Support For Enzyme Immobilization Research Articles

A critical review of enzymes immobilized on chitosan composites: characterization and applications.

Enzymes with industrial significance are typically used in biological processes. However, instability, high sensitivity, and impractical recovery are the major drawbacks of enzymes in practical applications. In recent years, the immobilization technology has attracted wide attention to overcoming these restrictions and improving the efficiency of enzyme applications. Chitosan (CS) is a unique functional substance with biocompatibility, biodegradability, non-toxicity, and antibacterial properties. Chitosan composites are anticipated to be widely used in the near future for a variety of purposes, including as supports for enzyme immobilization, because of their advantages. Therefor this review explores the effects of the chitosan's structure, molecular weight, degree of deacetylation on the enzyme immobilized, effect of key factors, and the enzymes immobilized on chitosan based composites for numerous applications, including the fields of biosensor, biomedical science, food industry, environmental protection, and industrial production. Moreover, this study carefully investigates the advantages and disadvantages of using these composites as well as their potential in the future.

Chitosan with modified porosity and crosslinked with genipin: A dynamic system structurally characterized

In this work, a deep structural characterization of the chitosan-genipin complex was performed by techniques like small-angle X-ray scattering, dye absorption, atomic force microscopy, compression strength, nitrogen adsorption/desorption, scanning electronic microscopy, and thermogravimetric analysis to understand the changes in the structure through all steps of the process. For this, chitosan beads were modified with Na2CO3 to change porosity and then crosslinked with genipin. The prepared particles were used as support for enzyme immobilization. Our results demonstrated modification in the porosity of the chitosan beads, changing the number of pores and pore volume. After crosslinking process (pH 9), especially the microstructure of chitosan-genipin beads was modified, enhancing the fractality and, after enzyme immobilization (pH 4.5) the fractality increased at the higher scale (∼100 nm) and reduced at the lower scale (∼1–10 nm). β-galactosidase immobilized on the modified particles presented high stability on the continuous production of galactooligosaccharides for 30 days. Our results indicate that the chitosan-genipin complex can be characterized as a plastic and dynamic system due to its structural modifications, particularly in response to changes in the pH environment, even after the crosslinking process.

Insight into Bioactivity of In-situ Trapped Enzyme-Covalent-Organic Frameworks.

Selecting a suitable support material for enzyme immobilization with excellent biocatalytic activity and stability is a critical aspect in the development of functional biosystems. The highly stable and metal-free properties of covalent-organic frameworks (COFs) make them ideal supports for enzyme immobilization. Herein, we constructed three kinds of COFs via a biofriendly and one-pot synthetic strategy at room temperature in aqueous solution. Among the three developed COFs (COF-LZU1, RT-COF-1 and ACOF-1), the horseradish peroxidase (HRP)-incorporated COF-LZU1 is found to retain the highest activity. Structural analysis reveals that a weakest interaction between the hydrated enzyme and COF-LZU1, an easiest accessibility by the COF-LZU1 to the substrate, as well as an optimal conformation of enzyme together promote the bioactivity of HRP-COF-LZU1. Furthermore, the COF-LZU1 is revealed to be a versatile nanoplatform for encapsulating multiple enzymes. The COF-LZU1 also offers superior protection for the immobilized enzymes under harsh conditions and during recycling. The comprehensive understanding of interfacial interactions of COF host and enzyme guest, the substrate diffusion, as well as the enzyme conformation alteration within COF matrices represents an opportunity to design the ideal biocatalysts and opens a broad range of applications of these nanosystems.

