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Review

Enzyme Immobilization on Nanomaterials and Their Applications

by
Rahul Vikram Singh
,
Bakul Singh
,
Anurag Kumar
,
Krishika Sambyal
,
Karthikeyan Kugalur Karuppanan
and
Jung-Kul Lee
*
Department of Chemical Engineering, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
*
Author to whom correspondence should be addressed.
Materials 2025, 18(17), 4106; https://doi.org/10.3390/ma18174106
Submission received: 18 July 2025 / Revised: 22 August 2025 / Accepted: 27 August 2025 / Published: 1 September 2025
(This article belongs to the Special Issue Magnetic Nanomaterials: Synthesis, Characterization and Applications)
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Versions Notes

Abstract

Immobilizing enzymes onto nanomaterials is a promising approach for increasing the efficiency of industrial biotransformation processes. Nanomaterials have large surface areas and unique physicochemical characteristics, they increase enzyme stability and catalytic efficiency, and they can be reused multiple times, making them favorable over free enzymes. Various nanomaterials, including carbon-based materials, metal and metal oxide nanoparticles, and polymeric nanoparticles, have been explored for immobilizing enzymes. Immobilized enzymes are more reusable than free enzymes, which are suitable for industrial applications such as in pharmaceuticals, as drug intermediates, and for synthesizing fine chemicals. Using immobilized enzymes multiple times enables numerous catalytic reactions, substantially increasing product yield and minimizing enzyme consumption, thus optimizing process efficiency and cost-effectiveness of manufacturing processes. This review explores recent developments in nanomaterials for immobilizing enzymes and biotransformation.

1. Introduction

Immobilizing enzymes on nanomaterials is a promising approach for increasing the efficiency of biotransformation processes in various industries. This technique combines the catalytic properties of enzymes with the unique properties of nanomaterials, resulting in increased enzyme stability, activity, and reusability, and provides large surface area for efficient immobilization, offering advantages over traditional enzymatic applications [1,2]. Enzymes are biological (natural and engineered) catalysts that play crucial roles in numerous industrial processes, including biofuel production, food processing, and pharmaceutical manufacturing [1]. However, the limited stability, recovery and reuse difficulties, and susceptibility to environmental factors hinder the use of free enzymes in industrial applications [2]. Enzymes have been immobilized on solid supports, particularly nanomaterials, to address these limitations and to improve the overall efficiency of enzymatic processes [3].
Nanomaterials have received considerable attention as support for immobilizing enzymes owing to their large surface-area-to-volume ratio, high biocompatibility, and physicochemical characteristics [4]. Using nanomaterials as enzyme carriers can considerably increase enzyme stability, catalytic activity, and reusability compared with enzymes used without these carriers, enabling the development of more cost-effective and sustainable industrial processes [1,2]. Various types of nanomaterials have been tested for their potential in enzyme immobilization, including carbon-based materials such as carbon nanotubes (CNTs) and graphene, silver nanoparticles, magnetic nanoparticles (MNPs), metal–organic frameworks (MOFs), and polymeric nanoparticles, as illustrated in Figure 1 [2,4]. Each type of nanomaterial offers distinct advantages and disadvantages, as described in Table 1, and each can be tailored to suit specific enzyme immobilization requirements [3].
Enzymes and substrates must interact to produce catalytic activity. The induced-fit hypothesis states that the enzyme and substrate undergo conformational changes upon binding, ensuring catalysis. Immobilizing enzymes can affect these interactions. Although the activity of immobilized enzymes may be lower owing to mass transfer barriers, immobilized enzymes are often more stable across broader pH and temperature ranges. A specific distance is required between the immobilized enzymes and the support surface to ensure structural stability [12]. The immobilization method, such as covalent attachment or adsorption, influences the enzyme–substrate interactions [13]. The hydrophobic nature of the carriers stabilizes enzyme, and multipoint attachment increases thermal stability, which ensures enzyme–substrate interactions as well as increasing enzyme stability and reusability [14,15,16,17]. Understanding the interactions between the enzyme, substrate, and immobilized matrix is crucial, as matrix compatibility influences how the matrix binds to the enzyme and determines the type of bonding that provides high stability during enzyme–substrate reactions. For example, computational studies have examined nitrilase (pdb:3wuy) immobilized on single-walled carbon nanotubes (swCNTs) functionalized with glutaraldehyde (GA) in this regard, as shown in Figure 2. In this setup, the immobilization matrix (yellow-green) offers structural support while preserving enzyme activity. The binding pocket of the enzyme accommodates the 3-cyanopyridine substrate, which interacts with key amino acid residues, such as P194, M197, V198, F202, C169, W170, H141, Y140, E142, K135, N118, and E53. Notably, aromatic residues, such as W170, F64, and F202, form pi–pi interactions with the benzene ring of the substrate, positioning the cyanocarbon near the active-site residue C169, which then initiates catalysis through nucleophilic attack. Additional residues, including E53 and K135, help stabilize the transition state, and the optimal positioning of the substrate within the active site highlights the complementarity and multiple noncovalent interactions that facilitate efficient catalytic conversion of the nitrile compound [18,19]. Magnetic nanoparticles (MNPs) are a particular subject of interest for enzyme immobilization because of their superparamagnetic properties. Magnetite (Fe3O4) is the most widely used MNP for immobilizing enzymes owing to its cost-effectiveness, biocompatibility, low toxicity, and high magnetic susceptibility [20]. The use of MNPs facilitates the separation and recovery of immobilized enzymes from reaction mixtures using an external magnetic field, simplifying downstream processing [16].
Several immobilization techniques have been developed to attach enzymes to nanomaterials, including covalent, noncovalent, and encapsulation methods [21]. Each immobilization method has advantages and limitations (Table 2), and the choice of method depends on the specific enzyme, nanomaterial, and intended application [2]. For example, covalent binding provides strong attachment but alters the structure of the enzyme, whereas noncovalent methods preserve the enzyme structure, but enzyme leaching is problem [2,20]. Immobilizing enzymes on nanomaterials has improved enzyme performance in various biotransformation processes. Immobilized enzymes are often more stable under harsh conditions, such as at extreme pH or temperature, compared with their free counterparts [1,3]. This increased stability allows for the use of enzymes in a broader range of industrial applications and extends their operational lifetimes [11]. Moreover, immobilizing enzymes on nanomaterials increases catalytic activity and efficiency [22]. The large surface area of nanomaterials allows for the loading of large amounts of enzyme, and the properties of nanomaterials create a favorable microenvironment for enzyme function [4,23]. In some cases, the catalytic activity of immobilized enzymes is several-fold higher than that of free enzymes [3]. The field of immobilizing enzymes on nanomaterials continues to evolve, with advancements focusing on developing multifunctional nanomaterials and the co-immobilization of multiple enzymes [6,24] to create more efficient and versatile biocatalytic systems for complex biotransformation processes [23]. The number of applications of enzyme–nanomaterial conjugates is annually expanding across a wide range of industries. Figure 3 depicts the keyword networks and the focus of nanomaterial applications in different sectors since 2001. In Figure 3, the applications of nanoparticles are divided into four sections: (a) biotransformation, (b) pharmaceutical sector, (c) biosensor development, and (d) bioremediation. Therefore, on the PubMed platform, keywords, such as the “application of nanoparticle,” in all four sectors were searched, and all available data (research, reviews, and other articles) were analyzed using VOSviewer (version_1.6.20_exe) to generate the network. Initial application searches focused on biotransformation and pharmaceuticals because article searches revealed that most published studies on enzyme immobilization focused on the synthesis of pharmaceutically relevant drug intermediates, precursors, and fine chemicals. Subsequently, nanoparticles have been utilized in drug delivery systems in the pharmaceutical sector. The development of nanomaterial-based biosensors has gained traction, particularly in bioremediation, highlighting their expanding role in environmental solutions. This evolution reflects the broadening scope and advancing sophistication of nanomaterial research across various industries.
Nanomaterials have been used in biofuel production, and immobilized enzymes have shown promise in increasing the efficiency of biomass conversion and biocatalytic processes [26]. Immobilized enzymes are used in various food industry applications, such as the production of functional foods and modification of food components [28]. However, several challenges remain despite the progress in this field: optimizing immobilization protocols to maintain enzyme activity, increasing the long-term stability of immobilized enzymes, and addressing potential environmental and safety concerns associated with nanomaterials [3,8].
Classical supports generally provide strong stability and reliable interactions with enzymes but often suffer from limited surface area and less tunable properties. In contrast, nanomaterials offer significantly higher surface areas, customizable sizes and shapes, and enhanced surface chemistry. These features improve enzyme loading, activity, and stability by enabling stronger interactions and better substrate accessibility. The advanced properties of nanomaterials thus lead to superior efficiency over classical supports in catalytic and biotransformation processes, aligning with recent findings in nano-biocatalysis research [6,8,10].

2. Nanomaterials for Immobilizing Enzymes

Various types of nanomaterials with properties appropriate for immobilizing enzymes have been synthesized. Nanomaterials are classified according to their dimensionality, morphology, and chemical composition (Figure 4) [29]. These classes include zero-dimensional (0D) structures, such as nanoparticles and quantum dots; one-dimensional (1D) structures, such as nanotubes and nanowires; two-dimensional (2D) structures, such as graphene; and three-dimensional (3D) nanostructures [6,8,9,29]. The chemical composition of these nanomaterials ranges from carbon-based (fullerenes and CNTs) to metal-based materials (gold and silver nanoparticles), metal oxides (titanium dioxide and iron oxide), and organic materials such as dendrimers [15,25,30]. The classification can also be based on specific properties such as optical, electrical, and magnetic characteristics. However, defining nanomaterials remains challenging because of their dynamic nature and the limitations of measurement methods, leading to ongoing debates regarding their classification criteria [13,31,32].

2.1. Carbon-Based Nanomaterials

Carbon-based nanomaterials, particularly CNTs and graphene, have emerged as appropriate supports for immobilizing enzymes because of their properties. CNTs, both single-walled (swCNTs) and multiwalled (mwCNTs), have large surface areas and are chemically inert as well as biocompatible, so are suitable for immobilizing enzymes [33,34,35,36]. Functionalization of carbon nanotubes (CNTs) with different groups, such as amino, carboxyl, or silane groups, enhances their surface properties for enzyme immobilization. These modifications enable covalent binding or crosslinking; improve enzyme stability, activity, and reusability; and reduce leaching, making CNTs effective supports for biocatalysts, as described in Table 3 along with the nanomaterial, functional groups, enzymes, enzyme sources, and their reusability cycles in the reaction. CNTs have been commercialized because of their biocompatibility, biodegradability, and strong film-forming ability; therefore, CNTs have received considerable research attention [37,38]. CNT applications are not limited to enzyme immobilization but also include other fields, such as biosensor development. Integrating molecularly imprinted polymers (MIPs) with CNTs has expanded their potential for use in sensor development. CNT-based sensors show promise in medical diagnostics [39,40,41], environmental monitoring [42,43], and industrial processing [44,45]. CNT-based biosensors offer innovative solutions for immobilizing enzymes and detecting biomolecules. Carbon nanotube (CNT)-based nanomaterials have significant potential for applications in biotransformation by leveraging enzymatic and microbial processes to modify their structures and reduce toxicity. For instance, fungi such as Penicillium chrysogenum and Pleurotus ostreatus utilize ligninolytic enzymes (e.g., laccases and peroxidases) to oxidize and shorten multi-walled CNTs (MWCNTs), alter surface functional groups (C=O and O–H), and enhance biocompatibility. Similarly, microalgae, such as Desmodesmus subspicatus can remove up to 55% of absorbed CNTs from aquatic environments, indicating their potential for applications in environmental remediation. Surface engineering techniques such as PEGylation further optimize the stability and biodegradability of CNTs, which are crucial for biomedical and ecological applications. These biotransformation strategies enable safer CNT integration in drug delivery, pollution mitigation, and regenerative medicine [46,47]. Superparamagnetic mwCNTs with four-armed polyethylene glycol were used to covalently coimmobilize Rhizopus oryzae and Candida rugosa lipases for producing biodiesel from waste oils. Biodiesel is a renewable fuel produced by the enzymatic transesterification of waste oils using lipases such as Rhizopus oryzae and Candida rugosa. In this process, waste oil serves as a substrate for immobilized enzymes, which are carried on modified CNTs to enhance their efficiency and stability. When applied to biodiesel production from waste cooking oil under ultrasound assistance, this system achieved a 97.64% conversion rate in 120 min, owing to the synergistic action of both the lipases and ultrasound. Notably, the system maintained a yield of over 78.55% after 10 cycles, demonstrating excellent operational stability and significant potential for industrial biodiesel production and other biocatalytic applications [48]. Other studies have proved that enzymes immobilized on CNTs retain high catalytic activity and multiple cycles of enzyme reusability (Table 3). The curvature of the nanotubes affects the enzyme immobilization yield and behavior, with enzymes covalently immobilized on amine-functionalized CNTs having higher catalytic activity and operational stability than physically adsorbed enzymes.

2.2. Graphene and Derivatives

Graphene and its derivatives have diverse applications in biomedicine, energy science, and material sciences. Because of their unique properties, they are used in biosensing, bioimaging, drug delivery, and tissue engineering. Graphene-based materials have been used in energy applications to enhance the performance of solar cells, batteries, and supercapacitors. They are suitable for nanoelectronics, sensors, and composite materials owing to their mechanical and electrical properties [70,71,72]. Functionalization of graphene and its derivatives with various groups (e.g., carboxyl, hydroxyl, and epoxy) enhances its surface chemistry, enabling effective enzyme immobilization. These modifications improve enzyme stability, activity, and reusability by promoting strong physical or covalent attachment, making graphene-based materials excellent carriers for biocatalysts, and such materials are presented in Table 4, including enzymes and their sources, functionalization groups, recyclability, and retained activity. Chloroperoxidase (CPO) and glucose oxidase (GOx) were co-immobilized on magnetic graphene oxide (MGO) to increase its catalytic activity. The activity of MGO-GOx-CPO was higher (96.6%) than that of separately immobilized enzymes (86.2%) in orange G decolorization. The co-immobilized system was more thermally stable and 38.5% of the activity was maintained after six reuse cycles. This approach demonstrates potential for environmental applications like wastewater treatment, combining efficient catalysis with easy magnetic recovery [73]. Metal–organic frameworks (MOFs) and graphene oxide (GO) have been combined with single-stranded DNA as a linker to co-immobilize glucose oxidase (GOx) and horseradish peroxidase (HRP). This approach had an enzyme stability of <70% of the initial activity and achieved activity through π–π stacking as well as coordination bonding. The immobilized GOX–HRP system showed enhanced in cascade reaction kinetics, substrate selectivity, and a wide linear range (50–750 μM) for detecting glucose. The reusability of PCN-222 and GO@DNA@GOx, HRP (PGE), and PCN-222 and GO@enzymes was investigated. PGE retained more than 75% catalytic activity after 10 cycles, whereas PCN-222 and GO@enzymes retained less than 30% of their initial activities [73]. A capillary-electrophoresis-based dual-enzyme microreactor (THR-FXa IMER) was developed using a polydopamine/GO coating to co-immobilize thrombin and factor Xa. The system accurately measured enzyme kinetics and determined inhibition constants and maintained 98% activity after 30 cycles. The system successfully screened dual-target inhibitors from 30 compounds, and the results of molecular docking validated these interactions, leading to the design of a new anticoagulant compound. THR-FXa IMER is a reliable method for screening coagulation enzyme inhibitors and aided in the development of anticoagulant drugs [74]. Graphene-based systems, such as MGO-GOx-CPO and MOF-GO hybrids, are catalytically active, stable, and reusable when immobilizing enzymes, demonstrating potential for remediating the environment and detecting glucose. Additionally, the dual-enzyme microreactor (THR–FXa IMER) highlights the role of graphene in drug discovery, as the use of graphene has enabled the efficient screening of anticoagulant compounds. These findings underscore the importance of graphene-based materials for creating sustainable high-performance platforms for diverse scientific and industrial applications. Graphene-based materials exhibit versatility in immobilizing enzymes, having increased catalytic performance, stability, and reusability compared with prior materials. The success of enzymes co-immobilized on graphene derivatives (Table 4) highlights their synergistic effects and ability to increase the overall efficiency of these systems. The literature underscores the value of graphene in developing sustainable high-performance platforms for various scientific and industrial applications [75,76,77,78,79,80].

