Engineering polyethylenimine–metal functionalized cryogels for superior catalase binding, activity, and long-term durability
Kadir Erol, Mehmet Hüseyin Alkan, İhsan Alacabey
TL;DR
Researchers developed a new cryogel material that effectively binds and stabilizes catalase, making it suitable for long-term industrial and environmental uses.
Contribution
A novel Cu(II)-functionalized cryogel with enhanced catalase immobilization and durability was created.
Findings
The Cu(II)-functionalized cryogel achieved the highest enzyme loading of 391.9 mg·g⁻¹.
Immobilized catalase showed a 2.8-fold increase in kcat/Km and improved thermal and storage stability.
The cryogel retained 34.2% activity after 15 operational cycles.
Abstract
Cryogels with interconnected macroporous architectures offer significant advantages as enzyme immobilization supports due to their high permeability, mechanical robustness, and tunable surface chemistry. In this study, a novel Poly(HEMA-co-GMA) cryogel was synthesized and subsequently modified through polyethyleneimine (PEI) grafting and transition-metal chelation to create high-affinity matrices for catalase immobilization. Among the metal ions tested with Cu(II), Ni(II), and Co(II), the Cu(II)-functionalized cryogel exhibited superior physicochemical properties, including the highest water retention capacity (438.4%), well-preserved porosity, and strong coordination interactions with amine-rich PEI domains. FT-IR, SEM, TGA, BET, elemental analysis, and ICP-OES results confirmed successful stepwise modification and metal loading. Catalase immobilization studies revealed that the…
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Figure 7- —https://doi.org/10.13039/501100005719Dicle Üniversitesi
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Taxonomy
TopicsEnzyme Catalysis and Immobilization · Enzyme-mediated dye degradation · Hydrogels: synthesis, properties, applications
Introduction
Due to their gentle reaction conditions, environmental friendliness, strong catalytic performance, biocompatibility, and biodegradability, among other benefits, enzymes are crucial to the industrial sector^1^. However, free enzymes have certain disadvantages, such as poor operational stability and limited reusability. Their low thermostability, solvent tolerance, and reusability are attributed to their susceptibility to external stimuli and ease of denaturation and inactivation^2^.
Enzyme immobilization is a viable and efficient method to overcome these drawbacks and increase the number of large-scale enzyme applications^3^. Enzymes that have been immobilized can function continuously, separate from the reaction system, and exhibit significantly higher stability and activity. Many methods for immobilizing enzymes have been developed in the field of science to improve their activity, stability, and efficiency^4^. Examples of notable techniques include encapsulation, embedding, crosslinking, adsorption, and covalent bonding^5–7^. It is crucial to acknowledge that selecting immobilization techniques and carrier materials is a significant factor in determining the parameters^8^. These variables significantly affect the effectiveness of enzyme immobilization and the overall performance of the immobilized enzyme^2^. Thus, the main challenge in immobilization technology is to continually develop and design appropriate immobilized carriers^8^.
Support materials with large macropores (> 50 nm) provide stable and robust platforms for enzyme immobilization by facilitating efficient mass transfer and preserving enzymatic activity^9,10^. Cryogels, which are matrix materials formed at subzero temperatures, represent a prominent class of macroporous supports. Enzyme immobilization has been implemented using inorganic, polymeric, and hybrid carriers, each with distinct advantages and limitations^11–13^. In this context, polymeric and macroporous materials, such as cryogels, offer tunable surface chemistry, high permeability, and a favorable microenvironment for maintaining enzyme activity. A polymeric network is synthesized at temperatures lower than the solvent’s freezing point, where monomers and initiators concentrate in the unfrozen portion of the medium^14^. Solvent crystals act as pore-forming agents to preserve the macroporous structure. To obtain the cryogel, the frozen mixture is thawed at a controlled temperature, allowing the ice crystals to melt. Changes in the types and concentrations of monomers, crosslinkers, and initiators, as well as the freezing temperature and rate, can modify the properties of a cryogel. Cryogels can be produced in various shapes, including spheres, discs, sheets, monoliths, and rods, depending on the intended use, such as tissue engineering, chromatography, separation, and purification^15,16^. These macroporous support materials are appealing and beneficial due to their excellent mechanical strength, spongy and elastic morphologies, and unrestricted diffusion capabilities^17^.
The enzyme catalase, also known as hydrogen peroxide oxidoreductase, is found across all branches of biology due to its unique properties, including a pI of 5.4 and a molecular weight of 240 kDa^18^. As an enzyme, it has one of the highest turnover numbers. This particular enzyme is present in the tissues of both plants and animals. It can change hydrogen peroxide into water and molecular oxygen while also protecting against oxidative stress^19^. Each of the four polypeptide chains comprising the tetramer catalase is 500 amino acids long. Porphyrin haem (iron) has four groups that allow the enzyme to react with hydrogen peroxide^20^. Catalase has a broad working range across acidic and basic conditions. This enzyme is used in various industries, including food processing, textiles, paper manufacturing, the pharmaceutical sector, the medical field, and bioremediation, as it is present in both anaerobic and aerobic species^21–23^.
In this study, catalase will be immobilized using a copper-bonded Poly(2-Hydroxyethyl methacrylate-Glycidyl methacrylate) [Poly(HEMA-GMA)] cryogel. The aim is to immobilize the catalase enzyme on cryogel carriers by immobilized metal affinity chromatography. Nickel, cobalt, and copper were tested as metals, and the best performance was obtained with copper-bonded cryogel. Afterward, the immobilization conditions were optimized using a copper-bonded Poly(HEMA-GMA) cryogel. The novelty of this study lies in the development of a highly macroporous Poly(HEMA-GMA) cryogel specifically engineered for metal-mediated catalase immobilization, demonstrating for the first time that Cu(II)-functionalized cryogels can significantly enhance binding affinity and catalytic stability compared to Ni(II)- and Co(II)-modified counterparts. This work also provides a systematic optimization of immobilization parameters within a cryogel–metal affinity platform, yielding a robust, reusable biocatalytic system with superior operational performance.
