of Mnt derivatives was added to 4.9 mL of 5% phenol solution, and then 100 μL of 30% H₂O₂ was added. After 24 and 48 h, the absorbances of the reaction supernatants at 600 nm were measured (higher absorbance corresponds to higher phenol polymerization), and the results are presented in Table 1. Native Mnt exhibited rather low phenol-polymerizing activity; however, this activity increased substantially after Mnt interaction with iron and copper ions. Interestingly, Cu-Mnt from Sigma-Aldrich demonstrated substantially higher phenol-polymerizing activity than the iron derivative after 24 h of incubation, but after 48 h the results were comparable. For other Mnt types, the iron modification was more effective for phenol polymerization than the copper modification. In any case, an increase in phenol-polymerization activity in vineyard soils containing higher copper concentrations due to the regular application of copper-based fungicides (Droz et al., Reference Droz, Payraudeau, Rodríguez Martín, Tóth, Panagos and Montanarella2021) is to be expected.

In addition, the abovementioned activity was displayed not only by free particles of Mnt and its derivatives, but also by immobilized particles. Figure 3 shows the example of Cu-Mnt (Sigma-Aldrich) particles bound to a low-cost colour catcher sheet (Color & Dirt Catcher Sheets, Brauns-Heitmann GmbH & Co. KG, Germany) before and after interaction with phenol in the presence of H₂O₂ (see above). The dark-coloured reaction product (polymerized phenol) was efficiently bound to the sheet, thus representing the combination of both enzyme-like and adsorbent properties. This result has interesting potential implications for environmental technology applications.

Figure 3. Pieces of colour catcher sheet (Color & Dirt Catcher Sheets, Brauns-Heitmann GmbH & Co. KG, Germany): A = native sheet; B = sheet after binding Cu-Mnt (Sigma-Aldrich); C = modified sheet after 24 h of interaction with phenol in the presence of H₂O₂.

Commercially available Mnt preparations have been tested for their P-L activity. It is expected that the potential P-L activity of soil Mnt and its complexes with metal cations will be utilized for activities usually performed by peroxidases, namely lignin breakdown and the decontamination of soils polluted with phenol-containing molecules (Hassan et al., Reference Hassan, Hamid, Auta, Pariatamby, Ossai, Barasarathi, Ahmed, Maddela, Abiodun and Prasad2022). Even at a low reaction rate, these long-term catalytic processes may lead to substantial decontamination of polluted soils.

In conclusion, specific mineral nanoparticles present in soils and exhibiting intrinsic enzyme-like activities could play important roles in the environmental biogeochemical cycles of elements, nutrients and pollutants (Chi & Yu, Reference Chi and Yu2021). Our study could encourage further research in the area of soil nanozymology to detect other interesting soil-derived enzyme-mimetic materials.