Insight into Bioactivity of In‐situ Trapped Enzyme‐Covalent‐Organic Frameworks

AbstractSelecting a suitable support material for enzyme immobilization with excellent biocatalytic activity and stability is a critical aspect in the development of functional biosystems. The highly stable and metal‐free properties of covalent‐organic frameworks (COFs) make them ideal supports for enzyme immobilization. Herein, we constructed three kinds of COFs via a biofriendly and one‐pot synthetic strategy at room temperature in aqueous solution. Among the three developed COFs (COF‐LZU1, RT‐COF‐1 and ACOF‐1), the horseradish peroxidase (HRP)‐incorporated COF‐LZU1 is found to retain the highest activity. Structural analysis reveals that a weakest interaction between the hydrated enzyme and COF‐LZU1, an easiest accessibility by the COF‐LZU1 to the substrate, as well as an optimal conformation of enzyme together promote the bioactivity of HRP‐COF‐LZU1. Furthermore, the COF‐LZU1 is revealed to be a versatile nanoplatform for encapsulating multiple enzymes. The COF‐LZU1 also offers superior protection for the immobilized enzymes under harsh conditions and during recycling. The comprehensive understanding of interfacial interactions of COF host and enzyme guest, the substrate diffusion, as well as the enzyme conformation alteration within COF matrices represents an opportunity to design the ideal biocatalysts and opens a broad range of applications of these nanosystems.

Framework for Optimizing Polymeric Supports for Immobilized Biocatalysts by Computational Analysis of Enzyme Surface Hydrophobicity

Immobilization is a powerful strategy for improving enzyme usability and stability in various technologies that employ biocatalysis. However, the interactions leading to stabilization or destabilization remain poorly understood, and a support that may stabilize one enzyme may destabilize another. Employing chemically heterogeneous and complex random copolymer brushes as supports, we demonstrate a rational approach toward estimating the chemical composition of an optimally stabilizing enzyme immobilization support by computational analysis of enzyme surface hydrophobicity. This approach was tested by immobilizing a range of enzymes with diverse functions and hydrophobicity on tunable statistical random copolymer brush supports composed of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA). Remarkably, we observed greatly improved enzyme performance as a function of brush composition with enhancements in the retention of catalytic activity at temperatures as high as 90 °C. Additionally, we observed an increase in activity at the optimal temperature by as much as 20-fold relative to the activity at the optimal temperature of the unimmobilized form of the enzyme. Most significantly, our results showed that the optimal composition of the brush support correlated with the overall hydrophobicity of the enzyme surface (ΔGsolv,total/area), which was determined from computational analysis. This correlation provides a framework for the choice of polymer brush supports based on enzyme structure and stabilizing enzymes using complex synthetic materials.

One-pot synthesis of rod-like lignin@zeolitic imidazolate framework-8 with enhanced immobilization of β-glucosidase

Developing value-added lignin-based biomaterials for enzyme immobilization has drawn great attention. This study developed a cost-effective and eco-friendly one-pot strategy to synthesize the rod-like lignin@zeolitic imidazolate framework-8 (ZIF-8) hybrid nanomaterial by using lignin derived from lignocellulosic biofuel production. The incorporation of lignin into the ZIF-8 network not only altered the morphology, but also modified the surface properties of the material, making it an ideal support for enzyme immobilization. The synthesized nanoscale hybrid materials were used to immobilize beta-glucosidase (BG) with high immobilization capacity and about 92–166 mg/g of BG was immobilized through physical adsorption. The immobilized BG exhibited good stability, catalytic activity and recycling properties, and was reused under acidic conditions for more than 8 cycles with more than 60% activity kept. The as-prepared hybrid material can serve as a great carrier for immobilizing various biomolecules.

MXene/Fluoropolymer‐Derived Laser‐Carbonaceous All‐Fibrous Nanohybrid Patch for Soft Wearable Bioelectronics

AbstractWhile state‐of‐the‐art skin‐adhering fibrous electrodes have distinct benefits in personal wearable bioelectronics, considerable challenges persist in the production of fibrous‐based soft conductive biosensing nanomaterials and their integration into efficient multisensing platforms. Here, an electrochemical‐electrophysiological multimodal biosensing patch based on MXene/fluoropolymer nanofiber‐derived hierarchical porous TiO2 nanocatalyst interconnected 3D fibrous carbon nanohybrid electrodes is reported. The nanohybrid electrode is produced via a one‐step laser carbonaceous thermal oxidation, resulting in excellent elctroconductivity (sheet resistance = 15.6 Ω sq−1), rich active edges for effective electron transmission, and abundant support for enzyme immobilization. The features are attributed to three synergistic effects: i) conductivity of the interior, unoxidized MXene layers, ii) quick heterogeneous electron transmission of the exterior TiO2 nanoparticles, and iii) the porous disordered carbon's electron “bridge” effects. Based on the foregoing, the nanohybrid modified biosensing patch integrated into textile is demonstrated to be capable of simultaneously and precisely monitoring sweat glucose with pH adjustment (sensitivity of 77.12 µA mm−1 cm−2 within physiological concentrations of 0.01–2 × 10−3 m) and electrocardiogram signals (signal‐to‐noise ratio = 37.63 dB). This novel approach paves the way for controlled investigations of the nanohybrid, for several functionalization and design options, and for the mass manufacturing capabilities required in real‐world applications.

Highly interconnected porous monolithic and beaded polymers using high internal phase emulsion polymerization: tuning porous architecture through synthesis variables

AbstractOpen porous polymeric materials have gained popularity due to their exceptional properties and applications in tissue engineering scaffolds, drug delivery, enzyme immobilization and catalysis support. This study developed a novel two‐stage approach to create networked, crosslinked poly(2‐hydroxyethyl methacrylate‐co‐N,N′‐methylenebisacrylamide) HEMA‐MBA beads. The first part involves producing an oil‐in‐water‐in‐oil high internal phase emulsion (HIPE). This is followed by suspension polymerization using a redox initiator pair. In this study, a mixed surfactant combination with low and high hydrophilicity–lipophilicity balance surfactants was identified and successfully utilized to prepare a stable oil‐in‐water‐in‐oil HIPE. The effect of crosslinker concentration (i.e. crosslink density), surfactant concentration and monomer‐to‐porogen ratio on pore architecture and surface area were successfully evaluated. In addition, a new protocol was developed to synthesize HEMA‐MBA monoliths using an oil‐in‐water HIPE method at ambient temperature using a redox initiator pair. The effect of crosslink density and oil phase on pore architecture and surface area was evaluated. Key variables affecting the morphology of porous HEMA‐MBA beads and monoliths were identified and quantified, allowing future development of porous HEMA‐based polymer beads and monoliths with tunable morphologies which are suitable for numerous applications, especially in the biomedical field. © 2022 Society of Industrial Chemistry.

A {Cu6}-added polyoxometalate cluster-organic framework: synthesis, structure and application as a solid support for enzyme immobilization.

A new two-dimensional {Cu6}-added polyoxometalate cluster-organic framework (Cu-POMCOF) was prepared by a hydrothermal method from lacunary polyoxoanions and was applied as a solid support for immobilizing MP-11 and Cyt c. The biocomposite complex exhibits higher stability and catalytic activity than the original free enzyme.

Preparation and Characterization of Magnetic Metal-Organic Frameworks Functionalized by Ionic Liquid as Supports for Immobilization of Pancreatic Lipase.

Enzymes are difficult to recycle, which limits their large-scale industrial applications. In this work, an ionic liquid-modified magnetic metal–organic framework composite, IL-Fe3O4@UiO-66-NH2, was prepared and used as a support for enzyme immobilization. The properties of the support were characterized with X-ray powder diffraction (XRD), Fourier-transform infrared (FTIR) spectra, transmission electron microscopy (TEM), scanning electronic microscopy (SEM), and so on. The catalytic performance of the immobilized enzyme was also investigated in the hydrolysis reaction of glyceryl triacetate. Compared with soluble porcine pancreatic lipase (PPL), immobilized lipase (PPL-IL-Fe3O4@UiO-66-NH2) had greater catalytic activity under reaction conditions. It also showed better thermal stability and anti-denaturant properties. The specific activity of PPL-IL-Fe3O4@UiO-66-NH2 was 2.3 times higher than that of soluble PPL. After 10 repeated catalytic cycles, the residual activity of PPL-IL-Fe3O4@UiO-66-NH2 reached 74.4%, which was higher than that of PPL-Fe3O4@UiO-66-NH2 (62.3%). In addition, kinetic parameter tests revealed that PPL-IL-Fe3O4@UiO-66-NH2 had a stronger affinity to the substrate and, thus, exhibited higher catalytic efficiency. The results demonstrated that Fe3O4@UiO-66-NH2 modified by ionic liquids has great potential for immobilized enzymes.

Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis.

All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article.

Immobilization of a Bienzymatic System via Crosslinking to a Metal-Organic Framework

A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic linkers and inorganic nodes, MOFs feature empty void space with large surface areas and have the ability to be modified post-synthesis. Our target enzyme system for immobilization is glucose oxidase (GOx) and chloroperoxidase (CPO). Glucose oxidase catalyzes the oxidation of glucose and is used for many applications in biosensing, biofuel cells, and food production. Chloroperoxidase is a fungal heme enzyme that catalyzes peroxide-dependent halogenation, oxidation, and hydroxylation. These two enzymes work sequentially in this enzyme system by GOx producing peroxide, which activates CPO that reacts with a suitable substrate. This study focuses on using a zirconium-based MOF, UiO-66-NH2, to immobilize the enzyme system via crosslinking with the MOF’s amine group on the surface of the MOF. This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS), providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond CPO and GOx onto UiO-66-NH2, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx is discussed through assays and characterization analyses. The DSG-crosslinked composites displayed enhanced activity relative to the free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability, with at least 30% of the activity being retained after four catalytic cycles. The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability.

Electrospun aluminum silicate nanofibers as novel support material for immobilization of alcohol dehydrogenase

Ceramic materials with high surface area, large and open porosity are considered excellent supports for enzyme immobilization owing to their stability and reusability. The present study reports the electrospinning of aluminum silicate nanofiber supports from sol-gel precursors, the impact of different fabrication parameters on the microstructure of the nanofibers and their performance in enzyme immobilization. A change in nanofiber diameter and pore size of the aluminum silicate nanofibers was observed upon varying specific processing parameters, such as the sol-composition (precursor and polymer concentration), the electrospinning parameters and the subsequent heat treatment (calcination temperature). The enzyme, alcohol dehydrogenase (ADH), was immobilized on the aluminum silicate nanofibers by physical adsorption and covalent bonding. Activity retention of 17% and 42% was obtained after 12 d of storage and repeated reaction cycles for physically adsorbed and covalently bonded ADH, respectively. Overall, the immobilization of ADH on aluminum silicate nanofibers resulted in high enzyme loading and activity retention. However, as compared to covalent immobilization, a marked decrease in the enzyme activity during storage for physically adsorbed enzymes was observed, which was ascribed to leakage of the enzymes from the nanofibers. Such fibers can improve enzyme stability and promote a higher residual activity of the immobilized enzyme as compared to the free enzyme. The results shown in this study thus suggest that aluminum silicate nanofibers, with their high surface area, are promising support materials for the immobilization of enzymes.

The effect of organic coatings in the magnetization of CoFe2O4 nanoparticles

Cobalt ferrite has attracted considerable attention in recent years due to its unique physical properties, such as high Curie temperature, large magnetocrystalline anisotropy, high coercivity, moderate saturation magnetization, large magnetostrictive coefficient, and excellent chemical stability and mechanical hardness. This work focuses on the neutron scattering results of the magnetic response characteristics of polysaccharide fucan coated cobalt ferrite nanoparticles for their application as a solid support for enzyme immobilization and other biotechnology applications. Here, we unambiguously show that surfactant coating of nanoparticles can significantly affect their magnetic response throughout the nanoparticle volume. While it has been recently suggested that oleic acid may preserve nanoscale magnetism in ferrites, we present evidence that the influence of oleic acid on the magnetic response of CoFe2O4 nanoparticles is more than a surface effect, instead pervading throughout the interior of the nanoparticle.

Hydrophilic Nonwoven Nanofiber Membranes as Nanostructured Supports for Enzyme Immobilization.