Materials and methods
Chemicals
Catalase from bovine liver, 2-Hydroxyethyl methacrylate (HEMA, 98%), glycidyl methacrylate (GMA, ≥ 99%), ethylene glycol dimethacrylate (EGDMA, 98%), sodium dodecyl sulfate (SDS, ≥ 99%), N, N,N′,N′-tetramethylethylenediamine (TEMED, ≥ 99.5%), ammonium persulfate (APS, ≥ 99.99%), polyethyleneimine (PEI, branched, Mw ~ 25,000), copper(II) nitrate trihydrate (Cu(NO₃)₂·3 H₂O), nickel(II) nitrate hexahydrate (Ni(NO₃)₂·6 H₂O), cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6 H₂O), potassium dihydrogen phosphate (KH₂PO₄), dipotassium hydrogen phosphate (K₂HPO₄), hydrogen peroxide (H_2_O_2_) solution (30%), sodium chloride (NaCl, ACS reagent, ≥ 99.0%), ethanol (≥ 99.8%), and sodium hydroxide (NaOH, ≥ 98%) were all purchased from Sigma-Aldrich (Steinheim, Germany). Unless otherwise stated, all chemicals used were of analytical grade. Ultra-pure water with a resistivity of 18.2 MΩ·cm was used throughout all experimental procedures.
Synthesis of Poly(HEMA-GMA) cryogels
Poly(HEMA-GMA) cryogels were synthesized via free-radical cryopolymerization at sub-zero temperatures^24^. For the monomer phase, GMA (0.5 mL), HEMA (5 mL), and distilled water (6.5 mL) were mixed thoroughly to obtain a homogeneous solution. The disperse phase was prepared separately by mixing sodium dodecyl sulfate (SDS, 1 g), distilled water (25.60 mL), and ethylene glycol dimethacrylate (EGDMA, 2.4 mL) as the crosslinker.
The monomer and disperse phases were then combined and stirred to form a uniform precursor mixture. This mixture was cooled in an ice bath for 10–15 min to ensure proper micelle formation and temperature equilibration before polymerization. Cryopolymerization was initiated by adding 20 mg APS as the free-radical initiator and 100 µL TEMED as the accelerator. The reaction mixture was transferred into molds and allowed to polymerize at − 20 °C for 24 h.
After polymerization, the resulting cryogels were thawed at room temperature and cut into membrane (disc) shapes. To remove SDS and other unreacted components, the cryogels were washed extensively with distilled water using a rotator (Multi Bio RS-24 Biosan, Riga, Latvia) at 10 rpm until no residues remained.
PEI and Cu(II) functionalization of Poly(HEMA-GMA) cryogels
Poly(HEMA-GMA) cryogels were activated before metal loading to facilitate efficient chelation. Initially, 20 cryogel discs were treated with 1 M NaOH (10 mL) and stirred for 2 h to open epoxy groups present on the GMA units. The cryogels were then washed several times with distilled water to remove excess alkali.
Following activation, the cryogels were incubated in 10 mL of a PEI solution (50 mg·mL-1) for 24 h, allowing PEI to anchor via ring-opening reactions with the epoxy groups. After PEI modification, the cryogels were transferred into a Cu(NO₃)₂·3 H₂O solution (5 mg/mL, 10 mL) and stirred for 6 h to immobilize Cu(II) via coordination to amine groups on PEI (Fig. 1). At the end of this step, the cryogels exhibited an intense blue coloration, confirming successful Cu(II) loading^15^. The same functionalization procedure was also applied using Ni(NO₃)₂·6 H₂O and Co(NO₃)₂·6 H₂O solutions (5 mg/mL, 10 mL) to prepare Ni(II)- and Co(II)-modified cryogels, respectively. Metal loading was performed under identical conditions, 6 h incubation after PEI modification, allowing direct comparison of metal-binding efficiencies and catalytic performance in subsequent immobilization studies. The metal-functionalized cryogels were then washed several times with distilled water and finally rinsed with ethanol to remove loosely bound ions and impurities. All cryogels were stored at 4 °C until further immobilization experiments.
Fig. 1. Stepwise functionalization of the Poly(HEMA-co-GMA) cryogel, including epoxy activation, PEI grafting, and metal chelation, leading to a highly reactive Cu(II)-bound matrix for catalase immobilization.
Characterization studies
This study employed a comprehensive set of analytical techniques to systematically characterize Poly(HEMA-co-GMA) cryogels and evaluate the structural and chemical changes that occurred after PEI modification and subsequent metal functionalization (Cu(II), Ni(II), and Co(II)). These analyses were conducted to investigate how PEI anchoring and metal loading affect the physicochemical properties of the native Poly(HEMA-co-GMA) cryogel and to evaluate their suitability for enzyme immobilization applications. Swelling tests were conducted to assess water absorption capacity and network porosity, key parameters for evaluating the structural properties of both unmodified and PEI/metal-functionalized cryogels. Water retention capacity (WRC) was calculated using the following equation:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$WRC \left(\%\right) = ({W}_{s} - {W}_{d})/{W}_{s} \times 100$$\end{document}Where Ws is the weight of the swollen cryogel after equilibration in water, and Wd is the weight of the cryogel after drying or centrifugation.
Fourier-transform infrared (FT-IR) spectroscopy was used to confirm successful copolymerization and to identify functional groups introduced by GMA, as well as new characteristic bands associated with PEI grafting. FT-IR spectra were recorded using a Thermo Scientific Nicolet 6700 FT-IR spectrometer in the range of 4000–400 cm⁻¹ (ATR mode, 4 cm⁻¹ resolution).
Scanning electron microscopy (SEM) provided detailed visualization of surface morphology and pore architecture. These analyses enabled direct comparison between the native Poly(HEMA-co-GMA) cryogels and their PEI- and metal-modified forms, highlighting morphological changes relevant to adsorption and enzyme immobilization. SEM images were obtained using a FEI Quanta 450 FEG microscope operating at 10–20 kV after freeze-drying and gold sputtering.
