Magnetically recyclable co-immobilized laccase–glucose oxidase nanobiocatalyst for sustainable cleaning of fouled polypropylene membranes

Membrane fouling remains a major challenge limiting the efficiency and sustainability of membrane-based water and wastewater treatment systems. Therefore, the development of effective and reusable biocatalytic cleaning systems is essential for improving membrane performance and extending membrane operational lifespan. In this study, laccase and glucose oxidase were co-immobilized onto chitosan-coated magnetic iron oxide nanoparticles using the combined cross-linked enzyme aggregate (combi-CLEAs) approach for cleaning organically fouled polypropylene membranes. Maximum enzyme loading values of 189.56 U mg −1 for laccase and 185.69 U mg −1 for glucose oxidase were obtained under optimized immobilization conditions. The immobilized enzymes exhibited improved pH and thermal stability and retained 69.24% and 77.89% residual activity for laccase and glucose oxidase, respectively, after ten reuse cycles. Following enzymatic treatment, membrane volumetric flux increased nearly three-fold from 32.48 ± 0.31 to 108.40 ± 1.31 L m −2 h −1. In addition, significant reductions in adenosine triphosphate, total organic carbon, and chemical oxygen demand were observed after treatment, indicating effective removal of biomass-associated and organic foulants. The results demonstrate the potential applicability of co-immobilized oxidoreductase nanobiocatalysts as sustainable membrane-cleaning systems.

Membrane-based separation technologies have become indispensable in modern water and wastewater treatment processes because of their high separation efficiency, compact design, and ability to produce high-quality permeate (Osman et al., 2024). These systems are extensively employed in desalination, industrial wastewater reclamation, food processing, pharmaceutical purification, and municipal water treatment (Tripathy et al., 2025). Despite their widespread applicability, membrane fouling remains one of the most critical operational challenges limiting long-term process efficiency and economic sustainability (Ahmed et al., 2023). Fouling occurs through the accumulation of proteins, polysaccharides, humic substances, microorganisms, extracellular polymeric substances (EPS), colloidal particles, and other organic contaminants on membrane surfaces and within membrane pores, resulting in severe permeability decline, increased transmembrane pressure, elevated energy consumption, and shortened membrane lifespan (Lin et al., 2024).

Conventional fouling mitigation strategies primarily rely on physical flushing and chemical cleaning using acidic, alkaline, oxidizing, or surfactant-based reagents. Although these approaches can temporarily restore membrane permeability, repeated chemical exposure may accelerate membrane deterioration, alter membrane surface chemistry, increase operational costs, and generate environmentally hazardous secondary waste streams. Furthermore, aggressive cleaning conditions often fail to completely remove strongly adhered organic foulants and biofilm-associated matrix components, leading to irreversible fouling and progressive decline in membrane performance (Ahmed et al., 2025). Consequently, the development of environmentally sustainable and membrane-compatible cleaning strategies has become an important research priority in membrane process engineering (Rajendran et al., 2025).

In recent years, enzymatic membrane cleaning has emerged as a promising alternative owing to its substrate specificity, biodegradability, and effectiveness under mild operating conditions (Pant et al., 2023). Enzymes are capable of selectively degrading organic foulants and biofilm matrix components without requiring harsh cleaning conditions that may damage membrane materials (Al-Balushi et al., 2024). Recent studies have increasingly explored enzymatic membrane cleaning using lipase (Mulinari et al., 2024), α-amylases (Khani et al., 2023), and oxidoreductases (Cruz-Balladares et al., 2025) for degradation of proteinaceous, polysaccharide-rich, and biofilm-associated foulants. Matrix-degrading enzyme systems have demonstrated effective reduction of membrane biofilms and foulant accumulation, highlighting their potential as environmentally sustainable alternatives for membrane fouling mitigation (Myshkevych et al., 2025). However, currently reported systems predominantly rely on free enzymes or single-enzyme immobilization approaches, which often suffer from poor operational stability, enzyme leaching, limited reusability, and insufficient catalytic durability during repeated cleaning cycles (Fahim et al., 2025).

Among oxidoreductase enzymes, laccase and glucose oxidase have attracted considerable attention because of their broad catalytic versatility and environmental applications. Laccase catalyzes the oxidation of phenolic and aromatic compounds using molecular oxygen as the terminal electron acceptor, enabling degradation of structurally complex organic contaminants (Zhang et al., 2025). Glucose oxidase catalyzes oxidation of glucose into gluconic acid while generating hydrogen peroxide, thereby potentially contributing to oxidative destabilization of organic foulants (Behary et al., 2025). The selection of laccase and glucose oxidase in the present study was therefore based on their complementary oxidoreductase activities toward degradation of heterogeneous organic membrane foulants. Co-immobilization of both enzymes was intended to improve foulant degradation, membrane flux restoration, operational stability, and reusability compared with single-enzyme systems.