The high porosity, interconnected pore structure, and high surface area-to-volume ratio make the hydrophilic nonwoven nanofiber membranes (NV-NF-Ms) promising nanostructured supports for enzyme immobilization in different biotechnological applications. In this work, NV-NF-Ms with excellent mechanical and chemical properties were designed and fabricated by electrospinning in one step without using additives or complicated crosslinking processes after electrospinning. To do so, two types of ultrahigh-molecular-weight linear copolymers with very different mechanical properties were used. Methyl methacrylate-co-hydroxyethyl methacrylate (p(MMA)-co-p(HEMA)) and methyl acrylate-co-hydroxyethyl acrylate (p(MA)-co-p(HEA)) were designed and synthesized by reverse atom transfer radical polymerization (reverse-ATRP) and copper-mediated living radical polymerization (Cu0-MC-LRP), respectively. The copolymers were characterized by nuclear magnetic resonance (1H-NMR) spectroscopy and by triple detection gel permeation chromatography (GPC). The polarity, topology, and molecular weight of the copolymers were perfectly adjusted. The polymeric blend formed by (MMA)1002-co-(HEMA)1002 (Mw = 230,855 ± 7418 Da; Mn = 115,748 ± 35,567 Da; PDI = 2.00) and (MA)11709-co-(HEA)7806 (Mw = 1.972 × 106 ± 33,729 Da; Mn = 1.395 × 106 ± 35,019 Da; PDI = 1.41) was used to manufacture (without additives or chemical crosslinking processes) hydroxylated nonwoven nanofiber membranes (NV-NF-Ms-OH; 300 nm in fiber diameter) with excellent mechanical and chemical properties. The morphology of NV-NF-Ms-OH was studied by scanning electron microscopy (SEM). The suitability for enzyme binding was proven by designing a palette of different surface functionalization to enable both reversible and irreversible enzyme immobilization. NV-NF-Ms-OH were successfully functionalized with vinyl sulfone (281 ± 20 μmol/g), carboxyl (560 ± 50 μmol/g), and amine groups (281 ± 20 μmol/g) and applied for the immobilization of two enzymes of biotechnological interest. Galactose oxidase was immobilized on vinyl sulfone-activated materials and carboxyl-activated materials, while laccase was immobilized onto amine-activated materials. These preliminary results are a promising basis for the application of nonwoven membranes in enzyme technology.

Immobilization of Interfacial Activated Candida rugosa Lipase Onto Magnetic Chitosan Using Dialdehyde Cellulose as Cross-Linking Agent.

Candida rugosa lipase (CRL) was activated with surfactants (sodium dodecyl sulfate [SDS]) and covalently immobilized onto a nanocomposite (Fe3O4-CS-DAC) fabricated by combining magnetic nanoparticles Fe3O4 with chitosan (CS) using polysaccharide macromolecule dialdehyde cellulose (DAC) as the cross-linking agent. Fourier transform infrared spectroscopy, transmission electron microscope, thermogravimetric analysis, and X-ray diffraction characterizations confirmed that the organic–inorganic nanocomposite support modified by DAC was successfully prepared. Enzymology experiments confirmed that high enzyme loading (60.9 mg/g) and 1.7 times specific enzyme activity could be obtained under the optimal immobilization conditions. The stability and reusability of immobilized CRL (Fe3O4-CS-DAC-SDS-CRL) were significantly improved simultaneously. Circular dichroism analysis revealed that the active conformation of immobilized CRL was maintained well. Results demonstrated that the inorganic–organic nanocomposite modified by carbohydrate polymer derivatives could be used as an ideal support for enzyme immobilization.

The Chemistry and Applications of Metal-Organic Frameworks (MOFs) as Industrial Enzyme Immobilization Systems.

Enzymatic biocatalysis is a sustainable technology. Enzymes are versatile and highly efficient biocatalysts, and have been widely employed due to their biodegradable nature. However, because the three-dimensional structure of these enzymes is predominantly maintained by weaker non-covalent interactions, external conditions, such as temperature and pH variations, as well as the presence of chemical compounds, can modify or even neutralize their biological activity. The enablement of this category of processes is the result of the several advances in the areas of molecular biology and biotechnology achieved over the past two decades. In this scenario, metal–organic frameworks (MOFs) are highlighted as efficient supports for enzyme immobilization. They can be used to ‘house’ a specific enzyme, providing it with protection from environmental influences. This review discusses MOFs as structures; emphasizes their synthesis strategies, properties, and applications; explores the existing methods of using immobilization processes of various enzymes; and lists their possible chemical modifications and combinations with other compounds to formulate the ideal supports for a given application.