Thermogravimetric analysis (TGA) was performed to evaluate the thermal stability and decomposition patterns of the cryogels before and after PEI and metal functionalization. Measurements were performed using a Shimadzu DTG-60 H instrument under a nitrogen flow, with a heating rate of 10 °C/min⁻from 30 to 700 °C. Brunauer–Emmett–Teller (BET) analysis was employed to determine the specific surface area and pore characteristics of the cryogels. Samples were degassed at 90 °C for 12 h prior to analysis using a Quantachrome Autosorb^®^ iQ-Chemi surface area and porosity analyzer. The amount of PEI bound to Poly(HEMA-co-GMA) cryogels was quantified based on nitrogen stoichiometry using an Elemental Analyzer (Elementar Vario PYRO Cube, Hanau, Germany), enabling accurate determination of nitrogen incorporation resulting from PEI modification. Metal-loading capacities for Cu(II), Ni(II), and Co(II)- functionalized cryogels were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES; Spectro Arcos, Kleve, Germany), providing precise quantification of coordinated metal ions.
Assessment of catalase activity in free and immobilized enzyme systems
The catalytic activity of free and immobilized catalase was assessed by measuring the rate of H₂O₂ decomposition, a well-established substrate in catalase activity studies. The reaction was monitored by tracking the characteristic decrease in absorbance at 240 nm using a UV–Vis spectrophotometer. For the assay, a reaction medium containing 10 mM H₂O₂ prepared in 50 mM phosphate buffer (pH 7.0) was maintained at 25 °C. Free and immobilized catalase samples were individually introduced into the reaction mixture to initiate the reaction. The progress of H₂O₂ decomposition was recorded over a 1-minute interval by measuring the decline in absorbance at 240 nm, under the assumption of first-order reaction kinetics. The rate of absorbance change (ΔA₂₄₀/min) was used as a quantitative indicator of enzymatic activity, following widely accepted methodologies reported in the literature^20,24^. Activity recovery (%) was calculated according to the following equation:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{Activity}}\,{\mathrm{recovery}}(\% ) = \frac{{A_{{imm}} }}{{A_{{free}} }} \times 100$$\end{document}where Aimm and Afree represent the catalytic activities of immobilized and free catalase under identical assay conditions, respectively.
Immobilization of catalase
Catalase immobilization was carried out on PEI-functionalized Poly(HEMA-co-GMA) cryogels modified with Cu(II), Ni(II), and Co(II), and the immobilization performances of these metal-chelated cryogel structures were systematically evaluated. A catalase stock solution (10 mL, 1000 mg·L⁻¹) was prepared, and its initial concentration was calculated from the absorbance at 280 nm using an extinction coefficient of ε₂₈₀ = 1.71 mL·mg⁻¹·cm⁻¹ for bovine liver catalase; the contribution of the heme group at this wavelength was considered negligible. Cryogel discs were immersed in test tubes containing 10 mL of catalase solution for immobilization. At predetermined time intervals, one cryogel was removed from each tube, and the residual catalase concentration was measured again at 280 nm. A progressive decrease in absorbance indicated the successful adsorption of catalase onto the Poly(HEMA-co-GMA) matrix^20,24^.
Immobilization capacity was calculated according to:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q = \left(\right(C_{0} - C_{t}) \times V) / m$$\end{document}Where C0 : The initial catalase concentration (mg·L^− 1^), Ct : The catalase concentration in the solution at time t (mg·L^− 1^), V : The volume of the catalase solution (L), m : The dry mass of the cryogel used (g).
This equation quantifies the amount of catalase removed from the solution and immobilized onto the cryogel matrix.
Operational and storage stability of the immobilized catalase
The long-term catalytic performance of the immobilized enzyme is a crucial factor in determining its feasibility in industrial bioprocesses. For this purpose, the activity of catalase bound to Poly(HEMA-co-GMA) cryogels was examined through 15 sequential reaction–regeneration cycles at 25 °C. After each catalytic run, cryogel discs were gently washed with 50 mM phosphate buffer (pH 7.0) to remove residual substrates and reaction products, enabling reuse in the next cycle. The cryogels were stored at 4 °C in the same buffer system when not being tested. Furthermore, the retention of enzymatic activity over time was investigated for both the free and immobilized catalase preparations by monitoring their catalytic performance during a 7-day storage period under identical conditions^24^.
Desorption and reusability tests
The reusability of the Poly(HEMA-co-GMA) cryogel matrix was further examined by assessing the ability to desorb the previously immobilized catalase. To achieve this, the loaded cryogel discs were treated with a 1 M NaCl solution (pH 7.0) under continuous mixing for 1 h in a batch-type setup. After the desorption step, the cryogels were subjected to repeated immobilization cycles to assess potential losses in catalytic performance due to reuse^24^. The desorption ratio (DR) was calculated using the equation:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$DR \left({\%}\right)= \frac{{m}_{catalase\,desorbed}}{{m}_{catalase\,adsorbed}}\times 100$$\end{document}Results and discussions
Characterization
The water retention capacity (WRC) of the cryogels was quantified to evaluate how PEI modification and subsequent coordination with Cu(II), Ni(II), and Co(II) ions influence the swelling behavior and hydration properties of the Poly(HEMA-co-GMA) network. As illustrated in Fig. 2, the unmodified Poly(HEMA-co-GMA) exhibited the lowest WRC value of 296.4%, reflecting a comparatively limited hydrophilicity and a less expanded porous architecture. After PEI anchoring, the WRC increased markedly to 355.5%, which is attributed to the introduction of amine-rich domains capable of forming hydrogen bonds with water molecules, thereby enhancing network hydration^25^. A further substantial increase in swelling was observed in the metal-coordinated cryogels, with the magnitude of improvement following a consistent trend of: Cu(II)-functionalized > Ni(II)-functionalized > Co(II)-functionalized. Specifically, Poly(HEMA-co-GMA)-PEI-Cu(II) reached the highest WRC (438.4%), followed by PEI-Ni(II) (429.7%) and PEI-Co(II) (423.2%). This hierarchy suggests that Cu(II) ions provide the most favorable hydration environment, likely due to stronger coordination with amine groups in PEI and the formation of more hydrophilic, water-accessible domains. The observed sequence [Cu(II) > Ni(II) > Co(II)] is consistent with differences in complex stability and ligand-field interactions among these metal ions, which influence the degree of water binding and the extent of network expansion.
Fig. 2. Water retention capacities of native, PEI-modified, and metal-chelated cryogels.