2.3. Metal and Metal–Oxide Nanoparticles

Metals such as Ag, Fe, Au, Cu, Pt, Zn, Mg, and Mn as well as metal–oxide nanoparticles such as TiO2, ZnO, Fe3O4, CuO, Al2O3, and MgO are suitable supports for immobilizing enzymes owing to their properties [109,110]. These nanoparticles have large surface-area-to-volume ratios, show superparamagnetic behavior (Fe3O4), and are easy to functionalize, suitable for increasing enzyme stability and catalytic activity. Fe3O4 nanoparticles are particularly popular owing to their biocompatibility, low toxicity, and low cost. Fe3O4 is easily separated and recovered using external magnetic fields when used for immobilizing enzymes, increasing the reusability of the enzymes used in industrial processes and protecting enzymes from harsh environmental conditions [111]. Surface modifications, such as coatings with various functional groups including thiols, amines, and carboxyls, enhance their stability, solubility, and biocompatibility, enabling efficient enzyme immobilization. These modifications facilitate strong enzyme attachment, improve catalytic performance, and expand applications in biosensing and biocatalysis, which are described in Table 5, along with enzymes, their sources, functionalization groups, and recyclability with retained activity. The enzyme immobilization on these nanoparticles are more thermally stable, tolerant to pH, and stable during storage than free enzymes, with some retaining up to 80% activity after extended storage periods (Table 5) [112]. The use of metal and metal–oxide nanoparticles, such as Fe3O4, TiO2, and CuO, has advanced enzyme immobilization by increasing enzyme stability, reusability, and catalytic activity. Their properties, including large surface area and functionalization potential, enable their applications in biosensing, environmental remediation, and biocatalysis. Advanced platforms that integrate nanoparticles with enzymes are highly sensitive, selective, and practical for industrial and biomedical applications [17,113,114,115,116,117,118,119,120].

2.4. MNPs

MNPs are composed of elements such as iron, cobalt, nickel, gadolinium, manganese, and chromium, and exhibit unique magnetic properties when exposed to external magnetic fields. These nanoparticles offer substantial advantages over other nanomaterials primarily because of their ease of separation from reaction mixtures by applying magnetic fields. This feature enables the efficient recovery and reuse of MNPs in various applications [130]. MNPs also have a large surface area-to-volume ratio, which enhances their catalytic activity and suitability for immobilizing enzymes. Magnetic nanoparticles (MNPs) are functionalized with various groups, such as amines, carboxyls, aldehydes, or peptide-based linkers, to enhance enzyme immobilization. These modifications improve the enzyme binding, stability, and reusability, enabling efficient catalytic applications and easy magnetic recovery in industrial processes. A few significant studies are reported in Table 6, along with various enzymes and their sources, functionalization groups, recyclability, and retained activity. Their superparamagnetic behavior, biocompatibility (especially with iron-oxide-based MNPs), and versatile surface functionalization have further contributed to their widespread use in fields such as biomedicine, industrial catalysis, environmental remediation, wastewater treatment, and pollutant removal [131,132,133]. More recent advancements have focused on developing MNPs tailored for specific applications, optimizing enzyme immobilization protocols, and creating enzyme-based bioreactors for industrial use [20].
The versatility of MNPs continues to drive innovation in fields ranging from biosensing to environmental remediation. Patil et al., (2022) [148] studied the co-immobilization of glucoamylase and α-amylase enzymes onto magnetic nanoparticles (MNPs) using glutaraldehyde as a cross-linking agent for the pre-treatment of Curcuma longa powder. The process was optimized for maximum enzyme activity, achieving the best results at a 1:4 MNPs-to-enzyme ratio, 60 mM glutaraldehyde concentration, and 120 min of cross-linking. The resulting enzymes@AMNPs were spherical with an average size of 100 nm and exhibited strong superparamagnetic properties (36.1 emu/g). When combined with low-power ultrasound, the biocatalyst significantly enhanced curcuminoid extraction yields by 1.3–1.5 times compared to individual methods. Subsequent crystallization led to a 54% (w/w) isolation of curcuminoids with 91% purity. The biocatalyst also demonstrated excellent reusability, maintaining about 50% activity after 10 cycles and over 95% activity after 30 days of storage [148]. Cascade reactions catalyzed using multienzyme systems offer considerable advantages over single-enzyme systems in industrial applications. Hydroxylase monooxygenase components (HpaB and HpaC) were co-immobilized on Ni-NTA-functionalized magnetic silica nanoparticles (Ni-NTA/H2N-SiO2@Fe3O4) to increase enzyme stability and activity. The co-immobilized system exhibited 2.6-fold higher activity than that with free enzymes, retained 76.6% activity after 12 days of storage, and maintained over 60% activity after seven cycles. This approach shows potential for industrial biocatalytic applications [149]. Thermomyces lanuginous lipase (TLL), Candida antarctica lipase B (CALB), and Rhizomucor miehei lipase (RML) were co-immobilized on amine-functionalized silica-coated magnetic nanoparticles via multicomponent reactions. The stability and specific activity of the co-immobilized enzymes were higher than those of the free enzymes. These co-immobilized enzymes were used for catalyzing biodiesel production from waste cooking oil, with Fe3O4@SiO2-NH2-RML-CALB and Fe3O4@SiO2-NH2-TLL-CALB achieving maximum FAME yields of 99% and 80%, respectively, under the optimized conditions determined using the response surface methodology and a central composite rotatable design [150].
MOFs are suitable carriers for immobilizing enzymes, although challenges such as enzyme conformational changes and substrate mass transfer resistance remain. Researchers developed a magnetic, hierarchical porous MOF (mH-UiO-66(Zr)) to co-immobilize enzymes horseradish peroxidase (HRP) and glucose oxidase (GOx. This design minimized enzyme leakage, reduced mass transfer resistance, and simplified handling via magnetism. The resulting bienzyme bioreactor exhibited exceptional stability (95.1% activity at 70 °C, >90% after 15 cycles), enhanced substrate affinity, and effectively degraded/transform pollutant 2,4-DCP, showcasing environmental remediation potential [151]. A novel magnetic four-enzyme nanobiocatalyst was developed through c-immobilizing cellulase (CelDZ1), β-glucosidase, GOx, and HRP onto amino-functionalized MNPs. The nanobiocatalyst exhibited high thermal stability, retaining up to 50% activity after five cycles and remaining active after 24 days at 5 °C. This nanobiocatalyst was successfully applied in a four-step cellulose hydrolysis cascade reaction, demonstrating its potential for industrial biocatalysis [152,153]. Qiu et al. (2021) [153] developed a novel biocatalyst by co-immobilizing laccase and 2,2-binamine-di-3-ethylbenzothiazolin-6-sulfonic acid on chitosan MNPs modified with an amino-functionalized ionic liquid (MACS-NIL-Cu-lac). The biocatalyst improved 1.7-fold activity as well as high stability and pollutant removal efficiency, achieving 100% 2,4-dichlorophenol and bisphenol A removal efficiency. The biocatalyst retained 93.2% efficiency after six cycles, demonstrating potential for practical water treatment applications [153]. MNPs effectively co-immobilize multiple enzymes and improved the catalytic activity, stability, and reusability (Table 6). MNP applications range from industrial biocatalysis to environmental remediation, showing potential for creating efficient and sustainable enzymatic systems in various fields [154,155,156,157,158].

2.5. Polymeric Nanoparticles

Polymeric nanoparticles are versatile platforms for immobilizing enzymes (Table 7), offering numerous advantages, such as high surface area, tunable properties, and biocompatibility. Polymeric nanoparticles can be synthesized from various natural and synthetic polymers, including polysaccharides, polystyrene, and polyurethane, providing a stable microenvironment for enzymes [159]. Functionalization of polymeric nanoparticles with various functional groups, such as amines, carboxyls, thiols, or biomolecular ligands, enables efficient enzyme immobilization. Techniques such as covalent binding, click chemistry, and surface adsorption allow precise attachment, enhancing the versatility and stability of NPs for biocatalysis, targeted delivery, and biosensing applications. Multiple functional groups can be incorporated in a single step for multifunctional use; for example, hyaluronic acid, chitin, chitosan, chitosan-cobalt oxide beads, and chitosan-coated superparamagnetic nanoparticles are described in Table 7, along with their enzymes, sources, functionalization groups, and recyclability with retained activity [160]. Suthiwangcharoen et al. (2014) developed novel core–shell nanoparticles using poly(4-vinylpyridine) to co-immobilize GOx and HRP, resulting in a 20% increase in enzyme activity compared with that of free enzymes [161]. Lipase was immobilized on polyurethane nanobiocatalysts via immobilizing CalB lipase on polyurethane (PU) nanoparticles functionalized with poly(ethylene glycol) (PU-PEG) synthesized via miniemulsion polymerization. The resulting p-NPB hydrolysis, enantioselective Mandelic acid hydrolysis, and were-3 ethyl ester production were analyzed. The kinetic parameters of PU-PEG6000-CalB exhibited the highest, whereas the activity of PU-PEG400-CalB was the highest, producing 43.72 and 16.83 mM.U−1 ethyl esters using EPA and DHA, respectively.
Trehalose has high thermal stability, and trehalose nanobiocatalysts show potential for use in sustainably synthesizing food and pharmaceuticals [162]. Polymeric nanoparticles show considerable potential in various applications for immobilizing enzymes, including biocatalysis, biosensing, and bioremediation. These systems exhibit high enzyme stability, reusability, and prolonged activity under harsh conditions. For example, Lee et al. (2008) [163] immobilized lipases on polyaniline nanofibers with iron oxide. The immobilized lipases were highly stable, easy to recover, and reusable, retaining over 80% activity after 32 days at room temperature. The system was applied for enantioselective esterification, yielding 18% prophilic ester of ibuprofen from racemic ibuprofen after 96 h, demonstrating an ability to efficiently and selectively perform biocatalysis [163]. However, challenges with mass transfer and scalability require ongoing research in the field of immobilizing enzyme using polymeric nanoparticles [164,165,166,167,168].
Table 7. Polymeric nanoparticles used for immobilizing enzymes.
Table 7. Polymeric nanoparticles used for immobilizing enzymes.
NanomaterialSkeleton MatrixFunctionalization GroupsEnzymeEnzyme SourceReusability (Retained Activity)Reference
Polymeric nanoparticlesHyaluronic acid, chitin, and chitosanGlutaraldehydeCatalaseSunflower seeds25 cycles (73.80%) (79.55%)[169]
CS-ALG-Fe3O4 MNPsIonic gelation methodLaccaseTrametes versicolor10 cycles (81%)[164]
Hyaluronic acid, chitin, and chitosanGlutaraldehydeCatalaseBacillus subtilis25 cycles (73.80%) (79.55%)[122]
Fe3O4@chitosanGlutaraldehydeLaccaseRhus verniciflua10 cycles (75.8%)[170]
ZnO/chitosanGlutaraldehyde, APTESTrypsin-10 cycles (50%)[171]
Silica-coated magnetic nanoparticlesIsocyanatopropyltriethoxysilaneProlidaseEscherichia coli prolidase20 cycles (80%)[172]
Chitosan-Fe3O4 and chitosan-ZnO-α-Amylase-10 cycles (50%)[173]
Chitosan-cobalt oxide beadsCyanuric chloridePeroxidaseEuphorbia tirucalli10 cycles (60%)[174]
Chitosan coated superparamagnetic nanoparticles1,3,5-triazineGlucoamylaseAspergillus niger10 cycles (70%)[175]
APTES: (3-aminopropyl)triethoxysilane.

3. Enzyme Immobilization on Nanomaterials for Biotransformation

Immobilizing enzymes on nanomaterials involves anchoring enzyme molecules to solid support, increasing enzyme stability and reusability as well as facilitating product separation and purification. This process reduces the overall enzyme consumption and costs, which is economically feasible for industrial applications [176,177]. The interactions between enzymes and nanomaterial supports include physical interactions (van der Waals forces, hydrogen bonds, and hydrophobic interactions), covalent bonding, and affinity-based binding [28]. Immobilization techniques have received considerable attention because of their potential to increase enzyme performance and longevity in various industrial and biomedical applications [21]. The support matrix must be carefully selected for immobilizing enzymes, with options including inorganic, organic, and hybrid categories. Inorganic supports include silica, MNPs, and carbon-based materials, whereas organic supports include natural and synthetic polysaccharides [160,178,179]. Hybrid materials combine the advantages of both and offer enhanced properties compared with those of inorganic or organic materials alone. Nanomaterials provide significant advantages as enzyme supports over traditional organic and inorganic materials. Their exceptionally high surface area-to-volume ratio enables greater enzyme loading and stability, resulting in enhanced catalytic performance and reusability. Compared to current organic and inorganic supports, nanomaterials offer improved activity retention, broader operational conditions, and reduced mass transfer limitations, making them highly effective platforms for enzyme immobilization [80,116,117]. Research trends have shifted from traditional supports (alginate, chitosan, carrageenan, and starch) to newer materials such as CNTs and their derivatives, silica, magnetic particles, polyethylene glycol, and chitosan for immobilizing enzymes [180,181,182]. Various polymeric supports, magnetic composites, and nanomaterial-based carriers have been developed in the decade from 2015 to 2025. Nanomaterial-based enzyme immobilization is a powerful technique that increases enzyme stability, reusability, and catalytic efficiency in biotransformation processes.
Various nanomaterials, including CNTs, MNPs, and MOFs, have been used for immobilizing enzymes to increase performance and reusability. swCNTs and mwCNTs have shown considerable strengths in immobilizing enzymes owing to their large surface area, mechanical properties, and biocompatibility [183,184,185,186,187,188]. For example, Kumar et al. (2019) [63] immobilized lipase from mesoporous SiO2 microparticles, which were synthesized through spray pyrolysis using mwCNTs as a template, resulting in a template that could be used 12 times. Dense- and mesoporous-SiO2-bound lipases retained 74.2% and 95.4% of their initial activity, respectively [63]. MNPs, particularly Fe3O4, have received substantial attention owing to their superparamagnetic properties, which allow for easily separating and reusing immobilized enzymes [3]. Superparamagnetic properties are particularly advantageous for industrial applications because they enable efficiently recovering and recycling biocatalysts. For example, lipases immobilized on magnetic mwCNTs have increased activity and reusability in biotransformation processes in comparison of free enzyme. Lipase immobilized on CNTs filled with magnetic iron oxide and modified with polyamidoamine dendrimers had 17-fold higher specific activity than the free enzyme and retained 90% of its original activity after 20 cycles [183]. The lipase from Burkholderia sp. C20 was immobilized on alkyl-functionalized Fe3O4SiO2 magnetic nanoparticles for synthesizing biodiesel. The binding efficiency of the immobilized enzyme was 97%, and the maximum adsorption capacity was 29.45 mg g−1. The immobilized enzyme exhibited Michaelis–Menten kinetics for olive oil hydrolysis, with a Vmax of 6251 U g−1 and a Km of 3.65 mM. The immobilized enzyme converted >90% of the FAME from olive oil within 30 h using 11 wt% immobilized lipase for producing biodiesel. The biocatalyst maintained 90% of its activity after 10 use cycles, demonstrating high stability and reusability in transesterification reactions [189]. Polyamidoamine dendrimers were grafted onto magnetic mwCNTs to create a functionalized surface for the oriented immobilization of Rhizomucor miehei lipase (RML). The immobilized enzyme exhibited recovery activity up to 2808% and 27-fold higher esterification activity than the free enzyme.
Immobilized RML achieved 94% conversion of waste vegetable oil to biodiesel under optimized conditions. The immobilized RML enzyme maintained its catalytic efficiency over 10 cycles, with high stability and reusability. This immobilized RML system can be used as a robust and reusable catalyst for producing industrial biodiesel [69]. A packed-bed reactor was developed using a lipase-Fe3O4 nanoparticle biocomposite catalyst for producing biodiesel from soybean oil. Emulsification increased the reaction rate. Conversion remained at 45% after 240 h in the single-packed-bed reactor. A four-packed-bed system maintained >88% conversion for 192 h, dropping to 75% after 240 h, showing suitability for producing industrial-scale enzymatic biodiesel [190]. A lipase immobilization strategy using 5-aminoisophthalic acid grafted onto magnetic nanoparticles via Co2+ chelation. Pseudomonas fluorescens lipase had a 136.9 mg/g immobilization capacity and 2125% activity recovery, converting 95% of the waste cooking oil to biodiesel retaining 83% yield after 10 cycles. This support was easily regenerated and is therefore suitable for biotechnological applications [191]. The microfluidic bioreactor prototype enables small-scale enzymatic reactions in compartmented environments. Reactants immobilized on magnetic microcarriers facilitate separation and multiple reactions; permanent and alternating magnetic fields facilitate the separation and mixing. Kinetic studies using immobilized HRP revealed an enzyme activity of 89 U/g. Recycling experiments demonstrated the importance of magnetic resus pension, with yields of 65–95% over 10 cycles with resuspension compared with <10% without resuspension. The biochemical reactions in this system are efficient, controllable, and small in scale, so may be used in various applications [192]. Amano lipase from P. fluorescens was covalently immobilized on carbon nanomaterials (functionalized swCNTs and GO) for producing biodiesel. The most effective preparation using SWCNTNH2 derivatized with glycerol diglycidyl ether converted >99% of the sunflower oil in 4 h under optimal conditions. The biocatalyst retained over 99% of the initial activity after 20 reuse cycle in batch systems [193]. Salem et al. (2021) [123] immobilized microbial α-amylase (AmyKS) and xylanase (XAn11) on biomimetic MNPs (BMNPs) and inorganic MNPs using electrostatic interaction and covalent bonding. AmyKS was successfully immobilized on the BMNPs via electrostatic interactions, whereas XAn11 was immobilized on the MNPs via EDC/NHS crosslinking. A total of 92% of AmyKS and 87% of XAn11 were immobilized on BMNPs and MNPs–E/N under optimized conditions, respectively. AmyKS-BMNPs and XAn11-MNPs-E/N were highly reusable, retaining 82% and 64% of their initial activity after 15 and 11 reaction cycles, respectively [30]. MOFs have also been explored for immobilizing enzymes, with zeolitic imidazolate frameworks (ZIFs), showing promise for immobilizing enzymes on CNT surfaces [189]. The immobilization strategies include covalent binding, physical adsorption, and encapsulation, each offering unique advantages depending on the specific enzyme and its application. Nanomaterial-based immobilization techniques have substantially increased enzyme stability, activity, and reusability across various applications [194,195]. These examples demonstrate how enzyme immobilization on nanomaterials directly enhances biotransformation processes by improving the catalytic efficiency, stability, and reusability of biocatalysts [190,191]. For example, lipases immobilized on mesoporous SiO2 and magnetic nanoparticles (MNPs) enable efficient transesterification reactions for biodiesel production, achieving high conversion rates and sustained activity over multiple cycles [63]. Functionalized carbon nanotubes (CNTs) and MNPs not only increase enzyme loading and specific activity but also facilitate product separation and enzyme recovery, which are crucial for continuous biotransformation in industrial settings [183]. Microfluidic bioreactors with immobilized enzymes exemplify scalable, compartmentalized biotransformation, allowing precise control over reaction conditions and repeated substrate conversion [192]. These advancements in nanomaterial-based enzyme immobilization have implications for many industries, such as biofuel production, food processing, and pharmaceuticals, where efficient and reusable biocatalysts are highly desirable [3,196]. Further improvements in enzyme immobilization techniques are expected as research in this field continues, leading to more sustainable and cost-effective biotransformation processes.

4. Perspectives

Future research on nanomaterials will focus on three major areas: developing multifunctional nanomaterials, optimizing the immobilization methods, as well as biosafety and environmental concerns. First, the future of immobilizing enzymes on nanomaterials involves developing multifunctional supports that enhance enzyme stability, activity, and reusability. Researchers have focused on creating nanomaterials with properties tailored for specific enzymes and applications [3,17,23,28]. One future research direction involves synthesizing MNPs that are more biocompatible with functionalizable surfaces. These advanced MNPs will have higher enzyme-loading capacity as well as facilitate separation and reuse in industrial processes. Another future area of research includes focusing on developing hybrid nanomaterials that combine the advantages of different materials. For example, carbon-based nanomaterials coated with metal nanoparticles can have a large surface area and act as highly stable [30,63,169,197], producing synergistic effects and enhancing the overall performance of the immobilized enzymes. Second, future research should focus on refining immobilization techniques to maximize enzyme activity and stability. This could involve exploring new methods of attaching enzymes to supports, such as affinity-based binding and site-specific immobilization [160,198]. These approaches can help preserve the native structure and function of enzymes, thus increasing catalytic efficiency. Researchers are also developing more standardized and scalable immobilization protocols by optimizing parameters such as pH, temperature, and reaction time for different enzyme–nanomaterial combinations [80,196]. Computational modeling and high-throughput screening techniques could accelerate this optimization process, allowing for rapidly identifying the ideal immobilization conditions. Another avenue involves developing stimuli-responsive nanomaterials that modulate enzyme activity in response to external triggers [3,117]. This could increase the precision of the control of enzymatic reactions in industrial applications. Third, addressing safety and environmental concerns will be crucial as the industrial adoption of nanomaterial-immobilized enzymes becomes widespread. Future research should focus on developing green methods for synthesizing nanomaterials to minimize their environmental impact [119,199]. These may include the use of plant extracts, microorganisms, and other eco-friendly approaches for producing nanomaterials with reduced toxicity. Long-term studies must be conducted on the release of nanoparticles from immobilized enzyme systems and their environmental fate [157,200]. These studies will help with establishing guidelines for the safe use and disposal of nanobiocatalysts in industrial settings. Additionally, developing biodegradable nanomaterials as enzyme supports could address concerns regarding their environmental accumulation. These materials could naturally break down after their useful lives, reducing their long-term environmental impacts. VOSviewer constructs and visualizes bibliometric networks for analysis; shows research trends, hot topics, and keyword relationships; and helps to identify active areas, emerging directions, and core themes within a scientific research field. It also shows if the number of scientific studies on nanomaterial synthesis and their application in various sectors has increased annually, which aids researchers in understanding research advancements in the field. Another target for future research will likely be engineering nanomaterials that can mimic or enhance natural enzyme environments, improving selectivity and conversion rates for complex biotransformations. The integration of nanomaterials with synthetic biology enables the design of tailored biocatalysts for specific chemical conversions, including those relevant to pharmaceuticals, fine chemicals, and environmental remediation. Moreover, advances in in situ monitoring and real-time control of biotransformation reactions using nanomaterial-based sensors will facilitate process optimization. These developments are expected to expand the scope and efficiency of biotransformation applications, making them more sustainable and economically viable [157,160,198,199,200].

5. Conclusions

Nanomaterial-immobilized enzymes are promising for industrial and biomedical applications. Researchers can ensure the full potential of nanobiocatalysts can be exploited by developing multifunctional nanomaterials, optimizing immobilization protocols, and addressing safety and environmental concerns. These nanomaterial-immobilized enzymes could increase the efficiency and sustainability of biofuel production, food processing, pharmaceutical production, and environmental remediation processes. Collaboration between materials scientists, enzymologists, and environmental researchers will be crucial as the field advances to overcome the current limitations and realize the full potential of nanomaterial-immobilized enzymes. These innovative biocatalysts are expected to play a large role in shaping the future of sustainable industrial processes and biotechnological applications with continued research and development.

Author Contributions

R.V.S.—writing—original draft, conceptualization, review and editing, B.S.—collected data for the manuscript, A.K.—designed computational 3D enzyme-substrate-matrix interaction, K.S.—data collection, review and editing, K.K.K.—formal analysis and review, J.-K.L.—supervision, review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2022M3A9I3082366, RS-2023-00222078, RS-2024-00440681).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting this article have been collected from the following sources: Data for Figure 3 were obtained from the Pubmed database (https://pubmed.ncbi.nlm.nih.gov/), accessed in 10 February 2025, and were analyzed and visualized using VOSviewer (https://app.vosviewer.com/) accessed in 10 February 2025. All data used is publicly available from these databases.

