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Multi-enzyme biocatalysis is an important technology to produce many valuable chemicals in the industry. Different strategies for the construction of multi-enzyme systems have been reported. In particular, immobilization of multi-enzymes on the support materials has been proved to be one of the most efficient approaches, which can increase the enzymatic activity via substrate channeling and improve the stability and reusability of enzymes. A general overview of the characteristics of support materials and their corresponding attachment techniques used for multi-enzyme immobilization will be provided here. This review will focus on the materials-based techniques for multi-enzyme immobilization, which aims to present the recent advances and future prospects in the area of multi-enzyme biocatalysis based on support immobilization.

The current advancements in nanotechnology had exquisite impacts on biocatalysis. Enzymes are exceptional biocatalysts because of their excellent substrate selectivity, regio- and stereo-specificity, and capacity to accelerate the reaction rate up to several orders of magnitude. One of the significant challenges in biotechnology is the utilization and development of enzymes as reliable biocatalysts. Applications of enzymes have drawn a lot of attention as demand for environmentally friendly and sustainable operations increases. Enzymes are promising biocatalysts with a wide range of uses, including as a biosensor, in food, agricultural, and pharmaceutical industries. The distinctive catalytic chemistry of enzyme-linked compatible nanostructures put forward many applications, including biocatalysis. The most recent advancements in enzyme immobilization organic / inorganic supports—including carbon-based, polymeric or hybrid, metal–organic framework—as well as various nanomaterials comprised of metals and metal oxides are discussed. The methods for immobilizing enzymes onto carriers and their stability and catalytic properties are also highlighted. The synergistic coupling of nanotechnology with biotechnology covers a broad range of tremendous applications. Nanostructures have specific characteristics that can balance parameters like effective encapsulation of enzymes, porosity, etc., which present peculiar prospects for designing an ideal biocatalyst. For synthetic chemistry and bio-manufacturing, photo-enzymes are potentially attractive biocatalysts. This review highlights that efforts are underway to bring the benefits of combining two research-intensive fields, biocatalysis and photo-biocatalysis, in a meaningful way. Graphical Abstract

Multi-enzyme immobilization onto a capacitive field-effect biosensor by nano-spotting technique is presented. The nano-spotting technique allows to immobilize different enzymes simultaneously on the sensor surface with high spatial resolution without additional photolithographical patterning. The amount of applied enzymatic cocktail on the sensor surface can be tailored. Capacitive electrolyte-insulator-semiconductor (EIS) field-effect sensors with Ta2O5 as pH-sensitive transducer layer have been chosen to immobilize the three different (pL droplets) enzymes penicillinase, urease, and glucose oxidase. Nano-spotting immobilization is compared to conventional drop-coating method by defining different geometrical layouts on the sensor surface (fully, half-, and quarter-spotted). The drop diameter is varying between 84 µm and 102 µm, depending on the number of applied drops (1 to 4) per spot. For multi-analyte detection, penicillinase and urease are simultaneously nano-spotted on the EIS sensor. Sensor characterization was performed by C/V (capacitance/voltage) and ConCap (constant capacitance) measurements. Average penicillin, glucose, and urea sensitivities for the spotted enzymes were 81.7 mV/dec, 40.5 mV/dec, and 68.9 mV/dec, respectively.

Climate change and the energy crisis, driven by excessive CO2 emissions, have emerged as pressing global challenges. The conversion of CO2 into high-value chemicals not only mitigates atmospheric CO2 levels but also optimizes carbon resource utilization. Enzyme-catalyzed carbon technology offers a green and efficient approach to CO2 conversion. However, free enzymes are prone to inactivation and denaturation under reaction conditions, which limit their practical applications. Metal–organic frameworks (MOFs) serve as effective carriers for enzyme immobilization, offering porous crystalline structures that enhance enzyme stability. Moreover, their high specific surface area facilitates strong gas adsorption, making enzyme@MOF composites particularly advantageous for CO2 catalytic conversion. In this paper, we review the synthesis technologies and the application of enzyme@MOFs in CO2 catalytic conversion. Furthermore, the strategies, including the enhancement of CO2 utilization, coenzyme regeneration efficiency, and substrate mass transfer efficiency, are also discussed to further improve the efficiency of enzyme@MOFs in CO2 conversion. The aim of this review is to present innovative ideas for future research and to highlight the potential applications of enzyme@MOFs in achieving efficient CO2 conversion.

Glycerol-based biorefinery can be a highly profitable process by producing highly value-added products such as dihydroxyacetone via combined catalytic strategies. Here, two-enzyme system is adopted for the transformation of glycerol into highly valuable dihydroxyacetone as well as cofactor regeneration at the same time. Glycerol dehydrogenase (GDH) and alcohol dehydrogenase (ADH) are co-immobilized within magnetically separable and spherical mesocellular silica foam (Mag-S-MCF), to prepare NER-(GDH/ADH). In details, GDH and ADH are adsorbed into the mesopores of Mag-S-MCF, and further crosslinked within the mesopores of Mag-S-MCF. The resulting nanoscale enzyme reactors (NER) of crosslinked GDH and ADH molecules within the bottle-neck structured mesopores can effectively prevent larger sized crosslinked enzyme aggregates from being leached out of smaller mesopores, due to the bottle-neck mesopore structure of Mag-S-MCF, as well as stabilize the activity of GDH and ADH upon chemical crosslinking, effectively preventing the denaturation of enzyme molecules. More importantly, the proximity of GDH and ADH molecules within mesopores of NER improves the efficiency of cofactor-mediated dual-enzymatic reactions by relieving mass-transfer limitations and improving cofactor recycling in an effective way, expediting both glycerol oxidation and dihydroxyacetone generation at the same time. As a result, the DHA concentration of NER-(GDH/ADH) and the simple mixture of NER-GDH and NER-ADH were 410 μM and 336 μM, respectively. To the best of our knowledge, this report is the first demonstration of stabilized nanoscale multi-enzyme reactor system, equipped with efficient cofactor regeneration within confined mesopores, for efficient glycerol transformation to high-valued dihydroxyacetone.