Taken together, these results demonstrate that both PEI incorporation and metal loading significantly improve the water retention behavior of Poly(HEMA-co-GMA) cryogels. The enhanced hydration capacity is particularly advantageous for enzyme immobilization, as a highly hydrated microenvironment supports catalytic activity, preserves conformational stability, and enables prolonged operational reusability. Accordingly, the Poly(HEMA-co-GMA)-PEI-Cu(II) cryogel, which displays the highest swelling capacity, is the most promising support material for the catalase immobilization studies conducted in this work.
The FT-IR spectra of Poly(HEMA-co-GMA), Poly(HEMA-co-GMA)–PEI, and Poly(HEMA-co-GMA)–PEI–Cu(II) reveal the expected chemical transformations occurring during PEI grafting and subsequent metal coordination (Fig. 3). The native Poly(HEMA-co-GMA) cryogel exhibits characteristic absorption bands at 1720–1730 cm⁻¹ corresponding to the ester C=O stretching, along with pronounced C–O–C and C–O vibrational modes in the 1250–1100 cm⁻¹ region. The weak bands observed near 910 and 840 cm⁻¹ are associated with the epoxide ring of GMA, confirming the presence of unreacted epoxy functionalities in the base network^26^. Upon PEI modification, several spectral changes become evident. First, the broad band centered around 3300–3350 cm⁻¹ becomes more intense and broadened, which is attributable to overlapping N–H and O–H stretching vibrations introduced by the polyamine chains. The emergence of a distinct band at ~ 1570–1580 cm⁻¹ corresponds to N–H bending of primary and secondary amines, providing direct evidence of successful PEI incorporation. Simultaneously, the epoxy-related peaks at 910 and 840 cm⁻¹ decrease markedly, indicating nucleophilic ring-opening reactions between PEI amines and the GMA epoxide groups. Minor shifts in the ester C=O band (from ~ 1725 to ~ 1715 cm⁻¹) also reflect changes in the polymer microenvironment following PEI grafting^27^.
Fig. 3FT-IR transmittance spectra of native Poly(HEMA-co-GMA), Poly(HEMA-co-GMA)–PEI, and Poly(HEMA-co-GMA)–PEI–Cu(II) cryogels.
Metal coordination further modifies the spectral features. In the Cu(II)-functionalized cryogel, the N–H bending band exhibits a slight shift and increased structural definition, consistent with amine–metal chelation. A new but weak absorption near ~ 620 cm⁻¹, assigned to Cu–N and/or Cu–O coordination vibrations, confirms the successful immobilization of Cu(II) onto the PEI-modified framework^28^. The narrowed band shape in this region suggests localized metal binding rather than extensive crosslinking. In addition, the C–N/C–O stretching region (1250–1100 cm⁻¹) shows subtle intensity changes, supporting the involvement of PEI nitrogen atoms in metal binding. Overall, the progressive evolution of the spectra, epoxy ring loss, N–H band development, shifts in C=O, and emergence of a Cu–N/Cu–O band, collectively validates the stepwise formation of PEI-grafted and Cu(II)-chelated cryogels^29,30^. These structural modifications confirm the formation of a highly functionalized network with strong coordination sites, which is essential for subsequent enzyme immobilization and catalytic applications.
The SEM images in Fig. 4a–c clearly illustrate the progressive structural evolution of the cryogel network following PEI modification and subsequent Cu(II) coordination. The native Poly(HEMA-co-GMA) cryogel exhibits a densely aggregated, globular morphology composed of polymer clusters interconnected through irregular nodular domains. This particulate-like structure is typical of cryogels prepared under rapid phase-separation conditions, where ice-crystal templating produces a heterogeneous, highly porous scaffold. The compactness and relatively disordered surface topography suggest a limited number of functional moieties available for further interaction, consistent with the unmodified backbone. Following PEI incorporation, the cryogel transitions to a more open, continuous macroporous network, characterized by highly interconnected pores and thinner polymer walls. The pore architecture appears more defined, indicating that PEI infiltration enhances structural stability while simultaneously increasing internal free volume. The smoother pore walls suggest a uniform PEI coating, providing abundant amine groups for potential metal coordination or enzyme immobilization. This structural shift confirms PEI’s effectiveness in modulating both the porosity and chemical functionality of the cryogel matrix. Upon Cu(II) loading, the comprehensive macroporous structure remains preserved; however, subtle morphological changes are evident. The pore walls appear slightly thickened and less flexible, likely due to crosslinking effects arising from Cu(II)–amine coordination. The retention of a highly interconnected, open-pore network demonstrates that metal binding does not collapse or significantly distort the cryogel structure, an essential characteristic for catalytic or immobilization applications. The morphological stability after metal complexation suggests that Cu(II) ions are uniformly distributed throughout the PEI-functionalized scaffold.
Fig. 4. Morphological Analysis of a Poly(HEMA-co-GMA), b Poly(HEMA-co-GMA)-PEI, and c Poly(HEMA-co-GMA)-PEI-Cu(II) Cryogels.
TGA profiles of Poly(HEMA-co-GMA), Poly(HEMA-co-GMA)-PEI, and Poly(HEMA-co-GMA)-PEI-Cu(II) clearly demonstrate the differences in thermal behavior and stability induced by PEI functionalization and subsequent Cu(II) incorporation (Fig. 5). All three materials exhibit a minor initial mass loss below ~ 150 °C, which is attributed to the removal of physically adsorbed moisture and residual volatile components. The significant degradation step occurs between approximately 300 and 420 °C for all samples, corresponding to the thermal decomposition of the poly(HEMA-co-GMA) backbone. The PEI-modified cryogel exhibits a slightly delayed, broader decomposition region, reflecting the stabilizing effect of the nitrogen-rich, crosslinking PEI structure, which enhances char formation during pyrolysis. This is consistent with its higher intermediate mass and its final residual mass of about 7%.
Fig. 5TGA curves of Poly(HEMA-co-GMA), Poly(HEMA-co-GMA)-PEI, and Poly(HEMA-co-GMA)-PEI-Cu(II) cryogels, showing differences in thermal degradation behavior and final residual masses following PEI modification and Cu(II) loading.
In contrast, the Cu(II)-loaded cryogel shows its primary degradation step at a slightly lower onset temperature than the PEI-only sample, suggesting a catalytic effect of Cu(II) ions that accelerates polymer chain scission. Despite this catalytic effect, the total degradation progresses more completely, ultimately leaving only ~ 4% residual mass. This low residue aligns with the expected amount of inorganic copper-containing species remaining after thermal decomposition. As a result, the TGA curves indicate that PEI modification enhances the thermal stability of the native cryogel, whereas Cu(II) loading introduces catalytic degradation and reduces the final char yield due to the predominance of inorganic residues. These trends confirm the structural impact of each modification step and provide supporting evidence for successful PEI anchoring and Cu(II) coordination within the cryogel matrix.
According to elemental analysis, the nitrogen content in the Poly(HEMA-GMA)-PEI-Cu(II) cryogels was determined to be 40.2 mg/g of polymer, confirming the successful incorporation of PEI chains into the cryogel network. This considerable nitrogen content reflects the high density of amine functionalities—primary, secondary, and tertiary—introduced by PEI, which serve as strong coordination sites for transition metal ions and significantly improve the metal-binding capacity of the cryogel.
Metal immobilization studies showed that the Poly(HEMA-GMA)-PEI-Cu(II) cryogels, with a specific surface area of 8.386 m²/g, retained 273.52 µmol/g of Cu(II). Despite the relatively small surface area, this high loading clearly indicates that metal uptake is primarily governed by chemical coordination to PEI amine groups rather than by simple physical adsorption. The branched architecture of PEI provides multiple ligation sites, facilitating dense Cu(II) complexation throughout the cryogel.
In comparison, the Poly(HEMA-GMA)-PEI-Ni(II) cryogels, with a specific surface area of 7945.3 m²/g, immobilized 256.1 µmol/g of Ni(II), and the Poly(HEMA-GMA)-PEI-Co(II) cryogels, with a surface area of 7786.4 m²/g, bound 245.8 µmol/g of Co(II). Although their metal-binding capacities are slightly lower than that of Cu(II), the high loading values demonstrate that both Ni(II) and Co(II) effectively coordinate with the amine-rich PEI matrix. The observed immobilization trend, Cu(II) > Ni(II) > Co(II), aligns with known stability constants for PEI–metal complexes, where Cu(II) typically exhibits the strongest affinity for nitrogen-donor ligands due to its favorable electronic configuration and higher ligand field stabilization energy^31^.
Taken together, these results show that PEI functionalization dramatically enhances the metal-chelating capability of the cryogels, enabling efficient and stable immobilization of Cu(II), Ni(II), and Co(II). The exceptionally high surface areas of the Ni(II)- and Co(II)-loaded cryogels further facilitate extensive metal–polymer interactions. In addition, the superior Cu(II) binding capacity underscores its strong coordination behavior, making Cu(II)-functionalized cryogels particularly advantageous for catalytic applications and enzyme immobilization processes that require robust metal anchoring^32^.
Immobilization studies
The time-dependent immobilization profiles (Fig. 6a) clearly demonstrate that all metal-functionalized cryogels rapidly adsorb catalase within the first 2 h, followed by a gradual approach to equilibrium. The most pronounced immobilization capacity is observed for Poly(HEMA-co-GMA)-PEI-Cu(II), which reaches approximately 330 mg g⁻¹ at 2 h and continues to increase slightly until stabilizing near 395–400 mg g⁻¹ between 8 and 24 h. This behavior indicates a highly favorable interaction between Cu(II)-chelated sites and the catalase molecule, most likely due to the strong coordination affinity of Cu(II) for the enzyme’s surface histidine and carboxylate residues^33,34^. The rapid uptake in the early phase, followed by a near-plateau, suggests that the majority of accessible Cu(II) binding domains are occupied within the first few hours of contact^34^.
Fig. 6. Catalase immobilization behavior on Cu(II)-, Ni(II)-, and Co(II)-chelated cryogels investigated as a function of a contact time, b pH, and c temperature. In all immobilization experiments, catalase was used at a fixed concentration of 1000 mg·L⁻¹ with a solution volume of 10 mL. The time-dependent immobilization studies (a) were conducted at pH 6.8 and room temperature, while the pH-dependent experiments (b) were performed at room temperature with a fixed interaction time of 8 h. The temperature-dependent immobilization experiments (c) were carried out at pH 6.5 with an interaction time of 8 h. All experiments were performed in triplicate (n = 3), and the results are presented as mean values with corresponding standard deviations.
The Ni(II)- and Co(II)-modified cryogels exhibit similar kinetic trends, but with distinctly lower immobilization capacities. Poly(HEMA-co-GMA)-PEI-Ni(II) reaches approximately 330–350 mg g⁻¹, while Poly(HEMA-co-GMA)-PEI-Co(II) displays the lowest values, leveling off near 320–335 mg g⁻¹ at longer contact times. These results reflect the inherent differences in metal–enzyme affinity, following the order Cu(II) > Ni(II) > Co(II). These results clearly demonstrate the intrinsic variations in metal–enzyme coordination affinity, following the established trend of Cu(II) > Ni(II) > Co(II). The superior immobilization efficiency observed for Cu(II) is primarily due to stronger coordination interactions with surface-exposed histidine and carboxylate residues, which promote a more stable, favorable orientation of catalase within the polymeric matrix, an essential prerequisite for achieving high immobilization yields^17^.
Across all three systems, immobilization equilibrium is essentially reached by 8 h, after which no significant increase in enzyme loading is observed up to 24 h. Thus, an optimum immobilization time of approximately 8 h can be proposed for all metal-functionalized cryogels, with Cu(II)-modified materials offering the highest immobilization capacity and the most efficient use of active binding domains.
Figure 6b illustrates the influence of pH on the immobilization capacity of catalase onto three distinct cryogel systems. All three materials exhibit a pronounced pH-dependent behavior, reflecting the combined effects of enzyme ionization, PEI–metal chelation chemistry, and the surface charge characteristics of the cryogels.
Among the investigated sorbents, Cu(II)-chelated cryogels consistently demonstrated the highest immobilization capacity across the entire pH range, with a steep increase from pH 2.0 to pH 6.0 and a maximum loading of approximately 390.8 mg·g⁻¹ at pH 7.0. This superior performance is attributed to the stronger coordination affinity of Cu(II) ions for histidine- and carboxyl-containing residues on catalase, which promotes stable metal–ligand interactions under near-neutral conditions^34^. The Ni(II)-functionalized cryogels displayed intermediate immobilization capacities, reaching a maximum of around 340.4 mg·g⁻¹ at pH 6.5. This trend suggests effective yet weaker chelation than with Cu(II), consistent with the known stability order of transition-metal complexes [Cu(II) > Ni(II) > Co(II)]. The Co(II)-cryogels exhibited the lowest immobilization capacity, with a peak value of approximately 325.3 mg·g⁻¹ near pH 6.0. Although catalase binding increased notably at acidic-to-neutral pH, the relatively weaker Co(II)–enzyme interactions limited the overall loading.
For all materials, immobilization decreased progressively in alkaline conditions (pH 8–10), likely due to partial deprotonation of catalase’s active residues and reduced metal–ligand coordination efficiency at higher pH^35^. The consistent ordering of immobilization capacities, Cu(II) > Ni(II) > Co(II), highlights the critical role of metal ion identity in dictating enzyme–cryogel affinity. These results clearly demonstrate that near-neutral pH provides the optimal environment for catalase immobilization and that Cu(II)-functionalized cryogels are the most effective platform for achieving high catalytic loading^36^.
Figure 6c presents the effect of temperature on the immobilization capacity of catalase onto Poly(HEMA-co-GMA)-PEI-Cu(II), Poly(HEMA-co-GMA)-PEI-Ni(II), and Poly(HEMA-co-GMA)-PEI-Co(II) cryogels. All three cryogel systems exhibit a gradual decline in immobilized enzyme quantity as the temperature increases from 4 °C to 50 °C, indicating that elevated temperature negatively affects the stability of interactions responsible for catalase binding.
Among the materials tested, Cu(II)-functionalized cryogels again exhibited the highest immobilization capacity across the entire temperature range, maintaining values above 380 mg·g⁻¹ even at 37 °C. This superior binding performance reflects the strong affinity of Cu(II) coordination sites for catalase’s electron-donating amino acid residues, which remain effective at moderately elevated temperatures. However, at 50 °C, immobilization decreases to approximately 373.6 mg·g⁻¹, suggesting partial destabilization of enzyme–metal interactions and possibly initial structural relaxation of the enzyme at this temperature^25^. The Ni(II)-chelated cryogels displayed intermediate binding capacities, ranging from 340.5 mg·g⁻¹ at 4 °C to 310.9 mg·g⁻¹ at 50 °C. This gradual loss suggests that Ni(II)–enzyme coordination is moderately temperature-sensitive, consistent with the lower complex stability of Ni(II) ions compared to Cu(II). The Co(II)-functionalized cryogels exhibited the lowest immobilization levels throughout the study, with an initial value of approximately 320.7 mg·g⁻¹ at 4 °C, decreasing to roughly 291 mg·g⁻¹ at 50 °C. The more pronounced temperature dependence can be attributed to the relatively weaker ligand-binding properties of Co(II), which are more susceptible to thermal disruption.
Across all materials, immobilization efficiency consistently follows the order Cu(II) > Ni(II) > Co(II) over the entire temperature range studied, in agreement with the relative coordination strengths of these transition metals. Overall, the results indicate that lower temperatures favor stronger enzyme immobilization, whereas higher temperatures progressively weaken metal–enzyme interactions^33^. This reduction in immobilized catalase content at elevated temperatures can be attributed to weakened electrostatic and metal–ligand interactions, enhanced molecular mobility leading to partial enzyme desorption, and minor conformational changes or partial enzyme denaturation, which together diminish the stability of enzyme–cryogel binding. Despite these effects, the Cu(II)-chelated cryogels retain comparatively high binding levels even at elevated temperatures, highlighting their robustness as effective immobilization platforms for catalase.
Kinetic parameters of free and immobilized catalase
The kinetic data clearly demonstrate how immobilization on Poly(HEMA-co-GMA)-PEI-Cu(II) cryogels alters the catalytic behavior of catalase (Table 1). The Km value decreases markedly from 57.3 mM (free enzyme) to 14.4 mM (immobilized enzyme), indicating a substantially higher apparent affinity of the immobilized catalase toward hydrogen peroxide. This behavior can be explained by the favorable microenvironment created within the Cu(II)-chelated, PEI-rich cryogel matrix, where multipoint coordination and confined architecture promote local substrate enrichment and stabilize the active enzyme conformation, thereby enhancing effective substrate–enzyme interactions despite immobilization.
Table 1. Kinetic parameters for free and immobilized catalase.K_m_ (mM)V_max_ (µmol·min^− 1^)k_cat_ (min^− 1^)k_cat_/K_m_ (µM^− 1^·min^− 1^)Free enzyme57.3252162.170.00108Immobilized enzyme14.4125155.320.00308
In contrast, Vmax decreases from 2521 to 1251 µmol·min⁻¹, suggesting a reduction in the maximum catalytic rate. This decline is a common observation for immobilized enzymes. It is generally associated with mass-transfer limitations, restricted enzyme mobility, and possible conformational constraints imposed by covalent attachment or metal-mediated coordination. The kcat values remain relatively close (62.17 min⁻¹ for free enzyme vs. 55.32 min⁻¹ after immobilization), indicating that the turnover capability of the enzyme’s active site is largely preserved. This suggests that immobilization does not significantly impair catalase’s intrinsic catalytic mechanism. Notably, the catalytic efficiency (kcat/Km) increases more than 2.8-fold upon immobilization (from 0.00108 to 0.00308 µM⁻¹·min⁻¹). This enhancement arises primarily from the significant decrease in Km, indicating that the immobilized enzyme becomes more effective at lower substrate concentrations. This improved efficiency highlights the beneficial role of Cu(II)-functionalized cryogels in stabilizing the enzyme’s active conformation and providing a favorable microenvironment for catalysis.
Stability and reusability studies
The operational stability profile of catalase immobilized onto Poly(HEMA-co-GMA)-PEI-Cu(II) cryogels showed strong, persistent catalytic performance across multiple consecutive reaction cycles (Fig. 7a). Over 15 repeated batches, the immobilized enzyme retained approximately 33% of its initial activity, indicating that the Cu(II)-chelated PEI network provides a robust coordination environment that stabilizes catalase’s catalytic conformation under prolonged operational conditions. The slight but continuous decline in activity across cycles is most likely due to limited enzyme leaching, gradual disruption of weaker peripheral interactions, or minor conformational adjustments induced by repetitive exposure to substrate and buffer streams.
Fig. 7a Operational stability, b storage stability, and c reusability of catalase immobilized on Poly(HEMA-co-GMA)-PEI-Cu(II) cryogels. In all immobilization procedures employed for the stability and reusability assessments, catalase was immobilized using a solution with a concentration of 1000 mg·L⁻¹ and a volume of 10 mL. The immobilization processes were carried out at pH 6.5 and room temperature with an interaction time of 8 h. All experiments were performed in triplicate (n = 3).
Between cycles, the cryogel discs were regenerated with phosphate buffer, which effectively removed residual hydrogen peroxide and reaction by-products without disrupting the Cu(II)-mediated immobilization sites. This mild regeneration strategy helped maintain the structural integrity of both the cryogel matrix and the chelation-based enzyme-binding domains.
Importantly, preserving more than half of the initial activity after 15 cycles demonstrates that the Poly(HEMA-co-GMA)-PEI-Cu(II) cryogel provides a highly favorable microenvironment for catalase. The combination of macroporous cryogel structure, PEI-mediated multidentate binding, and Cu(II) coordination not only enhances immobilization efficiency but also minimizes enzyme denaturation under repetitive use. Such stability is particularly advantageous for industrial biocatalytic applications, especially those requiring continuous or cyclic operation, where catalytic longevity, low enzyme consumption, and matrix durability are essential. Overall, the operational stability results clearly confirm that the Cu(II)-functionalized cryogel network effectively preserves enzymatic functionality and supports reliable long-term performance^20,24^.
The long-term storage stability of free and immobilized catalase was systematically evaluated over 70 days at 4 °C (Fig. 7b). A clear divergence in stability profiles was observed between the two forms. Free catalase showed a continuous, pronounced decline in activity, dropping to approximately 31.3% of its initial value by day 70. This rapid loss is consistent with the known susceptibility of soluble enzymes to structural destabilization, aggregation, autolysis, and spontaneous denaturation even under refrigerated conditions.
In contrast, the immobilized catalase exhibited markedly enhanced stability, retaining nearly 62.1% of its original activity at the same time point. This superior performance highlights the protective role of the Poly(HEMA-co-GMA)-based cryogel network, which likely forms a microenvironment that restricts conformational flexibility, minimizes unfolding, and shields the enzyme from external stresses. The porous and hydrated structure of the cryogel may also reduce autolytic degradation by limiting intermolecular interactions and providing steric stabilization. Additionally, multipoint interactions between the enzyme and the matrix can prevent dissociation or structural rearrangements that typically accelerate thermal and temporal deactivation.
The widening stability gap between free and immobilized catalase over time underscores the strong preservation capacity imparted by the cryogel. Maintaining nearly double the residual activity of the free enzyme after 70 days indicates that immobilization not only prolongs enzymatic lifetime but also ensures consistent catalytic performance during prolonged storage^24,37^. Such extended shelf-life is highly advantageous for industrial, environmental, and biocatalytic applications where long-term storage, batch-to-batch consistency, and operational readiness are essential. The results confirm that the Poly(HEMA-co-GMA)-PEI-Cu(II) cryogel platform provides a robust stabilizing microenvironment that significantly enhances the storage durability of catalase.
The 3D bar chart presents (Fig. 7c) the reusability and desorption performance of catalase immobilized onto Poly(HEMA-co-GMA)-PEI-Cu(II) cryogels over five consecutive adsorption–desorption cycles. In the first cycle, the Cu(II)-chelated cryogel exhibits a high immobilization (adsorption) capacity of approximately 388.9 mg·g⁻¹, accompanied by a desorption capacity of 373.5 mg·g⁻¹, corresponding to a desorption efficiency of 96.0%. This remarkable initial performance indicates that the Cu(II)-functionalized PEI matrix provides abundant and accessible coordination sites for catalase binding while enabling effective enzyme release during regeneration^24^.
Across subsequent cycles, both adsorption and desorption capacities gradually decrease, with adsorption declining from 379.5 mg·g⁻¹ in the second cycle to 336.8 mg·g⁻¹ in the fifth cycle; desorption likewise decreases from 358.4 mg·g⁻¹ to 282.7 mg·g⁻¹. The desorption efficiencies reduce from 94.5 to 84.0%, suggesting a progressive reduction in reversible enzyme binding. This decrease can be attributed to partial retention of catalase within the cryogel pores, minor structural compaction, or a gradual weakening of Cu(II)–enzyme interactions after repeated washing steps^38,39^. Despite these reductions, the Poly(HEMA-co-GMA)-PEI-Cu(II) cryogel maintains over 85% of its initial immobilization capacity after five cycles, demonstrating notable operational stability. The sustained performance underscores the robustness of the Cu(II)-chelated PEI network and its retention of enzyme-binding functionality across multiple regeneration cycles.
Comparative evaluation of catalase immobilization performance across different cryogel systems
A cross-comparison of various cryogel matrices demonstrates marked differences in immobilization capacity, catalytic efficiency, and operational stability of immobilized catalase (Table 2). Among the reported systems, Poly(HEMA-co-GMA)–PEI–Cu(II) exhibits the highest immobilization capacity (391.9 mg g⁻¹), surpassing both metal-free cryogels and previously reported functional materials such as p(HEMA)-BPCGO^18^ and Fe³⁺-poly(AAm-GMA)-IDA^40^. This superior loading capacity can be attributed to the synergistic effects of PEI’s high amine density and the strong coordination interactions provided by Cu(II), which together generate abundant, high-affinity binding sites for catalase.