Acknowledgments

This work was supported by the KU Research Professor Program of Konkuk University.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Bhavaniramya, S.; Vanajothi, R.; Vishnupriya, S.; Premkumar, K.; Al-Aboody, M.S.; Vijayakumar, R.; Baskaran, D. Enzyme immobilization on nanomaterials for biosensor and biocatalyst in food and biomedical industry. Curr. Pharm. Des. 2019, 25, 2661–2676. [Google Scholar] [CrossRef]
  2. Nawaz, A.F.; Samra Zafar, S.L.F.; Shahzadi, K.; Fatima, Z.; Siddique, I. Use of nanomaterials for the immobilization of industrially important enzymes. J. Nanotechnol. 2021, 3, 45–57. [Google Scholar]
  3. Cavalcante, A.L.G.; Dari, D.N.; da Silva Aires, F.I.; de Castro, E.C.; Dos Santos, K.M.; Dos Santos, J.C.S. Advancements in enzyme immobilization on magnetic nanomaterials: Toward sustainable industrial applications. RSC Adv. 2024, 14, 17946–17988. [Google Scholar] [CrossRef]
  4. Kondaveeti, S.; Park, G.D.; Shanmugam, R.; Pagolu, R.; Patel, S.K.; Bisht, A.; Kim, D.R.; Kang, Y.C.; Lee, J.K. Investigating the role of metals loaded on nitrogen-doped carbon-nanotube electrodes in electroenzymatic alcohol dehydrogenation. Appl. Catal. B Environ. 2022, 307, 121195. [Google Scholar] [CrossRef]
  5. Borzouee, F.; Varshosaz, J.; Cohan, R.A.; Norouzian, D.; Pirposhteh, R.T. A comparative analysis of different enzyme immobilization nanomaterials: Progress, constraints and recent trends. Curr. Med. Chem. 2021, 28, 3980–4003. [Google Scholar] [CrossRef] [PubMed]
  6. Zhang, J.; Lovell, J.F.; Shi, J.; Zhang, Y. Nanomaterials for co-immobilization of multiple enzymes. BMEMat 2024, 3, e12080. [Google Scholar] [CrossRef]
  7. Sharifi, M.; Karim, A.Y.; Mustafa Qadir Nanakali, N.; Salihi, A.; Aziz, F.M.; Hong, J.; Khan, R.H.; Saboury, A.A.; Hasan, A.; Abou-Zied, O.K.; et al. Strategies of enzyme immobilization on nanomatrix supports and their intracellular delivery. J. Biomol. Struct. Dyn. 2020, 38, 2746–2762. [Google Scholar] [CrossRef]
  8. Mohidem, N.A.; Mohamad, M.; Rashid, M.U.; Norizan, M.N.; Hamzah, F.; Mat, H.B. Recent advances in enzyme immobilisation strategies: An overview of techniques and composite carriers. J. Compos. Sci. 2023, 7, 488. [Google Scholar] [CrossRef]
  9. Onoka, I.; Makangara, J.J. Micro-and nanocarriers for immobilization of enzymes. In Marine Biopolymers, 1st ed.; Elsevier: San Diego, CA, USA, 2025; pp. 545–572. ISBN 978-0-443-15606-9. [Google Scholar]
  10. Ayub, J.; Saeed, M.U.; Hussain, N.; Zulfiqar, I.; Mehmood, T.; Iqbal, H.M.; Bilal, M. Designing robust nano-biocatalysts using nanomaterials as multifunctional carriers-expanding the application scope of bio-enzymes. Top. Catal. 2023, 66, 625–648. [Google Scholar] [CrossRef]
  11. Tan, W.Y.; Gopinath, S.C.; Anbu, P.; Yaakub, A.R.W.; Subramaniam, S.; Chen, Y.; Sasidharan, S. Bio-enzyme hybrid with nanomaterials: A potential cargo as sustainable biocatalyst. Sustainability 2023, 15, 7511. [Google Scholar] [CrossRef]
  12. Yuan, Y.; Shen, J.; Salmon, S. Developing enzyme immobilization with fibrous membranes: Longevity and characterization considerations. Membranes 2023, 13, 532. [Google Scholar] [CrossRef]
  13. Reis, C.L.B.; Sousa, E.Y.A.D.; Serpa, J.D.F.; Oliveira, R.C.; Santos, J.C.S.D. Design of immobilized enzyme biocatalysts: Drawbacks and opportunities. Química Nova 2019, 42, 768–783. [Google Scholar] [CrossRef]
  14. Jiang, Y.; Zheng, J.; Wang, M.; Xu, W.; Wang, Y.; Wen, L.; Dong, J. Pros and cons in various immobilization techniques and carriers for enzymes. Appl. Biochem. Biotechnol. 2024, 196, 5633–5655. [Google Scholar] [CrossRef] [PubMed]
  15. Bouguerra, O.M.; Wahab, R.A.; Huyop, F.; Al-Fakih, A.M.; Mahmood, W.M.A.W.; Mahat, N.A.; Sabullah, M.K. An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications. Appl. Biochem. Biotechnol. 2024, 196, 5711–5739. [Google Scholar] [CrossRef] [PubMed]
  16. Fan, X.; Zhai, S.; Xue, S.; Zhi, L. Enzyme immobilization using covalent organic frameworks: From synthetic strategy to COFs functional role. ACS Appl. Mater. Interfaces 2024, 16, 40371–40390. [Google Scholar] [CrossRef] [PubMed]
  17. Bai, H.; Yu, D.; Du, X. Review of porous microspheres for enzyme immobilization: Strategies, applications, and prospects. Int. J. Biol. Macromol. 2025, 295, 139627. [Google Scholar] [CrossRef]
  18. Zhang, L.; Yin, B.; Wang, C.; Jiang, S.; Wang, H.; Yuan, Y.A.; Wei, D. Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803. J. Struct. Biol. 2014, 188, 93–101. [Google Scholar] [CrossRef]
  19. Teixeira, S.; Sousa, S.F.; NMFSA, C. An Unsual Cys-Glu-Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2. ChemPhysChem 2021, 22, 796–804. [Google Scholar] [CrossRef]
  20. Bhattacharya, A.; Pandit, S.; Lee, S.; Ebrahimi, S.B.; Samanta, D. Modulating enzyme activity using engineered nanomaterials. ChemBioChem 2025, 26, e202400520. [Google Scholar] [CrossRef]
  21. Mishra, S.D.; Kawadkar, J.; Joshi, P.A.; Vats, K.; Srivastava, A.; Mishra, R.K.; Rai, V. Amphoteric Cross-Linked Cellulose Nanoparticles as a Platform for Immobilization of Proteins and Cells, Enabling Bioanalyte Sensing. ACS Appl. Mater. Interfaces 2025, 17, 7262–7274. [Google Scholar] [CrossRef]
  22. Patil, P.D.; Gargate, N.; Tiwari, M.S.; Nadar, S.S. Defect metal-organic frameworks (D-MOFs): An engineered nanomaterial for enzyme immobilization. Coord. Chem. Rev. 2025, 531, 216519. [Google Scholar] [CrossRef]
  23. Zaixing, L.; Chao, L.; Qinqin, Z.; Chen, H.; Wenjing, Z.; Bingbing, X.; Xue, Q.; Guixia, L.; Zhifang, N. Magnetic nanocomposites as multifunctional carriers for enzymes immobilization: A review. Chem. Pap. 2024, 78, 1353–1365. [Google Scholar] [CrossRef]
  24. Dakal, T.C.; Bhushan, R.; Dhakar, R.; Kumar, A. Classification and Applications of Bio-nanomaterials. In Bio-Nanomaterials in Environmental Remediation: Industrial Applications; Wiley-VCH: Weinheim, Germany, 2025; pp. 47–73. [Google Scholar]
  25. Da Costa, F.P.; Cipolatti, E.P.; Furigo, A., Jr.; Oliveira Henriques, R. Nanoflowers: A new approach of enzyme immobilization. Chem. Rec. 2022, 22, e202100293. [Google Scholar] [CrossRef] [PubMed]
  26. Kumari, P.; Khatik, A.G.; Patil, P.D.; Tiwari, M.S.; Nadar, S.S.; Jain, A.K. Recent immobilization techniques for ketoreducases: Its design and their industrial application. Biocatal. Agric. Biotechnol. 2024, 56, 103027. [Google Scholar] [CrossRef]
  27. Heinks, T.; Montua, N.; Teune, M.; Liedtke, J.; Höhne, M.; Bornscheuer, U.T.; Fischer von Mollard, G. Comparison of Four Immobilization Methods for Different Transaminases. Catalysts 2023, 13, 300. [Google Scholar] [CrossRef]
  28. Anboo, S.; Lau, S.Y.; Kansedo, J.; Yap, P.S.; Hadibarata, T.; Jeevanandam, J.; Kamaruddin, A.H. Recent advancements in enzyme-incorporated nanomaterials: Synthesis, mechanistic formation, and applications. Biotechnol. Bioeng. 2022, 119, 2609–2638. [Google Scholar] [CrossRef]
  29. Saleh, T.A. Nanomaterials: Classification, properties, and environmental toxicities. Environ. Technol. Innov. 2020, 20, 101067. [Google Scholar] [CrossRef]
  30. Salem, S.S.; Hammad, E.N.; Mohamed, A.A.; El-Dougdoug, W. A comprehensive review of nanomaterials: Types, synthesis, characterization, and applications. Biointerface Res. Appl. Chem. 2022, 13, 41. [Google Scholar] [CrossRef]
  31. Trotta, F.; Mele, A. Nanomaterials: Classification and properties. In Nanosponges: Synthesis and Applications; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2019; pp. 1–26. [Google Scholar]
  32. Rodríguez-Gómez, F.D.; Penon, O.; Monferrer, D.; Rivera-Gil, P. Classification system for nanotechnology-enabled health products with both scientific and regulatory application. Front. Med. 2023, 10, 1212949. [Google Scholar] [CrossRef]
  33. Patel, S.K.; Choi, H.; Lee, J.K. Multimetal-based inorganic–protein hybrid system for enzyme immobilization. ACS Sustain. Chem. Eng. 2019, 7, 13633–13638. [Google Scholar] [CrossRef]
  34. Kumari, A.; Rajeev, R.; Benny, L.; Sudhakar, Y.N.; Varghese, A.; Hegde, G. Recent advances in carbon nanotubes-based biocatalysts and their applications. Adv. Colloid. Interface Sci. 2021, 297, 102542. [Google Scholar] [CrossRef]
  35. Zhang, Y.; Gu, J.; Fu, Y.; Shi, W.; Wang, X.; Su, Y.; Wang, X.; Wang, X. Immobilization of lipase on mesoporous silica nanocarriers for efficient preparation of phytosterol esters. Int. J. Biol. Macromol. 2025, 286, 138310. [Google Scholar] [CrossRef]
  36. Prlainović, N.Ž.; Milovanović, J.S.; Milašinović, N.Z.; Bezbradica, D.I.; Mijin, D.Ž. Multi-walled carbon nanotubes as lipase carriers for organic synthesis: Current trends and recent update. Hem. Ind. 2024, 78, 1–16. [Google Scholar] [CrossRef]
  37. Zhang, M.; Luo, M.; Chen, G.; Chi, C.; Zhao, J. A novel Affi-Cova magnetic nanoparticles for one-step covalent immobilization of His-tagged enzyme directly from crude cell lysate. Int. J. Biol. Macromol. 2024, 280, 135811. [Google Scholar] [CrossRef] [PubMed]
  38. Meskher, H.; Ragdi, T.; Thakur, A.K.; Ha, S.; Khelfaoui, I.; Sathyamurthy, R.; Sharshir, S.W.; Pandey, A.K.; Saidur, R.; Singh, P.; et al. A review on CNTs-based electrochemical sensors and biosensors: Unique properties and potential applications. Crit. Rev. Anal. Chem. 2024, 54, 2398–2421. [Google Scholar] [CrossRef]
  39. Ranjbari, S.; Bolourinezhad, M.; Kesharwani, P.; Rezayi, M.; Sahebkar, A. Applications of Carbon Nanotube Biosensors: Sensing the Future. J. Drug Deliv. Technol. 2024, 97, 105747. [Google Scholar] [CrossRef]
  40. Li, L.; Wang, T.; Zhong, Y.; Li, R.; Deng, W.; Xiao, X.; Xu, Y.; Zhang, J.; Hu, X.; Wang, Y. A review of nanomaterials for biosensing applications. J. Mater. Chem. B 2024, 12, 1168–1193. [Google Scholar] [CrossRef] [PubMed]
  41. Acharya, R.; Patil, T.V.; Dutta, S.D.; Lee, J.; Ganguly, K.; Kim, H.; Randhawa, A.; Lim, K.T. Single-Walled Carbon Nanotube-Based Optical Nano/Biosensors for Biomedical Applications: Role in Bioimaging, Disease Diagnosis, and Biomarkers Detection. Adv. Mater. Technol. 2024, 9, 2400279. [Google Scholar] [CrossRef]
  42. Saguin, N.S.G.; Maulik, G.; Cao, X.; Luo, X.; Nag, A.; Gao, J.; Deng, S.; Wong, J.W. CNTs-based biosensors for enzyme detection. Sens. Actuators A Phys. 2024, 377, 115753. [Google Scholar] [CrossRef]
  43. Zhou, Y.; Zheng, S.; Qin, W. Electrochemical biochemical oxygen demand biosensors and their applications in aquatic environmental monitoring. Sens. Bio-Sens. Res. 2024, 44, 100642. [Google Scholar] [CrossRef]
  44. Liu, H.Y.; Zhu, Z.; He, J.; Yang, Y.; Liang, Y.; Li, Z.; Zhu, M.; Xiao, M.; Zhang, Z. Mass Production of Carbon Nanotube Transistor Biosensors for Point-of-Care Tests. Nano Lett. 2024, 24, 10510–10518. [Google Scholar] [CrossRef]
  45. Masood, M.; Albayouk, T.; Saleh, N.I.; El-Shazly, M.; El-Nashar, H.A. Carbon nanotubes: A novel innovation as food supplements and biosensing for food safety. Front. Nutr. 2024, 11, 1381179. [Google Scholar] [CrossRef]
  46. Pikula, K.; Johari, S.A.; Santos-Oliveira, R.; Golokhvast, K. Toxicity and biotransformation of carbon-based nanomaterials in marine microalgae Heterosigma akashiwo. Int. J. Mol. Sci. 2023, 24, 10020. [Google Scholar] [CrossRef]
  47. Bhattacharya, K.; Mukherjee, S.P.; Gallud, A.; Burkert, S.C.; Bistarelli, S.; Bellucci, S.; Bottini, M.; Star, A.; Fadeel, B. Biological interactions of carbon-based nanomaterials: From coronation to degradation. Nanomed. Nanotechnol. Biol. Med. 2016, 12, 333–351. [Google Scholar] [CrossRef]
  48. Abdulmalek, S.A.; Li, K.; Wang, J.; Ghide, M.K.; Yan, Y. Co-immobilization of Rhizopus oryzae and Candida rugosa lipases onto mMWCNTs@ 4-arm-PEG-NH2—A novel magnetic nanotube–polyethylene glycol amine composite—And its applications for biodiesel production. Int. J. Mol. Sci. 2021, 22, 11956. [Google Scholar] [CrossRef]
  49. Moradpour, H.; Forootanfar, H.; Ameri, A.; Beitollahi, H. Fabrication of the carbon paste electrode modified with Trametes versicolor laccase immobilized on carboxyl functionalized multi-walled carbon nanotubes and its application for measurement of dopamine. Int. J. Biol. Macromol. 2024, 283, 137891. [Google Scholar] [CrossRef] [PubMed]
  50. Perera, M.; Ibrahim, G.G.; Yang, M.; Yan, J.; Yan, Y. Improved performance of Pseudomonas fluorescens lipase immobilized on a magnetic mesoporous solvothermal hybrid-nanocarrier and its application in ultrasonication assisted estolide synthesis. J. Ind. Eng. Chem. 2024, 140, 258–268. [Google Scholar] [CrossRef]
  51. Yang, Y.Y.; Jing, S.L.; Shao, J.L.; Chen, J.X.; Zhang, W.F.; Wan, S.Y.; Shen, Y.P.; Yang, H.; Yu, W. Purification and immobilization of β-glucosidase using surface modified mesoporous silica Santa Barbara Amorphous 15 for eco-friendly preparation of sagittatoside A. Nat. Prod. Bioprospect. 2024, 14, 50. [Google Scholar] [CrossRef] [PubMed]
  52. Varan, N.E.; Yildirim, D.; Toprak, A.; Fernandéz-Lafuente, R.; Alagöz, D. Effect of the activation strategy of nickel oxide-multi-walled carbon nanotubes on the immobilization of xylanase for synthesis of xylooligosaccharides. Appl. Biochem. Biotechnol. 2024, 72, 911–923. [Google Scholar] [CrossRef]
  53. Kaur, P.; Jana, A.K. Candida rugosa lipase immobilization on Fe3O4 coated carboxyl functionalised multiwalled carbon nanotubes for production of food flavour esters. Biotechnol. Bioprocess. Eng. 2023, 28, 310–326. [Google Scholar] [CrossRef]
  54. Özdemir, F.İ.; Karaaslan, B.; Tülek, A.; Yucebilgic, G.; Yildirim, D. Immobilization of recombinant L-asparaginase from Geobacillus kaustophilus on magnetic MWCNT-nickel composites. Process Biochem. 2023, 127, 10–20. [Google Scholar] [CrossRef]
  55. Habimana, P.; Jiang, Y.; Gao, J.; Ndayambaje, J.B.; Darwesh, O.M.; Mwizerwa, J.P.; Zheng, X.; Ma, L. Enhancing laccase stability and activity for dyes decolorization using ZIF-8@ MWCNT nanocomposite. Chin. J. Chem. Eng. 2022, 48, 66–75. [Google Scholar] [CrossRef]
  56. Patila, M.; Athanasiou, P.E.; Kortessis, L.; Potsi, G.; Kouloumpis, A.; Gournis, D.; Stamatis, H. Immobilization of laccase on hybrid super-structured nanomaterials for the decolorization of phenolic dyes. Processes 2022, 10, 233. [Google Scholar] [CrossRef]
