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Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including adsorption, entrapment, encapsulation, and covalent bonding, as well as advanced site-specific methods that integrate enzyme engineering and bio-orthogonal chemistry, were discussed. These techniques enable precise control over enzyme orientation and interaction with carriers, optimizing catalytic activity and reusability. Key findings highlight the impact of immobilization on improving enzyme performance under various operational conditions and its role in reducing process costs through enhanced stability and recyclability. The review presents numerous practical applications of immobilized enzymes, including their use in the pharmaceutical industry for drug synthesis, in the food sector for dairy processing, and in environmental biotechnology for wastewater treatment and dye degradation. Despite the significant advantages, challenges such as activity loss due to conformational changes and mass transfer limitations remain, necessitating tailored immobilization protocols for specific applications. The integration of immobilization with modern biotechnological advancements, such as site-directed mutagenesis and recombinant DNA technology, offers a promising pathway for developing robust, efficient, and sustainable biocatalytic systems. This comprehensive guide aims to support researchers and industries in selecting and optimizing immobilization techniques for diverse applications in pharmaceuticals, food processing, and fine chemicals.

In recent years, enzyme immobilization technology has been developed, and studies on immobilized enzyme materials have become very prominent. With the immobilization technique, enzymes and compatible carrier materials are combined or enzyme crystals/aggregates are used in a carrier-free fashion, by physical, chemical, or biochemical methods. As a kind of biocatalyst, immobilized enzymes can catalyze certain chemical reactions with high selectivity and high efficiency under relatively mild reaction conditions and eliminate pollution to the environment. Considering the current status and applications of immobilized enzyme technology and materials emerging in the last 5 years, this mini-review introduces the advantages and disadvantages of various enzyme immobilization techniques with carriers as well as the pros and cons of different materials for immobilization. The future prospects of immobilization technology and carrier materials are outlined, aiming to provide a reference for further research and applications of sustainable technology. Graphical Abstract

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed.

Immobilization of enzymes enhances their properties for efficient utilization in industrial processes. Magnetic nanoparticles, due to their high surface area, large surface-to-volume ratio and easy separation under external magnetic fields, are highly valued. Significant progress has been made to develop new catalytic systems that are immobilized onto magnetic nanocarriers. This review provides an overview of recent developments in enzyme immobilization and stabilization protocols using this technology. The current applications of immobilized enzymes based on magnetic nanoparticles are summarized and future growth prospects are discussed. Recommendations are also given for areas of future research.

Metal–organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely β-glucosidase, invertase, β-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH 2 , or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes. Metal–organic frameworks (MOFs) are attractive for encapsulating enzymes for industrial purposes because they can increase selectivity, stability, and/or activity of the enzymes. Here, the authors developed an economical solid-state mechanochemical method to encapsulate enzymes during MOF synthesis.

Enzyme–electrode systems effectively integrate biological and non-biological components to facilitate ‘interfacial electron transfer’ (ET) between enzymes and electrodes.Comprehensive frameworks for enzyme–electrode designs are established according to interfacial ET mechanisms.Diverse enzyme–electrode interfacing strategies have been developed, taking into account the nature of the enzymatic reactions and the interfacial ET mechanisms involved.Cutting-edge protein-engineering approaches offer powerful and versatile tools to enhance the efficiency of enzyme–electrode wiring in bioelectronic systems. Advances in protein engineering-enabled enzyme immobilization technologies have significantly improved enzyme–electrode wiring in enzymatic electrochemical systems, which harness natural biological machinery to either generate electricity or synthesize biochemicals. In this review, we provide guidelines for designing enzyme–electrodes, focusing on how performance variables change depending on electron transfer (ET) mechanisms. Recent advancements in enzyme immobilization technologies are summarized, highlighting their contributions to extending enzyme–electrode sustainability (up to months), enhancing biosensor sensitivity, improving biofuel cell performance, and setting a new benchmark for turnover frequency in bioelectrocatalysis. We also highlight state-of-the-art protein-engineering approaches that enhance enzyme–electrode interfacing through three key principles: protein–protein, protein–ligand, and protein–inorganic interactions. Finally, we discuss prospective avenues in strategic protein design for real-world applications. Advances in protein engineering-enabled enzyme immobilization technologies have significantly improved enzyme–electrode wiring in enzymatic electrochemical systems, which harness natural biological machinery to either generate electricity or synthesize biochemicals. In this review, we provide guidelines for designing enzyme–electrodes, focusing on how performance variables change depending on electron transfer (ET) mechanisms. Recent advancements in enzyme immobilization technologies are summarized, highlighting their contributions to extending enzyme–electrode sustainability (up to months), enhancing biosensor sensitivity, improving biofuel cell performance, and setting a new benchmark for turnover frequency in bioelectrocatalysis. We also highlight state-of-the-art protein-engineering approaches that enhance enzyme–electrode interfacing through three key principles: protein–protein, protein–ligand, and protein–inorganic interactions. Finally, we discuss prospective avenues in strategic protein design for real-world applications.

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalytic systems. Enzymes, mainly hydrolytic class of enzyme, have been extensively immobilised on nanoscaffold support for long-term stabilisation by enhancing thermal, operational and storage catalytic potential. In the present report, novel nanoscaffold variants employed in the recent past for enzyme immobilisation, namely nanoparticles, nanofibres, nanotubes, nanopores, nanosheets and nanocomposites, are discussed in the context of lipase-mediated nanobiocatalysis. These nanocarriers have an inherently large surface area that leads to high enzyme loading and consequently high volumetric enzyme activity. Due to their high tensile strengths, nanoscale materials are often robust and resistant to breakage through mechanical shear in the running reactor making them suitable for multiple reuses. The optimisation of various nanosupports process parameters, such as the enzyme type and selection of suitable immobilisation method may help lead to the development of an efficient enzyme reactor. This might in turn offer a potential platform for exploring other enzymes for the development of stable nanobiocatalytic systems, which could help to address global environmental issues by facilitating the production of green energy. The successful validation of the feasibility of nanobiocatalysis for biodiesel production represents the beginning of a new field of research. The economic hurdles inherent in viably scaling nanobiocatalysts from a lab-scale to industrial biodiesel production are also discussed.

β-Galactosidase enzymes catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-galactosides. These enzymes are important in producing lactose-free dairy products, reducing the lactose content of whey in dairy products, and for production of galactooligosaccharides (GOS) as prebiotic additives to infant formula. To use β-galactosidases in industrial settings, enzyme immobilization procedures are used to enhance their activity and stability and to minimize enzyme quantities and cost. In this study, recombinant Bifidobacterium adolescentis β-galactosidase BgaC was immobilized in calcium alginate and gelatin cross-linked with glutaraldehyde. The kinetic parameters and stability properties of immobilized BgaC were characterized in comparison with free soluble enzyme. The K M for immobilized BgaC using ortho-nitrophenyl-β-galactoside (ONPG) was 810 ± 220 μM and the K M of free BgaC was 2500 ± 3 μM. The k cat and k cat / K M of immobilized BgaC were 802 s −1 and 990 s −1  mM −1 , respectively, compared to k cat and k cat / K M values of 209 s −1 and 84 s −1  mM −1 , respectively, for free BgaC. Immobilized BgaC β-galactosidase was active at all tested pH (pH 4–10), while the free enzyme had decreased activity at pH < 5.5 and > 8.0. The immobilized enzyme had optimum activity at 40 °C, while the free enzyme was most active at 37 °C. In addition, immobilization enhanced acidic pH and temperature stability compared to the free enzyme. Reutilization of the BgaC beads was assessed and the enzyme maintained 69% activity after 12 rounds of reutilization. Therefore, the enhanced performance properties of immobilized BgaC make it a promising candidate for industrial applications. Key points • Bifidobacterium adolescentis β-galactosidase BgaC was successfully immobilized • Immobilized BgaC has enhanced enzymatic activity and stability and allows recycling • Sustained activity of immobilized BgaC is advantageous for industrial applications Graphical Abstract

Robust immobilization of glucose oxidase (GOx) enzyme was achieved on poly(ethylene terephthalate) nonwoven fabric (PN) after integration of favourable surface functional groups through plasma treatments [atmospheric pressure-AP or cold remote plasma-CRP (N 2  + O 2 )] and/or chemical grafting of hyperbranched dendrimers [poly-(ethylene glycol)-OH or poly-(amidoamine)]. Absorption, stability, catalytic behavior of immobilized enzymes and reusability of resultant fibrous bio-catalysts were comparatively studied. Full characterization of PN before and after respective modifications was carried out by various analytical, instrumental and arithmetic techniques. Results showed that modified polyester having amine terminal functional groups pledged better surface property providing up to 31% enzyme loading, and 81% active immobilized enzymes. The activity of the enzyme was measured in terms of interaction aptitude of GOx in a given time to produce hydrogen peroxide using colorimetric assay. The immobilized GOx retained 50% of its original activity after being reused six (06) times and exhibited improved stability compared with the free enzyme in relation to temperature. The reaction kinetics, loading efficiency, leaching, and reusability analysis of enzyme allowed drawing a parallel to the type of organic moiety integrated during GOx immobilization. In addition, resultant fibrous bio-catalysts showed substantial antibacterial activity against pathogenic bacteria strains ( Staphylococcus epidermidis and Escherichia coli ) in the presence of oxygen and glucose. These results are of great importance because they provide proof-of-concept for robust immobilization of enzymes on surface-modified fibrous polyester fabric for potential bio-industrial applications.

The purpose of the study is to provide a method for mass production of nicotinamide mononucleotide (NMN) using an immobilized nicotinamide riboside kinase (NMRK). We synthesized a gene sequence comprising the gene encoding the enzyme. At the 3’ end of this NMRK-encoding gene, an optimized cellulose-binding domain (CBD) sequence was linked via a linker-encoding gene segment.Using the pET-32a plasmid as the vector and Escherichia coli BL21(DE3) as the host cell, the recombinant E. coli strain BL21/pET-32a-nmrk-1 was constructed and cultivated. This recombinant strain BL21/pET-32a-nmrk-1 secreted a significantly higher yield of the NMRK enzyme protein (32 mg/mL), and the secreted recombinant NMRK enzyme exhibited superior activity (80 U/g). Activated microcrystalline cellulose was mixed with the crude enzyme solution containing the recombinant NMRK enzyme. Following processing, the immobilized NMRK enzyme was obtained. This immobilized enzyme catalyzed the synthesis of NMN from 0.07 mol/L nicotinamide riboside (NR), achieving a conversion rate of 90% and a purity of 33%. The recombinant NMRK enzyme retained good activity after immobilization onto the carrier, enabling its reuse for more than 10 cycles. The initial activity of the immobilized NMRK enzyme was 540 U/g. After 11 cycles of use, the activity was 390 U/g, representing only a 27.8% decline in activity. This approach enhances the utilization efficiency of the NMRK enzyme while simultaneously reducing the production cost of NMN.