Immobilization of Urease onto Nanochitosan Enhanced the Enzyme Efficiency: Biophysical Studies and in Vitro Clinical Application on Nephropathy Diabetic Iraqi Patients

Immobilization of enzymes is an effective method for improving the properties and applications of modern enzymes. There are several supports for enzyme immobilization. Because of its unique features, such as inertness and high surface area, chitosan was widely used to immobilize enzymes. Immobilization of urease onto chitosan is a promising approach to treating high urea levels in the blood, however, the immobilization conditions for the best kinetics and enzyme efficiency are still challenging. Herein, we tried to immobilize urease onto nanochitosan (chitosan NPs) through a cross-linker and study the kinetics (km and v max values) and thermodynamics (Ea, ∆H, ∆S, and ∆G) parameters of the enzyme reaction before and after immobilization at different substrate concentration (50, 100, 150, 200, and 250 mg/dl) and incubation temperature (15, 20, 25, 30, 35, and 40°C) under selected optimum conditions. The immobilized urease chitosan NPs was characterized in our previous work using Fourier transform infrared infrared (FTIR), Atomic force force microscopy (AFM), and and imaged here by scanning electron microscopy microscopy (SEM). Results revealed that the highest efficiency % of immobilization (70.38%) was observed at 750 mg/ml chitosan NPs and phosphate buffer pH 7 at 40°C. With an increase of Km value for the immobilized enzyme, however, the efficiency of the enzyme was significantly higher than the free enzyme, p < 0.001 . In addition, the activation energy of the reaction catalyzed by the immobilized enzyme was lower than that of the free enzyme, which suggests that the active site geometry of the immobilized enzyme was more favorable to accommodate the substrate and thus required less energy than that of the free enzyme. The reaction was endothermic by means of positive ∆H. The immobilized urease enzyme was in vitro applied to blood samples of Iraq nephropathy diabetic patients (n = 35) to investigate the effect on serum urease activity and urea level compared to healthy volunteers. Interestingly, the activity of serum urease significantly increased after adding the immobilized enzyme and the level of urea significantly decreased ( p < 0.0001 ) by ∼1.5 folds. Thus, applying an immobilized urease urease to remove urea from blood could be effective in the blood detoxification or dialysis regeneration system of artificial kidney machines.

Development of a biocomposite based on alginate/gelatin crosslinked with genipin for β-galactosidase immobilization: Performance and characteristics

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for β-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized β-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

Enzymes immobilized in wood-derived cellulose scaffold for constructing a novel modular bioreactor

Modular bioreactors can provide a flexible platform for constructing complex multi-step pathways, which may be a solution for maximizing reactions and overcoming the complexity of multi-enzyme systems. Here, we selected wood-derived cellulose scaffold as a support for enzyme immobilization and constructed the modular bioreactor. Cellulose scaffold was prepared after removing lignin from wood, followed by citric acid functionalization and the addition of glutaraldehyde finally allowed the cross-linking of enzymes. Three enzymes, horseradish peroxidase (HRP), glucose oxidase (GOD), and catalase (CAT), were separately immobilized, resulting in the immobilized enzyme amount to over 40 mg/g. The introduction of carboxyl groups from citric acid facilitated the rapid enzyme adsorption on the support surface and immobilized enzymes possess ∼65% expressed activity. Modular bioreactors were constructed by using the immobilized enzymes. With the immobilized HRP module, reactor showed desired catalytic performance with the phenol degradation rate of > 90%. Also, a pH regulation can occur in the bioreactors for preserving enzyme activities and neutralizing acid products. In the GOD/CAT modular bioreactor, the cascade reaction with adjusting pH values can achieve a 95% yield of sodium gluconate and exhibit a favorable reusability of 5 operation cycles.