  57. Alagöz, D.; Toprak, A.; Yildirim, D.; Tükel, S.S.; Fernandez-Lafuente, R. Modified silicates and carbon nanotubes for immobilization of lipase from Rhizomucor miehei: Effect of support and immobilization technique on the catalytic performance of the immobilized biocatalysts. Enzym. Microb. Technol. 2021, 144, 109739. [Google Scholar] [CrossRef]
  58. Ji, S.; Liu, W.; Su, S.; Gan, C.; Jia, C. Chitosan derivative functionalized carbon nanotubes as carriers for enzyme immobilization to improve synthetic efficiency of ethyl caproate. LWT 2021, 149, 111897. [Google Scholar] [CrossRef]
  59. Khan, M.; Husain, Q. Multiwalled carbon nanotubes bound beta-galactosidase: It’s activity, stability and reusability. In Methods in Enzymology; Academic Press: Cambridge, MA, USA, 2020; Volume 630, pp. 365–405. [Google Scholar]
  60. Mahmoodi, N.M.; Saffar-Dastgerdi, M.H.; Hayati, B. Environmentally friendly novel covalently immobilized enzyme bionanocomposite: From synthesis to the destruction of pollutant. Compos. B Eng. 2020, 184, 107666. [Google Scholar] [CrossRef]
  61. Yadav, A.; Agrawal, D.C.; Srivastava, R.R.; Srivastava, A.; Kayastha, A.M. Nanoparticles decorated carbon nanotubes as novel matrix: A comparative study of influences of immobilization on the catalytic properties of Lens culinaris β-galactosidase (Lcβ-gal). Int. J. Biol. Macromol. 2020, 144, 770–780. [Google Scholar] [CrossRef] [PubMed]
  62. Ranjan, B.; Pillai, S.; Permaul, K.; Singh, S. Simultaneous removal of heavy metals and cyanate in a wastewater sample using immobilized cyanate hydratase on magnetic-multiwall carbon nanotubes. J. Hazard. Mater. 2019, 363, 73–80. [Google Scholar] [CrossRef]
  63. Kumar, A.; Park, G.D.; Patel, S.K.; Kondaveeti, S.; Otari, S.; Anwar, M.Z.; Kalia, V.C.; Singh, Y.; Kim, S.C.; Cho, B.K.; et al. SiO2 microparticles with carbon nanotube-derived mesopores as an efficient support for enzyme immobilization. Chem. Eng. J. 2019, 359, 1252–1264. [Google Scholar] [CrossRef]
  64. Asmat, S.; Anwer, A.H.; Husain, Q. Immobilization of lipase onto novel constructed polydopamine grafted multiwalled carbon nanotube impregnated with magnetic cobalt and its application in synthesis of fruit flavours. Int. J. Biol. Macromol. 2019, 140, 484–495. [Google Scholar] [CrossRef]
  65. Li, L.J.; Xia, W.J.; Ma, G.P.; Chen, Y.L.; Ma, Y.Y. A study on the enzymatic properties and reuse of cellulase immobilized with carbon nanotubes and sodium alginate. AMB Express 2019, 9, 112. [Google Scholar] [CrossRef]
  66. Szelwicka, A.; Boncel, S.; Jurczyk, S.; Chrobok, A. Exceptionally active and reusable nanobiocatalyst comprising lipase non-covalently immobilized on multi-wall carbon nanotubes for the synthesis of diester plasticizers. Appl. Catal. A Gen. 2019, 574, 41–47. [Google Scholar] [CrossRef]
  67. Ahmad, R.; Khare, S.K. Immobilization of Aspergillus niger cellulase on multiwall carbon nanotubes for cellulose hydrolysis. Bioresour. Technol. 2018, 252, 72–75. [Google Scholar] [CrossRef]
  68. Bencze, L.C.; Bartha-Vári, J.H.; Katona, G.; Toşa, M.I.; Paizs, C.; Irimie, F.D. Nanobioconjugates of Candida antarctica lipase B and single-walled carbon nanotubes in biodiesel production. Bioresour. Technol. 2016, 200, 853–860. [Google Scholar] [CrossRef]
  69. Fan, Y.; Wu, G.; Su, F.; Li, K.; Xu, L.; Han, X.; Yan, Y. Lipase oriented-immobilized on dendrimer-coated magnetic multi-walled carbon nanotubes toward catalyzing biodiesel production from waste vegetable oil. Fuel 2016, 178, 172–178. [Google Scholar] [CrossRef]
  70. Sahu, S.; Shera, S.S.; Banik, R.M. Enhanced reusability of horseradish peroxidase immobilized onto graphene oxide/magnetic chitosan beads for cost effective cholesterol oxidase assay. Open Biotechnol. J. 2019, 13, 93–104. [Google Scholar] [CrossRef]
  71. Banerjee, A.N. Graphene and its derivatives as biomedical materials: Future prospects and challenges. Interface Focus 2018, 8, 20170056. [Google Scholar] [CrossRef] [PubMed]
  72. Sun, J.; Du, S. Application of graphene derivatives and their nanocomposites in tribology and lubrication: A review. RSC Adv. 2019, 9, 40642–40661. [Google Scholar] [CrossRef]
  73. Gao, F.; Guo, Y.; Fan, X.; Hu, M.; Li, S.; Zhai, Q.; Jiang, Y.; Wang, X. Enhancing the catalytic performance of chloroperoxidase by co-immobilization with glucose oxidase on magnetic graphene oxide. Biochem. Eng. J. 2019, 143, 101–109. [Google Scholar] [CrossRef]
  74. Wu, Z.Y.; Zhang, H.; Yang, Y.Y.; Yang, F.Q. An online dual-enzyme co-immobilized microreactor based on capillary electrophoresis for enzyme kinetics assays and screening of dual-target inhibitors against thrombin and factor Xa. J. Chromatogr. A 2020, 1619, 460948. [Google Scholar] [CrossRef]
  75. Mahmood Khan, R.; Pervaiz, M.; Saeed, Z.; Majid, A.; Liaqat, M. Graphene-Based Enzymatic and Non-Enzymatic Electrochemical Glucose Sensors: Review of Current Research and Advances in Nanotechnology. ChemistrySelect 2023, 8, e202303952. [Google Scholar]
  76. Srivastava, N.; Singh, R.; Lal, B.; Mohammad, A.; Rai, A.K.; Ahmad, I.; Srivastava, M.; Choi, C.H.; Gupta, V.K. Graphene and its derivatives fabrication from paddy straw for improved and sustainable application in biofuels production: New Insight. Process Saf. Environ. Prot. 2024, 187, 1596–1605. [Google Scholar] [CrossRef]
  77. Krishna Perumal, P.; Chen, C.W.; Giri, B.S.; Singhania, R.R.; Patel, A.K.; Dong, C.D. Graphene-based functional electrochemical sensors for the detection of chlorpyrifos in water and food samples: A review. J. Food Sci. Technol. 2024, 61, 631–641. [Google Scholar] [CrossRef] [PubMed]
  78. Zhao, W.; Zhang, W.; Chen, J.; Li, H.; Han, L.; Li, X.; Wang, J.; Song, W.; Xu, C.; Cai, X.; et al. Sensitivity-Enhancing Strategies of Graphene Field-Effect Transistor Biosensors for Biomarker Detection. ACS Sens. 2024, 9, 2705–2727. [Google Scholar] [CrossRef]
  79. Joshi, R.; Ravindran, V.; Lahiri, I. Graphene-based materials and electrochemical biosensors: An overview. J. Phys. Condens. Matter 2025, 37, 143001. [Google Scholar] [CrossRef] [PubMed]
  80. Guha, S.; Chakrabarty, S. Graphene and its derivatives (GO, rGO and GQD): A comprehensive review of their role in combating COVID-19. J. Adv. Phys. 2025, 10, 2435278. [Google Scholar] [CrossRef]
  81. Shang, H.; Wang, J.; Guo, B.; Zhu, H.; Li, H. Immobilization of Phospholipase D on Fe3O4@SiO2-Graphene Oxide Nanocomposites: A Strategy to Improve Catalytic Stability and Reusability in the Efficient Production of Phosphatidylserine. Molecules 2025, 30, 912. [Google Scholar] [CrossRef]
  82. Monajati, M.; Ariafar, N.; Abedi, M.; Borandeh, S.; Tamaddon, A.M. Immobilization of L-Asparaginase on biofunctionalized magnetic graphene oxide nanocomposite: A promising approach for Enhanced Stability and reusability. Heliyon 2024, 10, e40072. [Google Scholar] [CrossRef]
  83. Hou, H.; Xu, F.; Ding, X.; Zheng, L.; Shi, J. Magnetic biocatalytic nanoreactors based on graphene oxide with graded reduction degrees for the enzymatic synthesis of phytosterol esters. Carbon. 2024, 226, 119170. [Google Scholar] [CrossRef]
  84. Pinto, G.C.; Lucena, G.N.; Piazza, R.D.; Costa, J.M.L.; e Silva, E.T.C.C.; Gu, Y.; de Paula, A.V.; Silva, N.J.O.; Marques, R.F.C. Evaluation of the alternating magnetic field (AMF) influence in catalytic activities of enzymes immobilized into magnetic graphene oxide: A new approach. Mater. Today Commun. 2023, 36, 106441. [Google Scholar] [CrossRef]
  85. Yu, D.; Li, Z.; Zhou, X.; Wang, W.; Wang, L.; Liu, T.; Du, J. Study on the modification of magnetic graphene oxide and the effect of immobilized lipase. Int. J. Biol. Macromol. 2022, 216, 498–509. [Google Scholar] [CrossRef]
  86. Badoei-Dalfard, A.; Tahami, A.; Karami, Z. Lipase immobilization on glutaraldehyde activated graphene oxide/chitosan/cellulose acetate electrospun nanofibrous membranes and its application on the synthesis of benzyl acetate. Colloids Surf. B Biointerfaces 2022, 209, 112151. [Google Scholar] [CrossRef] [PubMed]
  87. Paz-Cedeno, F.R.; Carceller, J.M.; Iborra, S.; Donato, R.K.; Godoy, A.P.; de Paula, A.V.; Monti, R.; Corma, A.; Masarin, F. Magnetic graphene oxide as a platform for the immobilization of cellulases and xylanases: Ultrastructural characterization and assessment of lignocellulosic biomass hydrolysis. Renew. Energy 2021, 164, 491–501. [Google Scholar] [CrossRef]
  88. Ariaeenejad, S.; Motamedi, E.; Salekdeh, G.H. Application of the immobilized enzyme on magnetic graphene oxide nano-carrier as a versatile bi-functional tool for efficient removal of dye from water. Bioresour. Technol. 2021, 319, 124228. [Google Scholar] [CrossRef]
  89. Li, Y.; Wang, B.; Wu, M.; Huan, W.; Li, J. Magnetic graphene oxide nanocomposites as an effective support for lactase immobilization with improved stability and enhanced photothermal enzymatic activity. New J. Chem. 2021, 45, 5939–5948. [Google Scholar] [CrossRef]
  90. Rong, J.; Zhou, Z.; Wang, Y.; Han, J.; Li, C.; Zhang, W.; Ni, L. Immobilization of horseradish peroxidase on multi-armed magnetic graphene oxide composite: Improvement of loading amount and catalytic activity. Food Technol. Biotechnol. 2019, 57, 260–271. [Google Scholar] [CrossRef]
  91. Wang, Y.; Wang, Q.; Song, X.; Cai, J. Hydrophilic polyethylenimine modified magnetic graphene oxide composite as an efficient support for dextranase immobilization with improved stability and recyclable performance. Biochem. Eng. J. 2019, 141, 163–172. [Google Scholar] [CrossRef]
  92. Lin, P.; Zhang, Y.; Ren, H.; Wang, Y.; Wang, S.; Fang, B. Assembly of graphene oxide-formate dehydrogenase composites by nickel-coordination with enhanced stability and reusability. Eng. Life Sci. 2018, 18, 326–333. [Google Scholar] [CrossRef]
  93. Vineh, M.B.; Saboury, A.A.; Poostchi, A.A.; Rashidi, A.M.; Parivar, K. Stability and activity improvement of horseradish peroxidase by covalent immobilization on functionalized reduced graphene oxide and biodegradation of high phenol concentration. Int. J. Biol. Macromol. 2018, 106, 1314–1322. [Google Scholar] [CrossRef]
  94. Samak, N.A.; Tan, Y.; Sui, K.; Xia, T.T.; Wang, K.; Guo, C.; Liu, C. CotA laccase immobilized on functionalized magnetic graphene oxide nano-sheets for efficient biocatalysis. Mol. Catal. 2018, 445, 269–278. [Google Scholar] [CrossRef]
  95. Besharati Vineh, M.; Saboury, A.A.; Poostchi, A.A.; Mamani, L. Physical adsorption of horseradish peroxidase on reduced graphene oxide nanosheets functionalized by amine: A good system for biodegradation of high phenol concentration in wastewater. Int. J. Environ. Res. 2018, 12, 45–57. [Google Scholar] [CrossRef]
  96. Asmat, S.; Husain, Q. Exquisite stability and catalytic performance of immobilized lipase on novel fabricated nanocellulose fused polypyrrole/graphene oxide nanocomposite: Characterization and application. Int. J. Biol. Macromol. 2018, 117, 331–341. [Google Scholar] [CrossRef] [PubMed]
  97. Patel, S.K.; Choi, S.H.; Kang, Y.C.; Lee, J.K. Eco-friendly composite of Fe3O4-reduced graphene oxide particles for efficient enzyme immobilization. ACS Appl. Mater. Interfaces 2017, 9, 2213–2222. [Google Scholar] [CrossRef]
  98. Gong, A.; Zhu, C.T.; Xu, Y.; Wang, F.Q.; Tsabing, D.A.K.; Wu, F.A.; Wang, J. Moving and unsinkable graphene sheets immobilized enzyme for microfluidic biocatalysis. Sci. Rep. 2017, 7, 4309. [Google Scholar] [CrossRef] [PubMed]
  99. Asmat, S.; Husain, Q.; Azam, A. Lipase immobilization on facile synthesized polyaniline-coated silver-functionalized graphene oxide nanocomposites as novel biocatalysts: Stability and activity insights. RSC Adv. 2017, 7, 5019–5029. [Google Scholar] [CrossRef]
  100. Chen, J.; Leng, J.; Yang, X.; Liao, L.; Liu, L.; Xiao, A. Enhanced performance of magnetic graphene oxide-immobilized laccase and its application for the decolorization of dyes. Molecules 2017, 22, 221. [Google Scholar] [CrossRef]
  101. Zhang, S.; Shi, J.; Deng, Q.; Zheng, M.; Wan, C.; Zheng, C.; Li, Y.; Huang, F. Preparation of carriers based on ZnO nanoparticles decorated on graphene oxide (GO) nanosheets for efficient immobilization of lipase from Candida rugosa. Molecules 2017, 22, 1205. [Google Scholar] [CrossRef]
  102. Ding, Y.; Cui, R.; Hu, M.; Li, S.; Zhai, Q.; Jiang, Y. Well-oriented bioarchitecture for immobilization of chloroperoxidase on graphene oxide nanosheets by site-specific interactions and its catalytic performance. J. Mater. Sci. 2017, 52, 10001–10012. [Google Scholar] [CrossRef]
  103. Li, W.; Wen, H.; Shi, Q.; Zheng, G. Study on immobilization of (+) γ-lactamase using a new type of epoxy graphene oxide carrier. Process Biochem. 2016, 51, 270–276. [Google Scholar] [CrossRef]
  104. Royvaran, M.; Taheri-Kafrani, A.; Isfahani, A.L.; Mohammadi, S. Functionalized superparamagnetic graphene oxide nanosheet in enzyme engineering: A highly dispersive, stable and robust biocatalyst. Chem. Eng. J. 2016, 288, 414–422. [Google Scholar] [CrossRef]
  105. Khan, M.; Husain, Q.; Naqvi, A.H. Graphene based magnetic nanocomposites as versatile carriers for high yield immobilization and stabilization of β-galactosidase. RSC Adv. 2016, 6, 53493–53503. [Google Scholar] [CrossRef]
  106. Singh, K.; Srivastava, G.; Talat, M.; Srivastava, O.N.; Kayastha, A.M. α-Amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization, kinetics and potential applications in starch based industries. Biochem. Biophys. Rep. 2015, 3, 18–25. [Google Scholar] [CrossRef]
  107. Patel, V.; Gajera, H.; Gupta, A.; Manocha, L.; Madamwar, D. Synthesis of ethyl caprylate in organic media using Candida rugosa lipase immobilized on exfoliated graphene oxide: Process parameters and reusability studies. Biochem. Eng. J. 2015, 95, 62–70. [Google Scholar] [CrossRef]
  108. Shao, Y.; Jing, T.; Tian, J.; Zheng, Y. Graphene oxide-based Fe3O4 nanoparticles as a novel scaffold for the immobilization of porcine pancreatic lipase. RSC Adv. 2015, 5, 103943–103955. [Google Scholar] [CrossRef]
  109. Yoon, Y.; Truong, P.L.; Lee, D.; Ko, S.H. Metal-oxide nanomaterials synthesis and applications in flexible and wearable sensors. ACS Nanosci. Au 2021, 2, 64–92. [Google Scholar] [CrossRef] [PubMed]