Previous studies have explored combined laccase–glucose oxidase systems in biosensing platforms, lignin transformation, contaminant degradation, and bioelectrochemical systems (Aljabri, 2025). However, their synergistic application as a co-immobilized oxidoreductase cascade system for membrane fouling mitigation and membrane cleaning remains insufficiently investigated. Multi-enzyme cascade systems offer important advantages over single-enzyme systems by enabling sequential and cooperative catalytic reactions within a unified reaction environment. Such systems improve catalytic efficiency, minimize intermediate accumulation, and facilitate degradation of structurally heterogeneous foulants (Cai et al., 2025). To replicate these synergistic catalytic interactions under heterogeneous conditions, combined cross-linked enzyme aggregates (combi-CLEAs) have emerged as an effective immobilization strategy (Stalter et al., 2026). Combi-CLEAs involve simultaneous immobilization of multiple enzymes into a cross-linked catalytic network, thereby improving enzyme stability, operational durability, catalytic retention, and recyclability while reducing enzyme leaching.

Furthermore, immobilization onto magnetic nanomaterials provides additional benefits including high surface area, improved enzyme dispersion, facile catalyst recovery, and enhanced operational reuse (Patti et al., 2025). Although previous studies have explored enzyme cocktails, oxidoreductase-assisted fouling mitigation, and immobilized nanobiocatalysts for wastewater treatment applications, investigations involving co-immobilized laccase–glucose oxidase Combi-CLEAs for reusable membrane-cleaning systems remain limited. Despite recent advances in enzymatic membrane cleaning, several important challenges remain unresolved. In particular, the relationship between co-immobilized enzymatic oxidation, fouling layer degradation, and membrane flux restoration remains insufficiently understood. Moreover, limited information is available regarding the operational stability, recyclability, and synergistic catalytic behavior of co-immobilized oxidoreductase systems during repeated membrane-cleaning cycles.

Therefore, the present study aimed to develop a co-immobilized laccase–glucose oxidase Combi-CLEA system using chitosan-coated magnetic Fe 3 O 4 nanoparticles and evaluate its effectiveness for cleaning organically fouled polypropylene membranes. Unlike previously reported single-enzyme cleaning systems, the proposed nanobiocatalytic platform integrates multi-enzyme cascade catalysis, Combi-CLEA stabilization, and magnetic nanocarrier-assisted recovery within a unified membrane-cleaning system. The study further evaluates the catalytic performance, operational stability, reusability, and membrane flux restoration capability of the co-immobilized enzyme system toward degradation of organic foulants. This work therefore addresses an important gap in the development of reusable oxidoreductase nanobiocatalysts for sustainable membrane fouling mitigation applications.

Chemicals and reagents

Fouled and fresh polypropylene membranes (molecular weight cut-off, MWCO: 200 Da) were procured from domestic water purification systems (Chennai, India). Laccase from Trametes versicolor (≥10 U mg -1) and glucose oxidase from Aspergillus niger (Type X-S, ≥100,000 U g -1 solid) were purchased from Sigma-Aldrich (United States). Ferrous sulfate heptahydrate (FeSO 4 ·7H 2 O, ≥99% purity), ferric chloride hexahydrate (FeCl 3 ·6H 2 O, ≥98% purity), chitosan (low molecular weight, ≥75% deacetylation), glutaraldehyde solution (25%, v/v), ammonium sulfate (≥99.5% purity), acetic acid (≥99.7% purity), sodium hydroxide (≥98% purity), ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ≥98% purity], p-benzoquinone (≥98% purity), D-glucose (≥99.5% purity), and bovine serum albumin (BSA) were obtained from Sigma-Aldrich, HiMedia, or SRL Chemicals. All chemicals used in the study were of analytical grade and used without further purification. All solutions and buffers were prepared using ultrapure deionized water.

Synthesis of chitosan-coated iron oxide nanoparticles

Chitosan-coated iron oxide nanoparticles (CS-IONs) were synthesized using a modified co-precipitation method. Briefly, FeSO 4 ·7H 2 O (0.69 g) and FeCl 3 ·6H 2 O (1.40 g) were dissolved in 25 mL deionized water under continuous stirring, followed by dropwise addition of ammonium hydroxide (1.75 mL) to induce formation of iron oxide nanoparticles. The resulting magnetic precipitates were magnetically separated, washed repeatedly with deionized water until neutral pH, and dried at 70 °C for 2 h. For chitosan coating, chitosan dissolved in 0.2 M acetic acid was mixed with iron oxide nanoparticles at a 1:1 (w/w) ratio and sonicated using a probe ultrasonicator (40 kHz, 30 min, 37 °C). Subsequently, 1 M NaOH was added to precipitate chitosan-coated nanoparticles. The resulting CS-IONs were dried at 60 °C, ground, and sieved through a 45 µm mesh prior to use (Rahmani et al., 2022).

Preparation of co-immobilized Lac–GOx Combi-CLEAs on CS-IONs

Laccase (10 mg mL -1) and glucose oxidase (0.1 mg mL -1) solutions were prepared in 0.1 M acetate buffer (pH 5.0). Co-immobilized cross-linked enzyme aggregates (Combi-CLEAs) were synthesized through enzyme adsorption onto CS-IONs followed by ammonium sulfate-induced aggregation and glutaraldehyde-mediated cross-linking. Briefly, 10 mg of CS-IONs were mixed with enzyme solution in acetate buffer and agitated at 20 °C for 90 min to facilitate enzyme–support interaction. Subsequently, ammonium sulfate precipitation (60% saturation) was carried out under continuous stirring for 120 min at 20 °C. Following enzyme aggregation, glutaraldehyde was added as cross-linking agent. Optimization studies were performed by varying glutaraldehyde concentration (1–10 mM), cross-linking duration (2–22 h), and enzyme-to-nanoparticle ratios (1:1–1:4, w/w). The optimized immobilization condition was selected based on maximum retained enzymatic activity and operational stability. The resulting Combi-CLEAs were recovered by magnetic decantation, washed three times with acetate buffer (0.1 M, pH 4.0) to remove unbound enzymes, and stored at 4 °C until further use (Talekar et al., 2013).

In the present study, immobilization optimization was primarily evaluated based on retained enzymatic activity and enzyme loading values (U mg -1) of laccase and glucose oxidase after immobilization under different experimental conditions, including glutaraldehyde concentration, cross-linking duration, and enzyme-to-support ratio. Enzyme loading was defined as the immobilized enzyme activity retained per unit mass of Combi-CLEA nanobiocatalyst and calculated using the following equation: where A i and A f represent initial and residual enzyme activities, respectively, and W s represents the weight of immobilized Combi-CLEA support.

Characterization of nanoparticles and Combi-CLEAs

Morphological characterization of CS-IONs and Combi-CLEAs was performed using scanning electron microscopy (SEM; Hitachi Ltd., Tokyo, Japan). Surface topography and roughness analysis were additionally examined using atomic force microscopy (AFM). Brunauer–Emmett–Teller (BET) analysis was conducted to determine surface area and porosity characteristics of CS-IONs. Elemental composition analysis was performed using FEI Quanta EDAX 200 FEG equipped with energy-dispersive X-ray spectroscopy (EDX). Functional group analysis was carried out using Fourier-transform infrared spectroscopy (FTIR; Agilent Cary 660 FTIR spectrometer) over the range of 4,000–400 cm -1 (He et al., 2021).

Characterization of free and immobilized enzymes

The optimum pH of free enzymes and Combi-CLEAs was evaluated over the pH range of 4.0–8.0 at 30 °C using appropriate buffer systems. Similarly, optimum temperature was determined by incubating enzyme preparations at temperatures ranging from 30 °C to 70 °C (Hürmüzlü et al., 2021). Thermal stability was evaluated by incubating free and immobilized enzymes at different temperatures for predetermined time intervals, followed by determination of residual enzyme activity under standard assay conditions. Thermal deactivation rate constant (k d) and half-life (t 1 / 2) were determined using first-order deactivation kinetics:

Kinetic parameters of free enzymes and Combi-CLEAs were determined using varying substrate concentrations (0.2–1.0 mM). ABTS was used as substrate for laccase, whereas glucose was used for glucose oxidase. Kinetic parameters including apparent Michaelis constant (K m), maximum reaction velocity (V max), turnover number (k c a t), and catalytic efficiency (k c a t / K m) were estimated using non-linear regression fitting of the Michaelis–Menten model. Lineweaver–Burk plots were used only for graphical representation of enzyme kinetics (Ademakinwa, 2021).

Laccase activity was determined spectrophotometrically at 420 nm using ABTS as substrate in 100 mM acetate buffer (pH 4.0). One unit (U) of laccase activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per minute under assay conditions (Kumar et al., 2014). Glucose oxidase activity was measured by monitoring hydroquinone formation during glucose oxidation using 0.1% (w/v) p-benzoquinone as electron acceptor in 50 mM sodium acetate buffer (pH 5.0) at 25 °C. One unit of glucose oxidase activity was defined as the amount of enzyme required to produce 1 µmol of product per minute (Ciucu and Pâtroescu, 1984).

Operational reusability of Combi-CLEAs was evaluated for ten consecutive cycles using 10 mg immobilized biocatalyst. After each cycle, Combi-CLEAs were magnetically recovered, washed three times with acetate buffer (0.1 M, pH 4.0), and reused under identical experimental conditions. Residual laccase and glucose oxidase activities were determined after each cycle using ABTS and glucose as respective substrates (Bai et al., 2022).

Residual activity (%) was calculated using: where A t represents activity after each reuse cycle and A 0 represents initial activity.

Enzymatic membrane cleaning

Fouled membranes were subjected to enzymatic cleaning using free enzymes and Combi-CLEAs separately. For free enzyme treatment, fouled membranes were incubated with mixed laccase and glucose oxidase solutions under agitation (150 rpm) for 6 h at room temperature. Similarly, immobilized enzyme treatment was performed by incubating fouled membranes with 10 mg Combi-CLEAs dispersed in 25 mL acetate buffer under identical conditions for 6 h. Hydrogen peroxide generation during enzymatic treatment was quantified spectrophotometrically at 260 nm using a UV–Visible spectrophotometer. Briefly, H 2 O 2 concentration was determined based on its reaction with sodium oxalate (16.34 mM) and cobalt chloride (67.8 mM) in the presence of saturated sodium bicarbonate at 25 °C (Ravi et al., 2020). After treatment, membranes were thoroughly washed with deionized water prior to further characterization and filtration analysis.

Membrane filtration and antifouling performance evaluation

Membrane filtration experiments were performed in a pressure-driven ultrafiltration system operated at 20 psi and 28 °C using an effective membrane area of 13.84 cm 2. Permeate flux was monitored under steady-state conditions at 10 min intervals (Luo et al., 2014).

Volumetric water flux (J w) was calculated using: where J w is volumetric flux (L m -2 h -1), Q is permeate volume (L), A is effective membrane area (m 2), and t is filtration time (h).

To evaluate membrane rejection performance, bovine serum albumin (BSA, 1 g L -1) prepared in phosphate-buffered saline (pH 6.0) was used as model foulant solution. Solute rejection (S R) was calculated using: where C p and C f represent permeate and feed concentrations, respectively.

Antifouling performance was evaluated through flux decline ratio (FDR) and flux recovery ratio (FRR). Initial pure water flux after membrane compaction was considered as J 0, whereas flux after fouling was represented as J i. FDR was calculated as:

Following enzymatic cleaning, membranes were rinsed and back-flushed with deionized water, and recovered flux (J r) was measured. FRR as cleaning efficiency was calculated as:

Evaluation of membrane fouling indicators

For ATP and TOC analyses, membrane samples were sectioned and suspended in 30 mL sterile water followed by sonication for 2 min using a bath sonicator to detach deposited organic matter (Sanawar et al., 2018). ATP extraction was performed using ATP extraction buffer, and luminescence was measured using a luminometer following reaction with luciferin–luciferase reagent. ATP concentration was quantified as pg cm -2 using calibration standards (Sanawar et al., 2018). ATP analysis was performed to evaluate biomass-associated fouling and microbial accumulation within the membrane fouling layer.

Total organic carbon was determined using a total organic carbon analyzer to quantify organic contaminants present on the membrane surface after cleaning treatment. Together, adenosine triphosphate and total organic carbon analyses were employed to assess the effectiveness of enzymatic cleaning toward removal and degradation of biomass-associated and organic foulants in addition to membrane flux restoration. Chemical oxygen demand was measured using the dichromate digestion method involving potassium dichromate and sulfuric acid digestion at 150 °C for 2 h, followed by absorbance measurement at 600 nm (da Silva et al., 2011).

Statistical analysis

All experiments were independently performed in triplicate (n = 3), and results are presented as mean ± standard deviation (SD). Significant differences among experimental groups were analyzed using one-way or two-way analysis of variance (ANOVA) followed by Tukey’s or Sidak’s post hoc multiple comparison test where appropriate. Statistical significance was considered at p < 0.05.

Optimization of immobilization parameters for Combi-CLEAs preparation

A preliminary sorption step was performed by incubating laccase, glucose oxidase, and chitosan-coated iron oxide nanoparticles (CS-IONs) in 0.1 M acetate buffer (pH 4.0) at 25 °C for 2 h under intermittent mixing prior to ammonium sulfate precipitation. Subsequently, glutaraldehyde-mediated cross-linking was carried out to form the Combi-CLEA nanobiocatalyst. Inclusion of the sorption step improved enzyme loading, yielding 144.48 U mg -1 for laccase and 123.53 U mg -1 for glucose oxidase, whereas omission of the sorption step reduced enzyme loading to 85.36 U mg -1 and 79.63 U mg -1, respectively. These results indicate that preliminary enzyme adsorption enhanced immobilization efficiency and catalytic retention on the CS-ION surface.

The effect of cross-linking duration on enzyme loading is presented in Figure 1a. The optimum cross-linking time was 2 h, resulting in maximum enzyme loading values of 189.56 U mg -1 for laccase and 185.69 U mg -1 for glucose oxidase. Increasing the cross-linking duration to 6 h and 22 h progressively reduced enzyme loading, likely due to excessive cross-linking, increased structural rigidity, and restricted substrate diffusion within the Combi-CLEA matrix. The influence of glutaraldehyde concentration on enzyme loading was also investigated (Figure 1b). The optimum concentration was 5 mM, producing the highest enzyme loading values for both enzymes. Lower glutaraldehyde concentration (1 mM) resulted in insufficient enzyme stabilization, whereas higher concentration (10 mM) reduced enzyme loading, possibly due to excessive cross-linking and steric hindrance affecting catalytic site accessibility. A study reported the maximum activity recovery of the laccase was obtained with 5 mM glutaraldehyde concentration. Utilizing glutaraldehyde concentrations of 10 and 20 mM, P. ostreatus laccase recovered its greatest amount of activity in combinations containing t-butanol and aqueous butanol (Barbosa et al., 2014).

The effect of enzyme-to-support ratio on Combi-CLEA formation was evaluated by varying the chitosan-coated magnetic nanoparticle-to-enzyme ratio from 1:1 to 4:1 (Figure 1c). The optimum ratio was observed at 2:1, yielding the highest enzyme loading values of 189.56 U mg -1 for laccase and 185.69 U mg -1 for glucose oxidase. Further increase in support concentration resulted in reduced enzyme loading. At a ratio of 3:1, enzyme loading decreased to 136.24 U mg -1 for laccase and 174.56 U mg -1 for glucose oxidase, while at 4:1, the loading values further declined to 132.45 U mg -1 and 161.28 U mg -1, respectively. The reduction in enzyme loading at higher support ratios may be attributed to excessive enzyme distribution over the support surface, leading to reduced enzyme packing density and lower effective catalytic interaction with substrates. The immobilization process was optimized based on retained catalytic activity and enzyme loading of the Combi-CLEA nanobiocatalyst under different immobilization conditions. Higher enzyme loading values obtained after the preliminary sorption step indicated improved enzyme adsorption and catalytic retention on the chitosan-coated magnetic nanoparticles.