Table 2. Comparative immobilization performance of catalase on various cryogel matrices.CryogelTypeIC(mg·g^− 1^) K m (mM)(Free / Imm) V max (µmol·min^− 1^)(Free / Imm)OSSSCRRefs.p(HEMA)-BPCGO261.7 ± 11.29.9 / 11357.1 / 769.2100% (1st use), 34.4% (15th use)65.12% (at 4 °C, 15 days)100% (1st use), 90.1% (5th use)^18^Poly(HEMA-GMA)298.7 ± 9.910.52 / 5.4310,000 / 2500100% (1st use); 31.0% (15th use)70.0% (at 4 °C, 7 days)100% (1st use), 87.21% (5th use)^20^p(HEMA-co-AGE)49.3 ± 1.246 / 19330 / 8.7–No significantdecrease after 45 days100% (1st use), 93.8 ± 1.2% (5thuse)^41^Fe³⁺-poly(AAm-GMA)-IDA12.99––––100% (1st use), 96.7% (40th use)^40^Poly(HEMA-co-AGE)-250356.3 ± 3.654.9 / 17.12433 / 1108100% (1st use); 33.1% (15th use)80.6% (at 4 °C, 35 days)100% (1st use), 85.69% (5th use)^24^Poly(HEMA-co-GMA)-PEI-Cu(II)391.9 ± 2.857.3 / 14.42521 / 1251100% (1st use); 34.2% (15th use)81.3% (at 4 °C, 35 days)100% (1st use), 83.95% (5th use)This studyIC: Immobilization capacity, Imm: Immobilized, OS: Operational Stability, SS: Storage Stability, CR: Cryogel Reusability, BPCGO: 4-biphenylchloroglyoxime, IDA: iminodiacetic acid, APTAC: (3-acrylamidopropyl) trimethylammonium chloride, AMPS: 2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt.
Kinetic analyses further highlight the efficiency of metal-chelated systems. The Km value of immobilized catalase on Poly(HEMA-co-GMA)–PEI–Cu(II) (14.4 mM) is significantly lower than that of the free enzyme (57.3 mM), indicating enhanced substrate affinity and a more favorable microenvironment. Although a decrease in Vmax is observed upon immobilization (2521 → 1251 µmol min⁻¹), this trend is consistent with immobilized enzyme systems and likely reflects diffusional restrictions within the cryogel network rather than intrinsic catalytic impairment. When compared to other cryogels, the kinetic enhancements in the Cu(II)-modified matrix are comparable to those of Poly(HEMA-co-AGE)-250^24^ and notably superior to those of p(HEMA-co-AGE)^41^, which exhibits a drastic Vmax reduction due to its less favorable pore structure and weaker binding interface.
In terms of operational stability, Poly(HEMA-co-GMA)-PEI-Cu(II) retains 34.2% activity after 15 consecutive cycles, a performance comparable to other cryogels, such as Poly(HEMA-GMA) (31.0%)^20^ and Poly(HEMA-co-AGE)-250 (33.1%)^24^. The observed decline is characteristic of repeated-use enzyme systems and may be attributed to partial leaching and conformational fatigue. Nevertheless, the sustained catalytic activity surpasses that of several metal-free matrices and indicates that Cu(II)-chelation provides structural reinforcement that slows activity loss during repeated cycling.
Storage stability exhibits similar trends. The Poly(HEMA-co-GMA)-PEI-Cu(II) cryogel preserves 81.3% of its initial activity after 35 days at 4 °C, outperforming the simpler Poly(HEMA-GMA) network (70% after 7 days)^20^ and closely matching the more robust Poly(HEMA-co-AGE)-250 (80.6% after 35 days)^24^. These results confirm that metal-ion crosslinking provides a stabilizing effect, protecting catalase from structural degradation during prolonged storage.
Reusability studies reveal high retention of catalytic activity during the first five cycles across all cryogels (≥ 83.95%), with Fe³⁺-poly(AAm-GMA)-IDA^40^ showing exceptional long-term reuse performance (96.7% activity after 40 cycles). Although Poly(HEMA-co-GMA)-PEI-Cu(II) does not reach this extreme durability, its strong performance within the first five cycles demonstrates reliable structural integrity and minimal enzyme desorption during repeated operations.
Collectively, these findings demonstrate that Poly(HEMA-co-GMA)-PEI-Cu(II) represents one of the most efficient cryogel systems for catalase immobilization reported to date, integrating high immobilization capacity with improved substrate affinity, robust storage stability, and competitive operational performance. The synergistic incorporation of PEI and Cu(II) into the Poly(HEMA-co-GMA) framework markedly enhances the physicochemical properties of the cryogel, rendering it particularly suitable for industrial biocatalytic applications that require repeated use, long-term storage, and high enzyme loading. Importantly, as summarized in Table 2, comparative analyses of activity, stability, and reusability reveal that catalase immobilized on Poly(HEMA-co-GMA)-PEI-Cu(II) consistently exhibits superior retained catalytic performance relative to the Ni(II)- and Co(II)-functionalized supports, indicating that the selection of this support was driven not only by immobilization capacity but also by favorable enzyme orientation and three-dimensional microenvironmental effects.
Conclusion
This study demonstrates the successful fabrication of a high-performance catalase immobilization platform based on Poly(HEMA-co-GMA) cryogels functionalized with polyethyleneimine and transition-metal ions. Extensive characterization confirmed that PEI grafting significantly increased the hydrophilicity and reactive amine density of the cryogel network. At the same time, metal chelation, especially with Cu(II), introduced strong coordination sites that preserved the macroporous architecture and enhanced binding affinity. Among the examined systems, the Cu(II)-modified cryogel exhibited the highest water retention capacity (438.4%) and the most significant catalase loading (391.9 mg·g⁻¹), outperforming both Ni(II)- and Co(II)-chelated matrices. Moreover, immobilized catalase on Poly(HEMA-co-GMA)-PEI-Cu(II) displayed markedly improved kinetic behavior, with Km decreasing from 57.3 to 14.4 mM, indicating enhanced substrate affinity. The immobilized enzyme also maintained 34.2% of its initial activity after 15 operational cycles. It retained 62.1% of its activity after 70 days of storage at 4 °C, demonstrating strong long-term stability and structural protection provided by the cryogel microenvironment.
Overall, these findings highlight Poly(HEMA-co-GMA)-PEI-Cu(II) as a robust, reusable, and high-capacity immobilization matrix that supports efficient catalysis under both operational and storage conditions. This cryogel platform shows significant potential for scale-up and integration into continuous bioprocesses, offering a promising foundation for next-generation immobilized enzyme technologies.