  110. Mujahid, M.H.; Upadhyay, T.K.; Khan, F.; Pandey, P.; Park, M.N.; Sharangi, A.B.; Saeed, M.; Upadhye, V.J.; Kim, B. Metallic and metal oxide-derived nanohybrid as a tool for biomedical applications. Biomed. Pharmacother. 2022, 155, 113791. [Google Scholar] [CrossRef] [PubMed]
  111. Bilal, M.; Iqbal, H.M.; Adil, S.F.; Shaik, M.R.; Abdelgawad, A.; Hatshan, M.R.; Khan, M. Surface-coated magnetic nanostructured materials for robust bio-catalysis and biomedical applications-A review. J. Adv. Res. 2022, 38, 157–177. [Google Scholar] [CrossRef] [PubMed]
  112. Li, C.; Jiang, S.; Zhao, X.; Liang, H. Co-immobilization of enzymes and magnetic nanoparticles by metal-nucleotide hydrogelnanofibers for improving stability and recycling. Molecules 2017, 22, 179. [Google Scholar] [CrossRef]
  113. Varshan, G.A.; Namasivayam, S.K.R. A green chemistry principle for the biotransformation of fungal biomass derived chitosan into versatile nano scale materials with high biocompatibility and potential biological activities—A review. BioNanoScience 2024, 14, 4145–4166. [Google Scholar] [CrossRef]
  114. Jacinto, C.; Javed, Y.; Lavorato, G.; Tarraga, W.A.; Conde, B.I.C.; Orozco, J.M.; Picco, A.S.; Garcia, J.; Dias, C.S.B.; Malik, S.; et al. Biotransformation and biological fate of magnetic iron oxide nanoparticles for biomedical research and clinical applications. Nanoscale Adv. 2025, 7, 2818–2886. [Google Scholar] [CrossRef]
  115. Huang, S.W.; Yao, Y.Y.; Zhang, H.X.; Guo, W.Y.; Fang, M.H.; Wang, H.B.; Sun, Y.J.; Li, M.H. Novel mechanisms for selenite biotransformation and selenium nanoparticles biogenesis in Acinetobacter sp. SX5 isolated from seleniferous soil. J. Hazard. Mater 2025, 489, 137694. [Google Scholar] [CrossRef]
  116. Perera, K.Y.; Pradhan, D.; Rafferty, A.; Jaiswal, A.K.; Jaiswal, S. A comprehensive review on metal oxide-nanocellulose composites in sustainable active and intelligent food packaging. Food Chem. Adv. 2023, 3, 100436. [Google Scholar] [CrossRef]
  117. Kyomuhimbo, H.D.; Feleni, U.; Haneklaus, N.H.; Brink, H. Recent advances in applications of oxidases and peroxidases polymer-based enzyme biocatalysts in sensing and wastewater treatment: A review. Polymers 2023, 15, 3492. [Google Scholar] [CrossRef] [PubMed]
  118. Mashhadban, K.F.; Gorgani, L.; Darzi, G.N. Enzymatic electrochemical biosensors for urea detection: A review. Sens. Actuators A Phys. 2024, 374, 115499. [Google Scholar] [CrossRef]
  119. Gupta, D.; Joseph, K.; Dixit, P.; Gupta, T.K. Industrial applications of nanoparticle immobilized enzymes. In Nano-Enzyme Incorporated Particles; Academic Press: Cambridge, MA, USA, 2024; pp. 187–220. [Google Scholar]
  120. Chaudhari, D.S.; Upadhyay, R.P.; Shinde, G.Y.; Gawande, M.B.; Filip, J.; Varma, R.S.; Zboril, R. A review on sustainable iron oxide nanoparticles: Synthesis and application in organic catalysis and environmental remediation. Green. Chem. 2024, 26, 7579–7655. [Google Scholar] [CrossRef]
  121. Sun, T.; Fu, M.; Xing, J.; Ge, Z. Magnetic nanoparticles encapsulated laccase nanoflowers: Evaluation of enzymatic activity and reusability for degradation of malachite green. Water Sci. Technol. 2020, 81, 29–39. [Google Scholar] [CrossRef]
  122. Atiroğlu, V.; Atiroğlu, A.; Özacar, M. Immobilization of α-amylase enzyme on a protein@ metal–organic framework nanocomposite: A new strategy to develop the reusability and stability of the enzyme. Food Chem. 2021, 349, 129127. [Google Scholar] [CrossRef]
  123. Salem, K.; Jabalera, Y.; Puentes-Pardo, J.D.; Vilchez-Garcia, J.; Sayari, A.; Hmida-Sayari, A.; Jimenez-Lopez, C.; Perduca, M. Enzyme storage and recycling: Nanoassemblies of α-amylase and xylanase immobilized on biomimetic magnetic nanoparticles. ACS Sustain. Chem. Eng. 2021, 9, 4054–4063. [Google Scholar] [CrossRef]
  124. Noma, S.A.A.; Ulu, A.; Acet, Ö.; Sanz, R.; Sanz-Pérez, E.S.; Odabaşı, M.; Ateş, B. Comparative study of ASNase immobilization on tannic acid-modified magnetic Fe3O4/SBA-15 nanoparticles to enhance stability and reusability. New J. Chem. 2020, 44, 4440–4451. [Google Scholar] [CrossRef]
  125. Ghannadi, S.; Abdizadeh, H.; Miroliaei, M.; Saboury, A.A. Immobilization of alcohol dehydrogenase on titania nanoparticles to enhance enzyme stability and remove substrate inhibition in the reaction of formaldehyde to methanol. Ind. Eng. Chem. Res. 2019, 58, 9844–9854. [Google Scholar] [CrossRef]
  126. Defaei, M.; Taheri-Kafrani, A.; Miroliaei, M.; Yaghmaei, P. Improvement of stability and reusability of α-amylase immobilized on naringin functionalized magnetic nanoparticles: A robust nanobiocatalyst. Int. J. Biol. Macromol. 2018, 113, 354–360. [Google Scholar] [CrossRef]
  127. Fortes, C.C.; Daniel-da-Silva, A.L.; Xavier, A.M.; Tavares, A.P. Optimization of enzyme immobilization on functionalized magnetic nanoparticles for laccase biocatalytic reactions. Chem. Eng. Process 2017, 117, 1–8. [Google Scholar] [CrossRef]
  128. Singh, V.; Rakshit, K.; Rathee, S.; Angmo, S.; Kaushal, S.; Garg, P.; Chung, J.H.; Sandhir, R.; Sangwan, R.S.; Singhal, N. Metallic/bimetallic magnetic nanoparticle functionalization for immobilization of α-amylase for enhanced reusability in bio-catalytic processes. Bioresour. Technol. 2016, 214, 528–533. [Google Scholar] [CrossRef]
  129. Motevalizadeh, S.F.; Khoobi, M.; Sadighi, A.; Khalilvand-Sedagheh, M.; Pazhouhandeh, M.; Ramazani, A.; Faramarzi, M.A.; Shafiee, A. Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions. J. Mol. Catal. B Enzym. 2015, 120, 75–83. [Google Scholar] [CrossRef]
  130. Meenakshi, M.; Bhaskar, R.; Kumar, S.A.; Kumar, R.S. A Concise Review on Magnetic Nanoparticles: Their Properties, Types, Synthetic Methods, and Current Trending Applications. Curr. Nanosci. 2025, 21, 2–17. [Google Scholar] [CrossRef]
  131. Oraby, K.R.; Villalonga, A.; Hassan, F.S.; Zayed, M.A.; Mubarak, M.F.; Ojeda, I.; Sánchez, A.; Villalonga, R. Immobilization of laccase on Fe3O4@ SiO2 core@ shell magnetic nanoparticles for methylene blue biodegradation. Process Biochem. 2025, 148, 10–16. [Google Scholar] [CrossRef]
  132. Seenuvasan, M.; Vinodhini, G.; Malar, C.G.; Balaji, N.; Kumar, K.S. Magnetic nanoparticles: A versatile carrier for enzymes in bio-processing sectors. IET Nanobiotechnol. 2018, 12, 535–548. [Google Scholar] [CrossRef]
  133. Kritika; Roy, I. Therapeutic applications of magnetic nanoparticles: Recent advances. Mater. Adv. 2022, 3, 7425–7444. [Google Scholar] [CrossRef]
  134. Yadav, A.; Kumar Yadav, D.; Rani, P.; Bhardwaj, N.; Gupta, A.; Bishnoi, N.R. Functionalized iron oxide nanoparticles for covalent immobilization of cellic CTec2 cellulase: Enabling enzyme reusability in cellulosic biomass conversion. Biofuels 2024, 15, 363–373. [Google Scholar] [CrossRef]
  135. Behshad, Y.; Pazhang, M.; Najavand, S.; Sabzi, M. Enhancing enzyme stability and functionality: Covalent immobilization of trypsin on magnetic gum arabic modified Fe3O4 nanoparticles. Appl. Biochem. Biotechnol. 2024, 196, 5283–5300. [Google Scholar] [CrossRef]
  136. Jimenez-Carretero, M.; Jabalera, Y.; Sola-Leyva, A.; Carrasco-Jimenez, M.P.; Jimenez-Lopez, C. Nanoassemblies of acetylcholinesterase and β-lactamase immobilized on magnetic nanoparticles as biosensors to detect pollutants in water. Talanta 2023, 258, 124406. [Google Scholar] [CrossRef]
  137. Da Silva Cavalcanti, M.H.; Alves, L.B.; Duarte, A.; Mendes, A.A.; da Silva, J.M.S.F.; da Silveira, N.J.F.; Escote, M.T.; Virtuoso, L.S. Immobilization of Thermomyces lanuginosus lipase via ionic adsorption on superparamagnetic iron oxide nanoparticles: Facile synthesis and improved catalytic performance. Chem. Eng. J. 2022, 431, 134128. [Google Scholar] [CrossRef]
  138. Zhou, Y.; Yang, Z.; Zhou, R.; Zeng, B.; Liu, X.; Li, X.; Zhang, G. Peptide-inspired one-step synthesis of surface-functionalized Fe3O4 magnetic nanoparticles for oriented enzyme immobilization and biocatalytic applications. ACS Appl. Nano Mater. 2022, 5, 8260–8270. [Google Scholar] [CrossRef]
  139. Cao, X.; Xu, H.; Li, F.; Zou, Y.; Ran, Y.; Ma, X.; Cao, Y.; Xu, Q.; Qiao, D.; Cao, Y. One-step direct transesterification of wet yeast for biodiesel production catalyzed by magnetic nanoparticle-immobilized lipase. Renew. Energy 2021, 171, 11–21. [Google Scholar] [CrossRef]
  140. Herman, R.A.; Zhu, X.; Ayepa, E.; Khurshid, M.; Zhang, Z.P.; You, S.; Qian, J.F.; Wang, J. Magnetic Janus SiO2 nanoparticles immobilized protease mutant T70I as a novel clarification agent for juice processing. Int. J. Biol. Macromol. 2025, 292, 139327. [Google Scholar] [CrossRef]
  141. Farhan, L.O.; Mehdi, W.A.; Taha, E.M.; Farhan, A.M.; Mehde, A.A.; Özacar, M. Various type immobilizations of Isocitrate dehydrogenases enzyme on hyaluronic acid modified magnetic nanoparticles as stable biocatalysts. Int. J. Biol. Macromol. 2021, 182, 217–227. [Google Scholar] [CrossRef] [PubMed]
  142. Zhu, Y.; Han, J.; Wu, J.; Li, Y.; Wang, L.; Mao, Y.; Wang, Y. A two-step method for the synthesis of magnetic immobilized cellulase with outstanding thermal stability and reusability. New J. Chem. 2021, 45, 6144–6150. [Google Scholar] [CrossRef]
  143. Coutinho, T.C.; Malafatti, J.O.; Paris, E.C.; Tardioli, P.W.; Farinas, C.S. Hydroxyapatite-CoFe2O4 magnetic nanoparticle composites for industrial enzyme immobilization, use, and recovery. ACS Appl. Nano Mater. 2020, 3, 12334–12345. [Google Scholar] [CrossRef]
  144. Suo, H.; Xu, L.; Xue, Y.; Qiu, X.; Huang, H.; Hu, Y. Ionic liquids-modified cellulose coated magnetic nanoparticles for enzyme immobilization: Improvement of catalytic performance. Carbohydr. Polym. 2020, 234, 115914. [Google Scholar] [CrossRef] [PubMed]
  145. Darwesh, O.M.; Matter, I.A.; Eida, M.F. Development of peroxidase enzyme immobilized magnetic nanoparticles for bioremediation of textile wastewater dye. J. Environ. Chem. Eng. 2019, 7, 102805. [Google Scholar] [CrossRef]
  146. Wang, X.Y.; Jiang, X.P.; Li, Y.; Zeng, S.; Zhang, Y.W. Preparation Fe3O4@ chitosan magnetic particles for covalent immobilization of lipase from Thermomyces lanuginosus. Int. J. Biol. Macromol. 2015, 75, 44–50. [Google Scholar] [CrossRef]
  147. Osuna, Y.; Sandoval, J.; Saade, H.; López, R.G.; Martinez, J.L.; Colunga, E.M.; de la Cruz, G.; Segura, E.P.; Arévalo, F.J.; Zon, M.A.; et al. Immobilization of Aspergillus niger lipase on chitosan-coated magnetic nanoparticles using two covalent-binding methods. Bioprocess. Biosyst. Eng. 2015, 38, 1437–1445. [Google Scholar] [CrossRef] [PubMed]
  148. Patil, S.S.; Rathod, V.K. Combined effect of enzyme co-immobilized magnetic nanoparticles (MNPs) and ultrasound for effective extraction and purification of curcuminoids from Curcuma longa. Ind. Crop. Prod. 2022, 177, 114385. [Google Scholar] [CrossRef]
  149. Liao, J.; Han, S.; Li, X.; He, J.; Secundo, F.; Liang, H. Co-immobilization of two-component hydroxylase monooxygenase by functionalized magnetic nanoparticles for preserving high catalytic activity and enhancing enzyme stability. Int. J. Biol. Macromol. 2020, 164, 3163–3170. [Google Scholar] [CrossRef] [PubMed]
  150. Alikhani, N.; Shahedi, M.; Habibi, Z.; Yousefi, M.; Ghasemi, S.; Mohammadi, M. A multi-component approach for co-immobilization of lipases on silica-coated magnetic nanoparticles: Improving biodiesel production from waste cooking oil. Bioprocess. Biosyst. Eng. 2022, 45, 2043–2060. [Google Scholar] [CrossRef]
  151. Gao, X.; Pan, H.; Wei, Y.; Ye, M.; Qiao, C.; Wang, J.; Liu, Q.; Zhou, C. A novel bienzymatic bioreactor based on magnetic hierarchical porous MOF for improved catalytic activity and stability: Kinetic analysis, thermodynamics properties and biocatalyst applications. Biochem. Eng. J. 2023, 197, 108995. [Google Scholar] [CrossRef]
  152. Giannakopoulou, A.; Patila, M.; Spyrou, K.; Chalmpes, N.; Zarafeta, D.; Skretas, G.; Gournis, D.; Stamatis, H. Development of a four-enzyme magnetic nanobiocatalyst for multi-step cascade reactions. Catalysts 2019, 9, 995. [Google Scholar] [CrossRef]
  153. Qiu, X.; Wang, S.; Miao, S.; Suo, H.; Xu, H.; Hu, Y. Co-immobilization of laccase and ABTS onto amino-functionalized ionic liquid-modified magnetic chitosan nanoparticles for pollutants removal. J. Hazard. Mater. 2021, 401, 123353. [Google Scholar] [CrossRef]
  154. Kaur, G.; Taggar, M.S.; Kalia, A. Cellulase-immobilized chitosan-coated magnetic nanoparticles for saccharification of lignocellulosic biomass. Environ. Sci. Pollut. Res. Int. 2023, 30, 111627–111647. [Google Scholar] [CrossRef]
  155. Bushra, R.; Ahmad, M.; Alam, K.; Seidi, F.; Shakeel, S.; Song, J.; Jin, Y.; Xiao, H. Recent advances in magnetic nanoparticles: Key applications, environmental insights, and future strategies. Sustain. Mater. Technol. 2024, 40, e00985. [Google Scholar] [CrossRef]
  156. Araújo, E.V.; Carneiro, S.V.; Neto, D.M.A.; Freire, T.M.; Costa, V.M.; Freire, R.M.; Fechine, L.M.U.D.; Clemente, C.S.; Denardin, J.C.; Dos Santos, J.C.S.; et al. Advances in surface design and biomedical applications of magnetic nanoparticles. Adv. Colloid. Interface Sci. 2024, 328, 103166. [Google Scholar] [CrossRef]
  157. Seghiri, R.; Haounati, R.; Rbaa, M. Multifunctional magnetic nanoparticles in biocatalysis. In Multifunctional Magnetic Nanoparticles in Therapy, Biology, and Pharmacy; CRC Press: Boca Raton, FL, USA, 2025; pp. 171–198. [Google Scholar]
  158. Chauhan, P.; Kumar, D.; Sharma, R. Biogenic Magnetic Iron Oxide Nanoparticles for Multifunctional Applications in Environmental Remediation and Biomedical Field. J. Environ. Chem. Eng. 2025, 13, 115646. [Google Scholar] [CrossRef]
  159. Lyu, X.; Gonzalez, R.; Horton, A.; Li, T. Immobilization of enzymes by polymeric materials. Catalysts 2021, 11, 1211. [Google Scholar] [CrossRef]
  160. Rodriguez-Abetxuko, A.; Sánchez-deAlcázar, D.; Muñumer, P.; Beloqui, A. Tunable polymeric scaffolds for enzyme immobilization. Front. Bioeng. Biotechnol. 2020, 8, 830. [Google Scholar] [CrossRef] [PubMed]
  161. Suthiwangcharoen, N.; Li, T.; Wu, L.; Reno, H.B.; Thompson, P.; Wang, Q. Facile Co-Assembly Process to Generate Core–Shell Nanoparticles with Functional Protein Corona. Biomacromolecules 2014, 15, 948–956. [Google Scholar] [CrossRef] [PubMed]
  162. Cipolatti, E.P.; Valério, A.; Henriques, R.O.; Pinto, M.C.C.; Lorente, G.F.; Manoel, E.A.; Guisán, J.M.; Ninow, J.L.; de Oliveira, D.; Pessela, B.C. Production of new nanobiocatalysts via immobilization of lipase B from C. antarctica on polyurethane nanosupports for application on food and pharmaceutical industries. Int. J. Biol. Macromol. 2020, 165, 2957–2963. [Google Scholar] [CrossRef]
  163. Lee, G.; Kim, J.; Lee, J.H. Development of magnetically separable polyaniline nanofibers for enzyme immobilization and recovery. Enzym. Microb. Technol. 2008, 42, 466–472. [Google Scholar] [CrossRef]
  164. Almulaiky, Y.Q.; Alkabli, J.; El-Shishtawy, R.M. Improving enzyme immobilization: A new carrier-based magnetic polymer for enhanced covalent binding of laccase enzyme. Int. J. Biol. Macromol. 2024, 282, 137362. [Google Scholar] [CrossRef]
  165. Chen, T.; Peng, Y.; Qiu, M.; Yi, C.; Xu, Z. Heterogenization of homogeneous catalysts in polymer nanoparticles: From easier recovery and reuse to more efficient catalysis. Coord. Chem. Rev. 2023, 489, 215195. [Google Scholar] [CrossRef]
  166. Khan, R.S.; Rather, A.H.; Wani, T.U.; Rather, S.U.; Amna, T.; Hassan, M.S.; Sheikh, F.A. Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis. Biotechnol. Bioeng. 2023, 120, 22–40. [Google Scholar] [CrossRef]
  167. Çalbaş, B.; Keobounnam, A.N.; Korban, C.; Doratan, A.J.; Jean, T.; Sharma, A.Y.; Wright, T.A. Protein–polymer bioconjugation, immobilization, and encapsulation: A comparative review towards applicability, functionality, activity, and stability. Biomater. Sci. 2024, 12, 2841–2864. [Google Scholar] [CrossRef]
  168. Wen, Y.; Wang, X.; Zhao, J.; Zhai, X.; Xia, W.; Li, P.; Lai, K.; Wu, L. Preparation and Application of Enzyme-based Hydrogels. Biosens. Bioelectron. X 2025, 23, 100594. [Google Scholar] [CrossRef]
  169. Atiroğlu, V.; Atiroğlu, A.; Atiroğlu, A.; Al-Hajri, A.S.; Özacar, M. Green immobilization: Enhancing enzyme stability and reusability on eco-friendly support. Food Chem. 2024, 448, 138978. [Google Scholar] [CrossRef] [PubMed]
  170. Zhang, K.; Yang, W.; Liu, Y.; Zhang, K.; Chen, Y.; Yin, X. Laccase immobilized on chitosan-coated Fe3O4 nanoparticles as reusable biocatalyst for degradation of chlorophenol. J. Mol. Struct. 2020, 1220, 128769. [Google Scholar] [CrossRef]
  171. Aggarwal, S.; Ikram, S. Zinc oxide nanoparticles-impregnated chitosan surfaces for covalent immobilization of trypsin: Stability & kinetic studies. Int. J. Biol. Macromol. 2022, 207, 205–221. [Google Scholar] [CrossRef] [PubMed]
  172. Wang, T.F.; Lo, H.F.; Chi, M.C.; Lai, K.L.; Lin, M.G.; Lin, L.L. Affinity immobilization of a bacterial prolidase onto metal-ion-chelated magnetic nanoparticles for the hydrolysis of organophosphorus compounds. Int. J. Mol. Sci. 2019, 20, 3625. [Google Scholar] [CrossRef]
  173. Bindu, V.U.; Shanty, A.A.; Mohanan, P.V. Parameters affecting the improvement of properties and stabilities of immobilized α-amylase on chitosan-metal oxide composites. Int. J. Biochem. Biophys. 2018, 6, 44–57. [Google Scholar] [CrossRef]
  174. Shukla, A.; Gundampati, R.K.; Jagannadham, M.V. Immobilization of Euphorbia tirucalli peroxidase onto chitosan-cobalt oxide magnetic nanoparticles and optimization using response surface methodology. Int. J. Biol. Macromol. 2017, 102, 384–395. [Google Scholar] [CrossRef]
  175. Amirbandeh, M.; Taheri-Kafrani, A.; Soozanipour, A.; Gaillard, C. Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization. Biochem. Eng. J. 2017, 127, 119–127. [Google Scholar] [CrossRef]
  176. Thakur, K.; Attri, C.; Seth, A. Nanocarriers-based immobilization of enzymes for industrial application. 3 Biotech 2021, 11, 1–12. [Google Scholar] [CrossRef]
  177. Hooe, S.L.; Breger, J.C.; Medintz, I.L. Enhancing enzymatic activity with nanoparticle display–an updated compendium and engineering outlook. Mol. Syst. Des. Eng. 2024, 9, 679–704. [Google Scholar] [CrossRef]
  178. Zdarta, J.; Meyer, A.S.; Jesionowski, T.; Pinelo, M. A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility. Catalysts 2018, 8, 92. [Google Scholar] [CrossRef]
  179. Yusuf, V.F.; Malek, N.I.; Kailasa, S.K. Review on metal–organic framework classification, synthetic approaches, and influencing factors: Applications in energy, drug delivery, and wastewater treatment. ACS Omega 2022, 7, 44507–44531. [Google Scholar] [CrossRef] [PubMed]
  180. Bisht, M.; Thayallath, S.K.; Bharadwaj, P.; Franklin, G.; Mondal, D. Biomass-derived functional materials as carriers for enzymes: Towards sustainable and robust biocatalysts. Green. Chem. 2023, 25, 4591–4624. [Google Scholar] [CrossRef]
  181. Ahmadi, S.; Pourebrahimi, S.; Malloum, A.; Pirooz, M.; Osagie, C.; Ghosh, S.; Zafar, M.N.; Dehghani, M.H. Hydrogel-based materials as super adsorbents and antibacterial agents for the remediation of emerging pollutants: A comprehensive review. Emerg. Contam. 2024, 10, 100336. [Google Scholar] [CrossRef]
  182. Qamar, S.A.; Junaid, M.; Riasat, A.; Jahangeer, M.; Bilal, M.; Mu, B.Z. Carrageenan-based Hybrids with Biopolymers and Nano-structured Materials for Biomimetic Applications. Starch-Stärke 2024, 76, 2200018. [Google Scholar] [CrossRef]
  183. Fan, Y.; Su, F.; Li, K.; Ke, C.; Yan, Y. Carbon nanotube filled with magnetic iron oxide and modified with polyamidoamine dendrimers for immobilizing lipase toward application in biodiesel production. Sci. Rep. 2017, 7, 45643. [Google Scholar] [CrossRef]
  184. Park, J.S.; Yang, S.Y.; Lee, J.K.; Kang, Y.C. A novel strategy for encapsulating metal sulfide nanoparticles inside hollow carbon nanosphere-aggregated microspheres for efficient potassium ion storage. J. Mater. Chem. A 2022, 10, 17790–17800. [Google Scholar] [CrossRef]
  185. Kim, S.J.; Hong, J.H.; Lee, J.K.; Kang, Y.C. Macroporous microspheres consisting of thickness-controlled bamboo-like CNTs and flower-like Co3O4 nanoparticles as highly efficient bifunctional oxygen electrocatalysts for Zn–air batteries. J. Mater. Chem. A 2021, 9, 25160–25167. [Google Scholar] [CrossRef]
  186. Hashempour, Y.; Mortezazadeh, F.; Rezaei, S.; Salehipour, M.; Gholami-Borujeni, F.; Ebrahimnejad, P.; Mogharabi-Manzari, M. Co-immobilization of laccase and zinc oxide nanoparticles onto bacterial cellulose to achieve synergistic effect of photo and enzymatic catalysis for biodegradation of favipiravir. Int. J. Biol. Macromol. 2025, 292, 139288. [Google Scholar] [CrossRef]
  187. Zheng, Y.; Zhang, Y.; Xue, Z.; Li, H.; Shen, S. Wearable biosensor with high specific surface area PGA-CNTs electrode for sweat glucose detection. Microchem. J. 2025, 210, 112965. [Google Scholar] [CrossRef]
  188. Jang, W.Y.; Kim, Y.J.; Chang, J.H. Comparative Study of Enzymatic Lipolysis Using Nanofructosome-Coated CalB Lipase Encapsulated in Silica and Immobilized on Silica-Coated Magnetic Nanoparticles. ACS Omega 2025, 10, 13319–13326. [Google Scholar] [CrossRef] [PubMed]
  189. Tran, D.T.; Chen, C.L.; Chang, J.S. Immobilization of Burkholderia sp. lipase on a ferric silica nanocomposite for biodiesel production. J. Biotechnol. 2012, 158, 112–119. [Google Scholar] [CrossRef] [PubMed]
  190. Wang, X.; Liu, X.; Zhao, C.; Ding, Y.; Xu, P. Biodiesel production in packed-bed reactors using lipase–nanoparticle biocomposite. Bioresour. Technol. 2011, 102, 6352–6355. [Google Scholar] [CrossRef] [PubMed]
  191. Wang, J.; Li, K.; He, Y.; Wang, Y.; Han, X.; Yan, Y. Enhanced performance of lipase immobilized onto Co2+ -chelated magnetic nanoparticles and its application in biodiesel production. Fuel 2019, 255, 115794. [Google Scholar] [CrossRef]
  192. Hübner, J.; Brakowski, R.; Wohlgemuth, J.; Brenner-Weiß, G.; Franzreb, M. Compartmented microfluidic bioreactor system using magnetic enzyme immobilisates for fast small-scale biotransformation studies. Eng. Life Sci. 2015, 15, 721–726. [Google Scholar] [CrossRef]
  193. Bartha-Vári, J.H.; Moisă, M.E.; Bencze, L.C.; Irimie, F.D.; Paizs, C.; Toșa, M.I. Efficient biodiesel production catalyzed by nanobioconjugate of lipase from Pseudomonas fluorescens. Molecules 2020, 25, 651. [Google Scholar] [CrossRef]
  194. Neupane, S.; Patnode, K.; Li, H.; Baryeh, K.; Liu, G.; Hu, J.; Chen, B.; Pan, Y.; Yang, Z. Enhancing enzyme immobilization on carbon nanotubes via metal–organic frameworks for large-substrate biocatalysis. ACS Appl. Mater. Interfaces 2019, 11, 12133–12141. [Google Scholar] [CrossRef]
  195. Kujawa, J.; Głodek, M.; Li, G.; Al-Gharabli, S.; Knozowska, K.; Kujawski, W. Highly effective enzymes immobilization on ceramics: Requirements for supports and enzymes. Sci. Total Environ. 2021, 801, 149647. [Google Scholar] [CrossRef]
  196. Guisan, J.M.; Fernandez-Lorente, G.; Rocha-Martin, J.; Moreno-Gamero, D. Enzyme immobilization strategies for the design of robust and efficient biocatalysts. Curr. Opin. Green. Sustain. Chem. 2022, 35, 100593. [Google Scholar] [CrossRef]
  197. Kotwal, N.; Pathania, D.; Singh, A.; Sheikh, Z.U.D.; Kothari, R. Enzyme immobilization with nanomaterials for hydrolysis of lignocellulosic biomass: Challenges and future Perspectives. Carbohydr. Res. 2024, 543, 109208. [Google Scholar] [CrossRef]
  198. Tang, K.H.D.; Lock, S.S.M.; Yap, P.S.; Cheah, K.W.; Chan, Y.H.; Yiin, C.L.; Ku, A.Z.E.; Loy, A.C.M.; Chin, B.L.F.; Chai, Y.H. Immobilized enzyme/microorganism complexes for degradation of microplastics: A review of recent advances, feasibility and future prospects. Sci. Total Environ. 2022, 832, 154868. [Google Scholar] [CrossRef]
  199. Khafaga, D.S.; Muteeb, G.; Elgarawany, A.; Aatif, M.; Farhan, M.; Allam, S.; Almatar, B.A.; Radwan, M.G. Green nanobiocatalysts: Enhancing enzyme immobilization for industrial and biomedical applications. PeerJ 2024, 12, e17589. [Google Scholar] [CrossRef]
  200. Cervantes, F.J.; Ramírez-Montoya, L.A. Immobilized nanomaterials for environmental applications. Molecules 2022, 27, 6659. [Google Scholar] [CrossRef]
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Figure 1. AI-generated 3D structures of different nanoparticles: (a) carbon nanotube (CNT); (b) graphene; (c) silver nanoparticle; (d) magnetic nanoparticle (MNP); (e) metal–organic framework (MOF); and (f) polymeric nanoparticle. Figure 1 was generated through deep artificial intelligence by authors.
Figure 1. AI-generated 3D structures of different nanoparticles: (a) carbon nanotube (CNT); (b) graphene; (c) silver nanoparticle; (d) magnetic nanoparticle (MNP); (e) metal–organic framework (MOF); and (f) polymeric nanoparticle. Figure 1 was generated through deep artificial intelligence by authors.
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Figure 2. Interaction between nitrilase, substrate, and immobilized matrix. (Nitrilase (pdb: 3wuy) is immobilized on a glutaraldehyde (GA)-functionalized single-walled carbon nanotube (swCNT). GA interacts with enzyme surface residues, including a hydrogen bond with the backbone carbonyl oxygen of A273 and hydrophobic contacts with residues K44, K43, and N40 (inset, top). The substrate, 3 cyanopyridine, binds productively in the enzyme pocket (inset, bottom), stabilized by π–π interactions with W170, F64, and F202, which orient the cyano group near the catalytic residue C169 for nucleophilic attack. The active site residues E53 and K135 further stabilize the transition state. This model highlights the molecular interactions enabling catalysis upon immobilization).
Figure 2. Interaction between nitrilase, substrate, and immobilized matrix. (Nitrilase (pdb: 3wuy) is immobilized on a glutaraldehyde (GA)-functionalized single-walled carbon nanotube (swCNT). GA interacts with enzyme surface residues, including a hydrogen bond with the backbone carbonyl oxygen of A273 and hydrophobic contacts with residues K44, K43, and N40 (inset, top). The substrate, 3 cyanopyridine, binds productively in the enzyme pocket (inset, bottom), stabilized by π–π interactions with W170, F64, and F202, which orient the cyano group near the catalytic residue C169 for nucleophilic attack. The active site residues E53 and K135 further stabilize the transition state. This model highlights the molecular interactions enabling catalysis upon immobilization).
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Materials 18 04106 g003
Figure 3. Evolution of nanomaterial application trends across four sectors (2001–2025). Network maps of keywords from PubMed reveal the growth and specialization of nanomaterial research in (a) biotransformation (highlighting enzymes, immobilization, and biological interfaces), (b) pharmaceutical sector (focusing on drug delivery and tumor targeting), (c) biosensor development (emphasizing biosensing, electrochemical techniques, and detection sensitivity), and (d) bioremediation (centered on environmental degradation and pollutant removal). The node size reflects the keyword importance, and the colors indicate the related groups, illustrating the expanding and diverse roles of nanomaterials over 24 years.
Figure 3. Evolution of nanomaterial application trends across four sectors (2001–2025). Network maps of keywords from PubMed reveal the growth and specialization of nanomaterial research in (a) biotransformation (highlighting enzymes, immobilization, and biological interfaces), (b) pharmaceutical sector (focusing on drug delivery and tumor targeting), (c) biosensor development (emphasizing biosensing, electrochemical techniques, and detection sensitivity), and (d) bioremediation (centered on environmental degradation and pollutant removal). The node size reflects the keyword importance, and the colors indicate the related groups, illustrating the expanding and diverse roles of nanomaterials over 24 years.
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Figure 4. Classification of nanomaterials according to different features and their categorization.
Figure 4. Classification of nanomaterials according to different features and their categorization.
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Table 1. Advantages and disadvantages of nanomaterials used for immobilizing enzymes.
Table 1. Advantages and disadvantages of nanomaterials used for immobilizing enzymes.
AdvantageDisadvantageSolution
High surface area and loading capacity
Nanomaterials, such as CNTs, MNPs, and graphene, have a large surface area-to-volume ratio, increasing enzyme loading and catalytic efficiency [3,5,6].		High cost of nanomaterials
Fabrication and functionalization of nanomaterials are expensive; large-scale applications are economically challenging [5,7,8].		Adopt low-cost fabrication techniques such as plasma reactors and sol–gel methods.
Enhanced stability
Immobilizing nanomaterials increases enzyme stability against denaturation caused by environmental factors such as temperature, pH, and solvents, which are suitable for industrial applications [3,6,9].		Diffusion limitations
Diffusion of substrates and products can be restricted within the immobilization matrix at high enzyme loading densities, reducing apparent activity [5,7,9].		Use of porous supports or optimize matrix design to increase diffusion.
Reusability and cost-effectiveness
Enzymes immobilized on nanomaterials can be easily separated from reaction mixtures (e.g., MNPs can be recovered using external magnetic fields) and reused for multiple cycles, reducing operational costs [3,6,10].		Enzyme leaching and instability
Noncovalent immobilization methods may result in enzyme leaching over time, reducing catalytic performance and contaminating the reaction medium [7,9].		Use covalent binding or crosslinking to enhance enzyme immobilization stability.
Increase catalytic activity
Nanoscale interactions between enzymes and nanomaterials enhance catalytic activity due to orientation and stabilization of the enzyme’s active site [3,5].		Complex preparation
Immobilization procedures often require specialized techniques and equipment, increasing process complexity compared with traditional methods [7,8].		Develop simplified protocols or use prefunctionalized, ready-to-use supports.
Versatility in applications
Enzymes immobilized on nanomaterials are used in various fields such as biosensors, biofuel production, drug delivery, and industrial biocatalysis due to their functionality [3,6].		Scalability issues and environmental sensitivity
Scalability of enzyme immobilization on nanomaterials is limited due to challenges with reproducibility and uniformity during large-scale production [8,11].
Some nanomaterials are prone to degrading or aggregating under specific conditions (e.g., MNPs in acidic or oxidative environments), which can compromise enzyme stability [3,7].		Optimization of processes and development of novel stable, environmentally resistant nanomaterials for immobilization.
Table 2. Methods of enzyme immobilization as well as their advantages and disadvantages.
Table 2. Methods of enzyme immobilization as well as their advantages and disadvantages.
MethodAdvantagesDisadvantagesReferences
AdsorptionSimple and cost-effective method.
Minimal chemical modification of enzyme.
Rapid immobilization process.		Adsorption immobilization relies on weak binding forces (van der Waals, ionic, and hydrogen bonds), making enzymes prone to desorption under shifts in pH, ionic strength, or temperature, resulting in low stability and activity loss.[20,21]
Covalent BindingStrong and stable linkage prevents enzyme leakage.
High thermal and operational stability.
Reusable for multiple cycles.		Covalent binding of enzymes presents significant drawbacks, primarily the risk of enzyme denaturation due to chemical modifications during immobilization. This method involves a complex process requiring expensive reagents and supports, increasing operational costs. Additionally, conformational changes induced by binding often lead to partial or complete loss of enzyme activity, reducing catalytic efficiency.[8,14,22,23,25].
EntrapmentProtects enzymes from environmental changes (e.g., pH and temperature).
Suitable for thermally and mechanically stable enzymes.		Diffusion limitations restrict substrate access to enzymes, reducing reaction rates. Recovering enzymes is challenging as they are trapped within the matrix, complicating reuse and process efficiency.[14]
EncapsulationHigh reproducibility and protection from shear forces.Encapsulation includes weak binding forces that can cause enzyme leakage, particularly under certain conditions such as high ionic strength. Enzyme inactivation due to mechanical stress during the encapsulation process is also a risk. Additionally, swelling of the capsules can lead to further leaching of the encapsulated enzymes. Low mechanical strength and large pore size in some encapsulation materials can also contribute to high enzyme leakage.[15,26]
CrosslinkingCarrier-free immobilization with high enzyme density.
High stability under industrial conditions.
Easy recycling and reuse.		Disadvantages of crosslinking include possible overcrowding of enzymes, which can reduce their activity. The process is irreversible, meaning that once enzyme activity is lost, it cannot be recovered. Additionally, crosslinking often requires highly pure, crystallized enzymes, making it an expensive technique. The chemicals used can also result in enzyme denaturation, further inhibiting activity.[23,27]
Table 3. CNTs and their derivatives used for immobilizing enzymes.
Table 3. CNTs and their derivatives used for immobilizing enzymes.
NanomaterialSkeleton MatrixFunctionalization GroupsEnzymeEnzyme SourceReusability (Retained Activity)Reference
CNT and derivativesMultiwalled CNT (mwCNT)HNO3/H2SO4LaccaseTrametes versicolor10 cycles (90%)[49]
mwCNTEDC/NHS crosslinkersLipasePseudomonas fluorescens10 cycles (49.2%)[50]
CNTN-aminoethyl-γ-aminopropyl trimethoxyβ-glucosidaseTerrabacter ginsenosidimutans14 cycles (76%)[51]
mwCNT-NiOGlutaraldehyde, APTESXylanaseThermomyces lanuginosus6 cycles (80%)[52]
mwCNTHNO3/H2SO4LaccaseTrametes versicolor10 cycles (90%)[49]
Fe3O4 mwCNTEDC/NHSLipaseCandida rugosa10 cycles (91%)[53]
Magnetic nickel oxide mwCNT3-aminopropyltriethoxysilaneL-asparaginaseGeobacillus kaustophilus10 cycles (90%)[54]
ZIF-8@MWCNTHNO3/H2SO4Laccases-10 cycles (68%)[55]
GO-CNTGlutaraldehydeLaccaseTrametes versicolor11 cycles (50%)[56]
Superparamagnetic mwCNTPolyethylene glycol amine polymerLipases-10 cycles (78.5%)[48]
CNTGlutaraldehyde/APTESLipaseRhizomucor miehei10 cycles (90%)[57]
CNTCinnamaldehyde ethanol solutionPorcine pancreatic lipasePorcine pancreatic lipase7 cycles (69%)[58]
Polyaniline cobalt CNTGlutaraldehydeβ-galactosidaseAspergillus oryzae10 cycles (74%)[59]
CNTGlutaraldehydeLaccaseNovo Nordisk Company, Bagsværd, Denmark10 cycles (69%)[60]
mwCNT-MoS2 NCGlutaraldehyde crosslinkerβ-galactosidaseLens culinaris21 cycles (>50%)[61]
mwCNTGlutaraldehydeCyanate hydratasePichia pastoris10 cycles (>94%)[62]
Mesoporous SiO2 microparticleGlutaraldehydeLipaseThermomyces lanuginosus12 cycles (95.4%)[63]
mwCNTAminated polydopamineLipaseCandida rugosa10 cycles (84%)[64]
mwCNTSodium alginateCellulaseTrichoderma7 cycles (70%)[65]
mwCNT-LipaseCandida antarctica7 cycles (95%)[66]
mwCNTCarbodiimide couplingCellulaseAspergillus niger10 cycles (85%)[67]
swCNTN,N′-carbonyldiimidazole, CH2Cl2Lipase BCandida antarctica10 cycles (>90%)[68]
Magnetic-mwCNTPolyamidoamineLipaseRhizomucor miehei10 cycles (94%)[69]
EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, APTES: (3-aminopropyl)triethoxysilane, NHS: N-hydroxysulfosuccinimide.
Table 4. Graphene and derivatives used for immobilizing enzymes.
Table 4. Graphene and derivatives used for immobilizing enzymes.
NanomaterialSkeleton MatrixFunctionalization GroupsEnzymeEnzyme SourceReusability (Retained Activity)Reference
Graphene and derivativesGraphene oxide (GO) Fe3O4@SiO2GlutaraldehydePhospholipase DStreptomyces chromofuscus10 cycles (78.3%)[81]
Graphene oxide (GO-Asp-Fe3O4)EDC/NHSL-asparaginase-8 cycles (80%)[82]
Magnetic GO3-Mercaptopropyl trimethoxysilaneLipaseCandida rugosa8 cycles 76.5%[83]
Magnetic GOGlutaraldehydeLipaseCandida rugosa10 cycles (50%)[84]
Magnetic GOγ-Ureapropyltrimethoxy silane, APTES, γ-mercapto propyltriethoxysilaneLipaseAspergillus oryzae8 cycles (80.2%)[85]
Activated GO/chitosan/celluloseGlutaraldehydeLipaseBacillus licheniformis (Lipase Km12)10 cycles (80%)[86]
Magnetic GOCarbodiimideCellulase/sylanase-10 cycles (70%)[87]
Magnetic GOGlutaraldehyde, APTESPersiManXyn1-15 cycles (94%)[88]
Magnetic GO PolyethylenimineLactaseEscherichia coli, Sangon Biotech (Shanghai, China)20 cycles (83.1%)[89]
Magnetic GOPolyethylene glycol (PEG)Horseradish peroxidase-8 cycles (68.1%)[90]
Magnetic GOGlutaraldehydeDextranase-20 cycles (85.7%)[91]
GOGlutaraldehydeHorseradish Peroxidase-12 cycles (90%)[70]
GOHorseradish peroxidase and oxalate oxidaseFormate dehydrogenaseCandida boidinii8 cycles (63.8%)[92]
Reduced GOGlutaraldehydeHorseradish peroxidase-10 cycles (70%)[93]
Magnetic GONα, Nα-Bis(carboxymethyl)-l-lysine hydrateLaccase-10 cycles (89.4%)[94]
GOAmine groupHorseradish peroxidase-10 cycles (60%)[95]
GO-LipaseCandida rugosa10 cycles (85%)[96]
Reduced GO-Laccase, horseradish peroxidaseTrametes versicolor10 cycles (92.6%)[97]
Graphene-Naringinase 10 cycles (85.4%)[98]
Polyaniline–silver functionalized GO nanocompositesGlutaraldehydeLipaseAspergillus niger11 cycles (86%)[99]
Magnetic GOxEDC/NHSLaccaseTrametes versicolor11 cycles (59.8%)[100]
GO(GO/ZnO)-LipaseCandida rugosa14 cycles (90%)[101]
GOEDAC, N-hydroxysulfosuccinimide sodium saltChloroperoxidaseCaldariomyces fumago8 cycles (52%)[102]
GOEpoxy chloropropaneγ-LactamaseEscherichia coli BL21(DE3)15 cycles (70%)[103]
GO nanosheets
Superparamagnetic iron oxide nanoparticles		Cyanuric chlorideXylanaseThermomyces lanuginosus10 cycles (70%)[104]
Graphene–iron oxide nanocomposites (Gr@Fe3O4 NCs)-β-galactosidaseAspergillus oryzae8 cycles (83%)[105]
Graphene sheetsCysteamine and glutaraldehydeα-AmylaseTriticum aestivum10 cycles (73%)[106]
Exfoliated graphene oxide (EGO)SilaneLipaseCandida rugosa30 cycles (50%)[107]
GO-based magnetic3-ChloropropyltriethoxysilaneLipase-10 cycles (87%)[108]
EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, APTES: (3-aminopropyl)triethoxysilane, NHS: N-hydroxysulfosuccinimide.
Table 5. Metal and metal oxide nanoparticles used for immobilizing enzymes.
Table 5. Metal and metal oxide nanoparticles used for immobilizing enzymes.
NanomaterialSkeleton MatrixFunctionalization GroupsEnzymeEnzyme SourceReusability (Retained Activity)Reference
Metal and metal oxide nanoparticlesMagnetic laccase nanoflowersGlutaraldehydeLaccaseTrametes versicolor18 times (90%)[121]
Metal–organic frameworks Glutaraldehydeα-AmylaseBacillus subtilis20 cycles (81%)[122]
Magnetic nanoparticlesGlutaraldehydeα-Amylase and xylanaseBacillus subtilis and Aspergillus niger15 cycles (82%)
11 cycles (64%)		[123]
Fe3O4/SBA-15Tannic acidL-asparaginaseEscherichia coli16 times (70%)[124]
Titania nanoparticlesGlutaraldehydeAlcohol dehydrogenaseSaccharomyces cerevisiae10 times (84%)[125]
-Naringinα-AmylaseBacillus subtilis10 times (60%)[126]
FeCl3APTES, GlutaraldehydeLaccaseAspergillus oryzae6 cycles (75%)[127]
Magnetic nanoparticles3-phosphono propionic acidα-AmylaseAspergillus oryzae10 cycles (60%)[128]
Fe3O4 magnetic nanoparticlesPolyethylenimineLipaseThermomyces lanuginosa10 cycles (60%)[129]
APTES: (3-aminopropyl)triethoxysilane.
Table 6. Magnetic nanoparticles (MNPs) used for immobilizing enzymes.
Table 6. Magnetic nanoparticles (MNPs) used for immobilizing enzymes.
NanomaterialSkeleton MatrixFunctionalization GroupsEnzymeEnzyme SourceReusability (Retained Activity)Reference
Magnetic nanoparticles (MNPs)Silica-coated amine functionalized iron oxide nanoparticle (IONP@SiO2-NH2)GlutaraldehydeCellulase-6 cycles
(80%)		[134]
Fe3O4, Affi-Cova beadsIDAr-BirA 10 cycles (76.1%)[37]
Fe3O4, SiO2 core@shell3-(Triethoxysilyl) propyl isocyanateLaccase Trametes versicolor13 cycles (88%)[131]
-GlutaraldehydeTrypsin-15 cycles (93%)[135]
Fe3O4N-hydroxysuccinimide, ethyl-3-(3-dimethylaminopropyl) carbodiimideβ-LactamaseBacillus cereus12 cycles (BC: 57%, IC: 65%)[136]
Fe3O4N-acetyl and N-methylamideLipaseThermomyces lanuginosus12 cycles (80%)[137]
Fe3O4SpyCatcher-fused elastin-like polypeptidesXylanase
Lichenase		-10 cycles (66.3 and 72.7%)[138]
Fe3O4, SiO2-CHOTetraethyl orthosilicate, APTES, and glutaraldehydeLipaseSaitozyma podzolica10 cycles (90%)[139]
Fe2O3, Fe3O4APTESLaccase 10 cycles (82.9%)[140]
Fe3O4Hyaluronic acidIsocitrate dehydrogenases-DH/HA/MNPs, IDH/HA/MNPs-CLEAs and IDH/BSA/HA/MNPs-CLEAs 15 cycles (76%, 80%, 85%)[141]
Super paramagnetic iron oxide nanoparticlesAPTESLaccaseThermomyces lanuginosus12 cycles (80%)[137]
Fe3O4@C@cellulase-SiO2-Cellulase-9 cycles (80%)[142]
Fe3O4Hydroxyapatite in cobalt ferrite (CoFe2O4)β-glucosides-10 cycle (70%)[143]
Fe3O4, carboxymethyl cellulosePPL-MCMC
PPL-IL-MCMC		Cellulose
-10 cycles (83.9%, 86.1%)[144]
Fe3O4, chitosanGlutaraldehydePeroxidasePseudomonas aeruginosa100 cycles (95%)[145]
His6-EcPepQ@NiNTASiMNPsNi2+LipaseThermomyces lanuginosus20 cycles (80%)[146]
FeCl2, FeCl3Glutaraldehyde, glycidolChitosanAspergillus niger15 cycles (80%)[147]
APTES: (3-aminopropyl)triethoxysilane.
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Singh, R.V.; Singh, B.; Kumar, A.; Sambyal, K.; Karuppanan, K.K.; Lee, J.-K. Enzyme Immobilization on Nanomaterials and Their Applications. Materials 2025, 18, 4106. https://doi.org/10.3390/ma18174106

AMA Style

Singh RV, Singh B, Kumar A, Sambyal K, Karuppanan KK, Lee J-K. Enzyme Immobilization on Nanomaterials and Their Applications. Materials. 2025; 18(17):4106. https://doi.org/10.3390/ma18174106

Chicago/Turabian Style

Singh, Rahul Vikram, Bakul Singh, Anurag Kumar, Krishika Sambyal, Karthikeyan Kugalur Karuppanan, and Jung-Kul Lee. 2025. "Enzyme Immobilization on Nanomaterials and Their Applications" Materials 18, no. 17: 4106. https://doi.org/10.3390/ma18174106

APA Style

Singh, R. V., Singh, B., Kumar, A., Sambyal, K., Karuppanan, K. K., & Lee, J.-K. (2025). Enzyme Immobilization on Nanomaterials and Their Applications. Materials, 18(17), 4106. https://doi.org/10.3390/ma18174106

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