SEM assessed the morphology of the synthesized chitosan-coated IONs and enzyme immobilized on chitosan-coated IONs (combi-CLEAs) with EDX. The SEM of the nanoparticles and combi-CLEAs revealed the dispersed patterns of particles in the form of aggregates that are not uniformly distributed. The tiny white dots on the particles’ surface may represent iron oxide embedded by the enzymes (laccase and glucose oxidase), which gives the immobilized enzymes their magnetic property (See Supplementary Material).

AFM analysis of the Combi-CLEA nanobiocatalyst revealed a rough, heterogeneous, and aggregated surface morphology with irregular elevated domains. The bright regions represent raised enzyme–nanoparticle aggregates, while the darker regions indicate surface depressions or inter-aggregate spaces. This uneven topography suggests successful formation of cross-linked enzyme aggregates on the chitosan-coated magnetic nanoparticle surface. The rough and compact morphology may provide increased surface contact area for enzyme–substrate interaction and contribute to improved catalytic stability and reusability of the immobilized system (See Supplementary Material).

The surface area of the chitosan-coated IONs was 49.8 m 2 /g, and the pore volume and pore size were evaluated as 1.11 cm 2 /g and 17.3 nm, respectively. A study evaluated the magnetic nanoparticle’s particle size between 10 and 20 nm, exhibiting a spherical or elliptical form at the nanoscale (Liu et al., 2022). The improved support has remained spherical or elliptical, but the nanoparticles’ average particle size is approximately 1.56 nm larger compared to ferrous nanoparticles at 23.48 nm.

Fourier transform infrared spectroscopy (FTIR), which is used to identify the chemical linkage in compounds and the functional groups on the nanoparticles. The FTIR spectra for chitosan-coated IONs and enzymes immobilized on the chitosan-coated IONs were determined to have a range of 4,000–400 cm -1 (See Supplementary Material). Peaks at 1,421 cm -1 and 1,153 cm -1 in the IR region of chitosan’s spectra are due to carbon-nitrogen stretches and the hydroxyl bending of ten alcohols. Chitosan has been effectively coated in magnetic nanoparticles, as evidenced by the peak at 1,615.15 cm -1, which is ascribed to the bond in the amino radical in nanoparticles. The carbonyl group’s (C=O) extension shows a peak at 1,650.13 cm -1. Peaks related to C-H linkages can be observed at 2,875 cm -1, N-H bonds at 1,650 cm -1, and ether bonds at 1,066 cm -1 for the analysis of chitosan.

Enzyme kinetics and stability studies of free and coimmobilized enzyme

The optimum pH for free laccase and glucose oxidase was observed at pH 5.0, whereas the immobilized combi-CLEAs exhibited maximum activity at pH 6.0 (Figure 2a). At pH 8.0, free laccase and glucose oxidase retained only 31.82% and 35.24% relative activity, respectively, while the immobilized enzymes retained substantially higher activities of 72.36% and 70.25%, respectively. The improved pH tolerance of the combi-CLEAs may be attributed to enhanced structural stabilization and protection of the enzyme microenvironment provided by the cross-linked immobilization matrix. In heeding the statement, combi-CLEAs laccase has a pH shift from 4.5 pH to 5.0 pH than the free enzyme shown by (Zheng et al., 2016). The pH shift is due to the conformational changes in the structure of the insolubilized enzyme, making them more resistant to pH changes than the free enzyme (Yuan et al., 2016).

The optimum temperature for free laccase and glucose oxidase was 30 °C, whereas the immobilized Combi-CLEAs exhibited maximum activity at 40 °C (Figure 2b). At 70 °C, free laccase and glucose oxidase retained only 32.58% and 36.58% relative activity, respectively, while the immobilized enzymes maintained significantly higher residual activities of 63.21% and 68.21%, respectively. The enhanced thermal stability of the Combi-CLEAs may be attributed to structural stabilization and reduced conformational flexibility provided by the cross-linked immobilization matrix, resulting in a broader operational temperature range compared with free enzymes (Mazlan and Hanifah, 2017). Enzymes experience enhanced energy uptake, leading to destructive collisions that cause enzyme denaturation and disrupt their attachment to carrier matrix structures (Mazlan and Hanifah, 2017). Another study reported that the free enzyme loses roughly 60% of its catalytic activity at 60 °C. Still, the activities of the immobilized glucose oxidase catalysts only decline by 4%–18%, depending on the catalyst type (Jaquish et al., 2018).

Over the temperature range of 30 °C, 40 °C, and 50 °C, the stability of free and immobilized enzymes for 780 min has been investigated. At 30 °C, the half-life of the combi-CLEA enzyme was more than the free enzyme. The free laccase and glucose oxidase half-life was 198 min and 130.75 min, respectively, whereas the co-immobilized enzyme had a half-life of 535 min for combi-CLEA laccase and 512 min for combi-CLEAs glucose oxidase. On increasing the temperature to 40 °C, the free enzyme’s half-life was 157.5 min and 97.6 min for laccase and glucose oxidase, respectively. In contrast, co-immobilized enzymes have a higher half-life than the free form, accounting for 433.12 min and 330 min for combi-laccase and combi-CLEA glucose oxidase, respectively (Figure 2c).

Even on increasing the temperature to 50 °C, the stability of co-immobilized CLEAs displays a half-life of 315 min and 256.6 for combi-laccase and combi-CLEAs glucose oxidase, respectively. In contrast, the free enzyme took a deep plunge in half-life rendering 110 min for laccase and 75.36 min for glucose oxidase. The Staphylococcus spp. enzyme was immobilized using the CLEA technique utilizing graphene oxide and magnetite nanoparticles which were cross-linked with glutaraldehyde (Doraiswamy et al., 2020). As a result, it has been demonstrated that magnetite and graphene oxide-CLEAs have improved stability throughout a broad temperature and pH range, as well as improved reusability and storage stability.

The substrate kinetic study for both forms of enzymes was studied (Table 1). For free enzyme, the Km value was found to be 0.12 mM (laccase) and 0.058 mM (glucose oxidase), while co-immobilized enzyme has 0.9 mM (combi-laccase) and 0.38 mM (combi-glucose oxidase). The decrease in enzyme flexibility required for binding substrates, which results in increased steric hindrance and diffusional substrate limitation, is depicted by the rise in Km value for immobilized enzymes (Rouhani et al., 2018). The Vmax value for the free enzyme was 250.23 U/min and 181.81 U/min for laccase and glucose oxidase, respectively, whereas the co-immobilized enzymes, namely combi-laccase and combi-glucose oxidase had 294.11 U/min and 250.31 U/min, respectively.

The turnover number (K cat) and the catalytic efficiency (K cat/ K m) have also been observed. The K cat and K cat/ K m values for the immobilized enzyme were higher than the free enzyme indicating the conformational changes due to the immobilization rendering the insolubilized enzymes more available to the substrate. The turnover number for free laccase and immobilized laccase was 0.92 min -1 and 1.09 min -1, respectively, whereas for the free and immobilized glucose oxidase was 2.09 min -1 and 1.5 min -1, respectively. The catalytic efficiency was found to be 1.03 mM/min, 2.86 mM/min, 17.39 mM/min, and 28.63 mM/min for free laccase, free glucose oxidase, combi-laccase, and combi-glucose oxidase, respectively. A similar study found that the K cat/ K m was increased by 14% more than the free enzyme, indicating that crosslinking increases the enzyme’s catalytic efficiency (Hong et al., 2021).

The increase in apparent K m values observed after immobilization suggests reduced substrate affinity compared with free enzymes, which may be associated with diffusional limitations and restricted substrate accessibility within the cross-linked Combi-CLEA matrix. The immobilized enzyme structure can introduce internal mass-transfer resistance, thereby limiting diffusion of substrates toward active catalytic sites. In addition, the cross-linked network and immobilization onto chitosan-coated Fe 3 O 4 nanoparticles may alter the local enzyme microenvironment, including conformational flexibility, local pH, and substrate transport behavior. Nevertheless, the higher apparent V max values observed for Combi-CLEAs may indicate improved structural stabilization and enhanced catalytic retention of the immobilized enzymes under operational conditions. Therefore, the observed kinetic behavior likely reflects the combined influence of enzyme stabilization, substrate diffusion resistance, and altered microenvironmental conditions within the immobilized enzyme matrix (Hong et al., 2021).

Immobilized enzymes may be reused repeatedly, continuously, or in batches, which lowers manufacturing costs and is a significant factor for industrial applications. The combi-CLEAs were able to retain their enzyme activity till 10th cycle. At the end of the 10th cycle, 69.24% and 77.89% of residual activity accounted for combi-CLEA laccase and combi-CLEA glucose oxidase, respectively (Figure 2d). The enzyme denaturation and active site deformation carried on by repeated substrate encounters may cause the immobilized enzymes’ reduced activity. The gradual decline in enzymatic activity during repeated cycles may be attributed to partial enzyme deactivation, conformational alterations, and reduced catalytic accessibility caused by repeated washing and operational handling. Beyond repeated reuse cycles, the catalytic activity progressively decreased toward lower operationally effective levels, which may potentially limit membrane-cleaning efficiency during prolonged operation. Nevertheless, the retained catalytic activity after ten cycles indicates that the Combi-CLEA system possesses considerable reusability and operational durability for membrane-cleaning applications. Further investigations involving extended cyclic operation and quantitative enzyme leaching analysis would provide deeper insight into long-term catalyst stability and industrial applicability of the immobilized nanobiocatalyst system.

Membrane filtration performance after enzymatic cleaning

Fresh membrane volumetric flux was determined as 114.22 ± 0.37 L m -2 h -1, whereas the fouled membrane exhibited a significantly reduced flux of 32.48 ± 0.31 L m -2 h -1 due to accumulation of organic foulants on the membrane surface (Figure 3a). Following enzymatic treatment, the flux of the laccase-treated membrane increased to 98.20 ± 0.29 L m -2 h -1. Similarly, treatment with free glucose oxidase restored the membrane flux to 81.97 ± 0.72 L m -2 h -1, accompanied by hydrogen peroxide generation of 544.21 mg L -1 during the enzymatic reaction. Among the enzymatic systems, the Combi-CLEA treatment exhibited the highest flux recovery, resulting in a volumetric flux of 108.40 ± 1.31 L m -2 h -1. The substantial increase in water flux after enzymatic treatment indicates effective restoration of membrane permeability following foulant removal.

The enzymatic treatment was further compared with conventional chemical cleaning methods. Acid and alkali treatments resulted in volumetric flux values of 113.14 ± 0.25 L m -2 h -1 and 117.40 ± 0.37 L m -2 h -1, respectively, with corresponding flux decline ratios of 21.2% ± 0.15% and 19.2% ± 0.31%. Although chemical cleaning exhibited comparatively higher immediate flux recovery, the enzymatic treatment demonstrated efficient membrane cleaning under comparatively milder operating conditions. Previous studies have similarly reported improved membrane permeability following enzymatic cleaning using enzyme cocktail systems for membrane fouling mitigation (Rudolph et al., 2018).

The solute rejection of the fouled membrane decreased to 43.26% ± 0.92% compared with 76.40% ± 1.33% for the fresh membrane. After enzymatic treatment, the solute rejection increased to 50.70% ± 0.78%, 59.65% ± 0.89%, and 57.60% ± 1.29% for free laccase, free glucose oxidase, and Combi-CLEAs, respectively. In comparison, acid- and alkali-treated membranes exhibited rejection values of 50.60% ± 0.24% and 51.10% ± 0.87%, respectively (Figure 3a). The increase in solute rejection after enzymatic treatment suggests partial restoration of membrane filtration performance following removal of deposited foulants. Similar improvements in membrane rejection after fouling mitigation treatments have also been reported previously (Krishnan et al., 2022).

Cleaning efficiency and antifouling behavior

The cleaning efficiency or flux recovery ratio of free and immobilized enzymes was evaluated using BSA solution (0.1%) as model foulant (Figure 3b). The fresh membrane exhibited a cleaning efficiency of 98.14% ± 0.28%, whereas the fouled membrane showed only 16.35% ± 0.91% flux recovery due to severe membrane fouling and increased hydraulic resistance. Following enzymatic treatment, the cleaning efficiency increased to 83.14% ± 0.35% for free laccase treatment. In the case of free glucose oxidase, the cleaning efficiency was determined as 82.71% ± 0.98%. Meanwhile, the Combi-CLEA system achieved the highest cleaning efficiency of 85.90% ± 0.48%, indicating improved foulant removal efficiency by the co-immobilized enzyme system.

The antifouling performance of the treated membranes was further evaluated through flux decline analysis. The fresh and fouled membranes exhibited flux decline values of 69.76% ± 0.21% and 71.20% ± 0.20%, respectively. After treatment, the flux decline ratios decreased to 28.2% ± 0.25% and 34.5% ± 0.36% for free laccase- and free glucose oxidase-treated membranes, respectively, whereas the Combi-CLEA-treated membrane exhibited the lowest flux decline of 24.5% ± 0.80%. The reduced flux decline after enzymatic treatment suggests effective removal of deposited foulants and reduced pore blockage on the membrane surface. Similar reductions in membrane fouling after enzymatic cleaning have been previously reported for protein-rich foulant systems (Li et al., 2020).

Compared with enzymatic cleaning, chemical treatments using acid and alkali solutions achieved cleaning efficiencies of 88.14% ± 0.65% and 83.10% ± 0.59%, respectively. Although chemical cleaning exhibited comparatively higher immediate cleaning performance, repeated exposure to aggressive chemical reagents may potentially contribute to membrane surface alteration and reduced membrane lifespan. In contrast, enzymatic cleaning provides an environmentally compatible cleaning strategy because of its biodegradable nature, catalytic specificity, and operation under relatively mild conditions. Therefore, the present findings suggest that Combi-CLEAs may serve as a sustainable alternative cleaning strategy with additional advantages including magnetic recoverability and operational reusability.

Mechanistic interpretation of Combi-CLEA membrane cleaning

The enhanced cleaning performance of the co-immobilized Combi-CLEA system can be attributed to the complementary catalytic activities of laccase and glucose oxidase toward degradation of organic membrane foulants. Membrane fouling layers typically consist of proteins, polysaccharides, humic-like substances, lipids, and aromatic compounds that reduce membrane permeability through surface deposition and pore blockage. In the present system, glucose oxidase catalyzed glucose oxidation, generating gluconic acid and hydrogen peroxide, thereby creating a localized oxidative environment capable of destabilizing proteinaceous and carbohydrate-rich foulants. Simultaneously, laccase catalyzed oxidation of phenolic and aromatic compounds, promoting structural modification and reduced foulant adhesion on the membrane surface.

Furthermore, co-immobilization on chitosan-coated Fe 3 O 4 nanoparticles improved enzyme stability, catalytic proximity, and operational durability, thereby contributing to enhanced foulant degradation and membrane flux recovery compared with free enzyme systems. The membrane cleaning performance observed in this study was closely associated with the retained catalytic activity of the immobilized enzyme system. The Combi-CLEAs retained 69.24% laccase activity and 77.89% glucose oxidase activity after the 10th reuse cycle, indicating substantial operational stability during repeated membrane-cleaning operations. The preserved catalytic activity enabled continuous degradation and destabilization of accumulated organic foulants, thereby contributing to the nearly three-fold increase in volumetric flux and reduction in total organic compounds after treatment.

Although hydrogen peroxide generation was detected during enzymatic treatment, the present study did not directly quantify hydroxyl radical formation or Fe 2+ /Fe 3+ redox cycling associated with Fenton-like reactions. Therefore, the involvement of reactive oxygen species in foulant degradation can only be considered as a possible contributing mechanism rather than a conclusively established pathway. The observed membrane cleaning performance is primarily attributed to the synergistic catalytic activities of laccase and glucose oxidase toward degradation and destabilization of organic foulants. Further mechanistic investigations involving radical detection assays, scavenger studies, and iron redox analysis would provide deeper understanding of the oxidative pathways associated with the Combi-CLEA system.

The improved membrane permeability, cleaning efficiency, and antifouling performance were observed after enzymatic treatment, several limitations remain in the present study. Detailed analyses of long-term fouling resistance, transmembrane pressure stability, and membrane surface morphology after repeated cleaning cycles were not performed. Therefore, further investigations involving SEM, AFM, cyclic fouling experiments, and long-term operational stability analyses are necessary to comprehensively evaluate membrane structural integrity and durability during repeated enzymatic cleaning operations. In addition, future studies involving advanced mechanistic investigations and techno-economic analysis would further strengthen the practical applicability of the Combi-CLEA membrane-cleaning system for large-scale membrane fouling mitigation applications.

Removal of organic compounds by enzymatic treatment

The accumulation of biomass and organic foulants on the membrane surface was evaluated using ATP, TOC, and COD analyses (Figure 4). The ATP concentration of the fouled membrane prior to cleaning was 8,100 ± 0.34 pg cm -2, indicating substantial biomass accumulation within the fouling layer. Following enzymatic treatment, the ATP concentration decreased to 3,128 ± 0.12 pg cm -2 and 3,057 ± 0.50 pg cm -2 for free laccase- and free glucose oxidase-treated membranes, respectively. The Combi-CLEA-treated membrane exhibited the lowest ATP concentration among the enzymatic systems, reaching 2,870 ± 0.35 pg cm -2. The reduction in ATP concentration after enzymatic treatment indicates effective removal and destabilization of biomass-associated foulants from the membrane surface. Similar reductions in membrane-associated ATP after cleaning treatments have also been reported previously Jafari et al. (2020).

The total organic carbon (TOC) concentration of the fouled membrane was determined as 16.84 ± 0.89 mg L -1 (Figure 4). Following enzymatic treatment, the TOC concentration decreased to 13.44 ± 0.54 mg L -1 and 12.17 ± 0.38 mg L -1 for free laccase and free glucose oxidase treatments, respectively, whereas the Combi-CLEA-treated membrane exhibited a TOC value of 12.24 ± 0.74 mg L -1. The reduction in TOC concentration suggests partial removal of deposited organic contaminants and restoration of membrane permeability after enzymatic cleaning.

The COD value of the fouled membrane was determined as 17.1 ± 0.21 mg L -1, indicating the presence of substantial oxidizable organic matter within the fouling layer. After enzymatic cleaning, COD values decreased to 14.55 ± 0.32 mg L -1, 13.81 ± 0.53 mg L -1, and 14.51 ± 0.32 mg L -1 for free laccase, free glucose oxidase, and Combi-CLEA treatments, respectively. The decrease in COD after treatment further supports degradation and removal of organic foulants from the membrane surface.

Chemical cleaning treatments using HCl and NaOH resulted in comparatively lower ATP concentrations of 1,096 ± 0.91 pg cm -2 and 1,032 ± 0.85 pg cm -2, respectively. Similarly, TOC values decreased to 2.67 ± 0.56 mg L -1 and 2.81 ± 0.61 mg L -1, while COD values decreased to 3.22 ± 0.89 mg L -1 and 3.96 ± 0.74 mg L -1 for HCl and NaOH treatments, respectively. These results indicate that chemical cleaning exhibited comparatively higher immediate removal of biomass and organic contaminants than enzymatic cleaning. Nevertheless, the Combi-CLEA system demonstrated effective membrane cleaning under comparatively milder operating conditions with additional advantages including catalytic specificity, magnetic recoverability, and operational reusability. Therefore, enzymatic cleaning may serve as an environmentally compatible complementary cleaning strategy rather than a direct replacement for conventional chemical cleaning methods based solely on immediate organic foulant removal efficiency.

This study demonstrated the potential applicability of co-immobilized laccase and glucose oxidase on chitosan-coated magnetic iron oxide nanoparticles for enzymatic cleaning of organically fouled polypropylene membranes. The co-immobilized enzyme system exhibited enhanced catalytic performance, operational stability, and reusability compared with free enzymes, with catalytic efficiency values of 17.39 mM -1 min -1 for laccase and 28.63 mM -1 min -1 for glucose oxidase under optimized immobilization conditions. The Combi-CLEA system effectively reduced membrane-associated biomass and organic foulants, as evidenced by the reduction in adenosine triphosphate concentration from 8,100 ± 0.34 to 2,870 ± 0.35 pg cm -2 following enzymatic treatment. In addition, membrane volumetric flux increased nearly three-fold after treatment, indicating substantial restoration of membrane permeability. The enhanced cleaning performance was attributed to the synergistic catalytic activity, improved stability, and magnetic recoverability of the co-immobilized enzyme system. These findings demonstrate the potential of co-immobilized oxidoreductase nanobiocatalysts as environmentally compatible and reusable membrane-cleaning systems. Future studies should focus on long-term operational stability, membrane surface integrity after repeated cleaning cycles, enzyme leaching analysis, advanced mechanistic investigations, and pilot-scale membrane applications to further evaluate the industrial applicability of the proposed nanobiocatalytic cleaning platform.