Enzymes, as nature’s precision biocatalysts, hold transformative potential across industrial, environmental, and biomedical sectors. However, their instability, solvent sensitivity, and limited reusability in their free form necessitate advanced immobilization strategies to enhance their robustness and scalability. This review critically examines cutting-edge advancements in enzyme immobilization, focusing on the integration of artificial intelligence (AI), novel nanomaterials, and dynamic carrier systems to overcome the traditional limitations of mass transfer, enzyme leakage, and cost inefficiency. Key innovations such as metal–organic frameworks (MOFs), magnetic nanoparticles, self-healing hydrogels, and 3D-printed scaffolds are highlighted for their ability to optimize enzyme orientation, stability, and catalytic efficiency under extreme conditions. Moreover, AI-driven predictive modeling and machine learning emerge as pivotal tools for rationalizing nanomaterial synthesis, multi-enzyme cascade design, and toxicity assessment, while microfluidic systems enable precise biocatalyst fabrication. This review also explores emerging carrier-free strategies, including cross-linked enzyme aggregates (CLEAs) and DNA-directed immobilization, which minimize diffusion barriers and enhance substrate affinity. Despite progress, challenges persist in regards to eco-friendly nanomaterial production, industrial scalability, and real-world application viability. Future directions emphasize sustainable hybrid material design, AI-aided lifecycle assessments, and interdisciplinary synergies between synthetic biology, nanotechnology, and data analytics. By connecting laboratory innovation with industrial needs, this work provides a forward-thinking framework to harness immobilized enzymes for achieving global sustainability goals, particularly in bioremediation, bioenergy, and precision medicine.

Oxidoreductases are a wide class of enzymes that can catalyze biological oxidation and reduction reactions. Nowadays, oxidoreductases play a vital part in most bioenergetic metabolic pathways, which have important applications in biodegradation, bioremediation, environmental applications, as well as biosensors. However, free oxidoreductases are not stable and hard to be recycled. In addition, cofactors are needed in most oxidoreductases catalyze reactions, which are so expensive and unstable that it hinders their industrial applications. Enzyme immobilization is a feasible strategy that can overcome these problems. Recently, metal–organic frameworks (MOFs) have shown great potential as support materials for immobilizing enzymes due to their unique properties, such as high surface-area-to-volume ratio, chemical stability, functional designability, and tunable pore size. This review discussed the application of MOFs and their composites as immobilized carriers of oxidoreductase, as well as the application of MOFs as catalysts and immobilized carriers in redox reactions in the perspective of the function of MOFs materials. The paper also focuses on the potential of MOF carrier-based oxidoreductase immobilization for designing an enzyme cascade reaction system.

We report the preparation, characterization and application of a novel magnetic four-enzyme nanobiocatalyst prepared by the simultaneous covalent co-immobilization of cellulase (CelDZ1), β-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) onto the surface of amino-functionalized magnetic nanoparticles (MNPs). This nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization process yielded maximum recovered enzymatic activity (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a 10% v/v cross-linker concentration, after 2 h incubation time and at 1:1 mass ratio of MNPs to total enzyme content. The immobilization process leads to an increase of Km and a decrease of Vmax values of co-immobilized enzymes. The thermal stability studies of the co-immobilized enzymes indicated up to 2-fold increase in half-life time constants and up to 1.5-fold increase in their deactivation energies compared to the native enzymes. The enhanced thermodynamic parameters of the four-enzyme co-immobilized MNPs also suggested increment in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant part of their activity (up to 50%) after 5 reaction cycles at 50 °C and remained active even after 24 d of incubation at 5 °C. The nanobiocatalyst was successfully applied in a four-step cascade reaction involving the hydrolysis of cellulose.

Multi-enzymatic cascade reactions, that is, the integration of several biocatalytic transformations occurring simultaneously, offer a wide range of possibilities and new ways to synthesize products with high added value. Such systems improve biocatalytic processes by saving time and reducing waste, while being self-sufficient in terms of requirements for related factors. As a more environmentally friendly and sustainable alternative method for the production of chemicals and biological products increases in importance, cascade reactions can be a very promising approach in this direction, eliminating the short traditional step-by-step synthesis. When using multi-enzyme systems in cascade reactions, local substrate concentration occurs around the second and all subsequent enzymes that enter the system. Such systems are particularly attractive because many commercially available enzymes operate under relatively similar environmental conditions (e.g., pH and temperature). In this work, various types of multi-enzyme systems have been studied using various enzymes, such as, for example, cellulase, peroxidase, glucose oxidase, etc. Various methods of immobilization of several enzymes on a single carrier for the production of gluconic acid and its derivatives are described. Examples of the processing of cellulose-containing raw materials using multi-enzyme systems and methods of enzyme compartmentalization are given. This research is particularly relevant because enzymatic cascade systems provide promising approaches to creating more flexible ways to produce various chemical products at a lower cost.

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate.