Approach For Enzyme Immobilization Research Articles

Exploring Enzyme Encapsulation Efficiency in MOFs Using Eco-Friendly Approaches.

The encapsulation of protein enzymes in metal-organic frameworks (MOFs) has been recognized as an effective enzyme immobilization approach. In this study, we demonstrated the influence of enzyme amount and the isoelectric points (pI) of different enzymes on the enzyme loading capacity in both mechanochemical (ball-milling) and water-based approaches. We found that increasing enzyme amounts enhances MOF enzyme loading without compromising activity, while the MOF shell protects encapsulated enzymes from proteinase K degradation through its size-sheltering mechanism. However, an excess of enzymes can hinder the formation of ZIF-90. Moreover, enzymes with low pI values (e. g., catalase, pI 5.4) facilitate encapsulation in MOFs, whereas enzymes with high pI values (e. g., lysozyme, pI 11.35) are more challenging to encapsulate. The simulation results revealed that increasing the enzyme amounts and pI values raises the activation energy necessary for MOF formation. This study highlights the crucial role of enzyme properties in the encapsulation process within MOFs, providing valuable insights for fabricating enzyme-MOF biocomposites for diverse applications, such as protein drug delivery.

Boosting catalytic efficiency of lipase by regulating amphiphilic microenvironment through reversible addition-fragmentation chain transfer polymerized modifications on polyacrylonitrile fiber

Lipases are increasingly attracting attention in green and sustainable biodiesel production. Currently, the research emphasis lies in immobilizing unstable lipase onto carriers to enhance its performance. Polyacrylonitrile fiber (PANF) is considered to be a promising material for lipase immobilization due to its excellent properties. In this study, functional carriers with regulated surface hydrophobicity were obtained by loading functional groups on PANF via reversible addition-fragmentation chain transfer (RAFT) polymerized modification, and Candida rugosa lipase (CRL) was covalently immobilized on the carrier with glutaraldehyde as a linker. By employing this optimized biocatalyst PANF@BMA&2VImBr-NH2-CRL in the transesterification process, the yield of biodiesel derived from soybean oil reached an impressive 92.7 %. The outstanding performance can be attributed to the activation of lipase interface induced by hydrophobic microenvironment derived from alkyl ester on the carrier skeleton. Moreover, the stability and storage performance of immobilized lipase were significantly improved. The immobilized lipase exhibited facile recovery and maintained a consistent biodiesel yield of 80.9 % even after undergoing 5 cycles of reuse, thereby highlighting its potential for sustainable production. To sum up, our research demonstrates that the designed and prepared process of PANF-supported lipase offers a promising approach for enzyme immobilization, thereby presenting extensive potential applications in the field of biotechnology.

Carbonic anhydrase immobilized on Zn(II)-geopolymer membrane for CO2 capture

Inorganic membranes are widely used in water filtration and gas separation due to their many advantages. In this work, we proposed an innovative approach for enzyme immobilization using a geopolymer based tubular inorganic membrane (GM) as a carrier. The GM has abundant mesopores, excellent mechanical strength and strong stability for continuous flow systems in biocatalysis field, and carbonic anhydrase (CA) was successfully loaded into this mesoporous membrane using adsorption-crosslinking method, relying on the excellent ion exchange capacity of geopolymers and large number of hydroxyl groups naturally present in the membrane's mesopores. The results of the immobilization experiments showed that the immobilized enzyme composite membrane (GZnM-CA) could achieve 63.7% enzyme recovery. This is a significant improvement over the enzyme recoveries obtained on organic membranes which are typically less than 50%. In stability tests, the membrane retained more than 90.0% relative activity after 10 consecutive uses. In the CO2 capture assay, the CO2 capture efficiency developed dramatically under the catalytic of GZnM-CA. To our delight, the membrane has excellent separation capability for CO2 gas mixtures, especially at low CO2 concentrations, achieving 99.90% CO2 removal rate, and this result can be maintained for a long period of time in a pH = 8.0 environment.

A Review on Approaches for Enzyme Immobilization

Abstract: Enzyme The enzymes are widely studied and explored area due to its abundant potential to cater wide application range which makes researchers to study more of this area. The high catalytic activity enabling ease in reactions formation of multiple products and byproducts or breakage of complex substances makes it an unavoidable option. However the cost of pure enzyme increases the economic burden on the operations obstructing its full fledged use. This leads to making enzyme restrict or immobilize within any inert matrix which can improve the reusability and also improve the tolerance against pH and temperature that can improve the activity of enzyme. Many different enzyme immobilization approaches are present in following article we have discussed some of the used approaches.

Film-shaped reusable smart polymer to produce lactose-free milk by simple immersion

In this study, we report the synthesis and characterization of a highly manageable polyacrylic film material for enzyme immobilization, using β-galactosidase (β-gal) as a model enzyme. The material is based on commercially available monomers and achieves efficient immobilization of β-gal through the formation of azo linkages between amino styrene groups in the polyacrylic material and the enzyme. The immobilized enzyme demonstrates superior performance compared to free enzyme in lactose hydrolysis of UHT milk, achieving lactose concentrations below 0.1% (<1 mg/mL), indicating its potential for lactose hydrolysis in dairy products. The film-shaped material is designed for easy submersion and removal, similar to a smart card, and offers reusability, with the ability to be reused at least 10 times without loss of enzymatic activity. Characterization of the immobilized enzyme on the polymeric material was performed using various techniques, including scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Protein release studies confirmed the stability of the immobilized enzyme during prolonged incubation in aqueous solution without significant enzyme leakage. Overall, the polyacrylic film material demonstrates promise as a simple and efficient approach for enzyme immobilization, with potential applications in various industries, including the food industry.

Invertase Immobilization on Magnetite Nanoparticles for Efficient Fructooligosaccharide Generation: A Comprehensive Kinetic Analysis and Reactor Design Strategy

This study investigated the effectiveness of immobilizing Saccharomyces cerevisiae invertase (SInv) on magnetite nanoparticles to produce fructooligosaccharides (FOSs). Based on the existing literature and accompanied by parameter estimation, a modified kinetic model was employed to represent the kinetics of sucrose hydrolysis and transfructosylation using SInv immobilized on magnetite nanoparticle surfaces. This model was utilized to simulate the performance of batch reactors for both free and immobilized enzymes. The maximum FOS concentration for the free enzyme was determined to be 123.1 mM, while the immobilized case achieved a slightly higher concentration of 125.4 mM. Furthermore, a continuous stirred-tank reactor (CSTR) model was developed for the immobilized enzyme, resulting in a maximum FOS concentration of 73.96 mM at the reactor’s outlet and a dilution rate of 14.2 h−1. To examine the impact of glucose inhibition on FOS production, a glucose oxidase reaction mechanism was integrated into the fitted immobilized theoretical model. In a batch reactor, the reduction or elimination of glucose in the reactive media led to a 2.1% increase in FOS production. Immobilizing the biocatalyst enhanced the overall performance of SInv. This enzyme immobilization approach also holds the potential for coupling glucose oxidase onto functionalized nanoparticles to minimize glucose inhibition, thereby improving FOS synthesis and facilitating optimal enzyme recovery and reuse.

Competition between enzymatic and non-enzymatic electrochemical determination of cholesterol

Cholesterol is considered an essential component in animal bodies that plays a vital role in hormone production and creating vitamin D. The elevated blood cholesterol level increases the possibility of heart disease. Consequently, the clinical diagnosis of cholesterol concentration is critical for human health care. The electrochemical sensor is one of the leading techniques for detecting cholesterol in biological fluids with high accuracy. Therefore, this review included comparative insights into the enzymatic and non-enzymatic techniques for electrochemical sensing. We discussed the recent progress in the electrochemical determination of cholesterol via various materials, e.g., metal composites, carbonaceous materials, ionic liquid crystals, and polymers. We explained electrochemical sensing techniques like amperometric, potentiometric, conductometric, and impedimetric methods. Furthermore, various enzyme-immobilization approaches were reported, like surface binding and encapsulation. The synergistic effect between the electrode surface and enzymes was demonstrated to clarify the detection mechanism. The review article summarized the outcome detection limits and linear range of detection for various surfaces for enzymatic and non-enzymatic electrochemical techniques within the last twenty years.

Mussel-Inspired Electro-oxidation-Modified Three-Dimensional Printed Carriers for a Versatile Enzyme Immobilization Approach

Conventional enzyme immobilization approaches can only immobilize certain specific enzymes with poor generality. Attempts to improve the universality of enzyme types tend to impart them with more enzymatic catalysis applications. Here, inspired by mussel adhesive proteins, we present a novel eco-friendly surface carrier that was 3D printed and modified by electro-oxidation for enzyme immobilization. The carrier was fabricated through 3D printing by transforming acrylonitrile butadiene styrene (ABS) material into a suitable structure (3DABS). Then, electro-oxidative modification was performed on the surface to form a polydopamine (PDA) coating (3DABS-PDA). The desired structures for the enzyme immobilization carriers were obtained through 3D printing technology, while electro-oxidation modification of the surface provided numerous and firmly covalent binding sites. Based on these features, we have demonstrated that 3D printed and electro-oxidation-modified carriers could be applied to immobilize different types of enzymes. The loading capacity of all immobilized enzymes (galV, EG5C-1, XynLK9, and kdcA) exceeded 25 mg·g–1 (37.7 mg·g–1 for galV), and after 10 reuse cycles, the substrate conversion rate of 3DABS-PDA@galV was still over 85%. The carriers can be reused after simple processing. These results indicate that 3DABS-PDA provides an efficient, sustainable, and versatile approach for enzyme immobilization and exhibits excellent value in various enzymatic catalysis applications.

Nanomaterial-based electrochemical enzymatic biosensors for recognizing phenolic compounds in aqueous effluents

With the rapid development of industrial society, phenolic pollutants already identified in water are severe threats to human health. Traditional detection techniques like chromatography are poor in the ability of cost-effectiveness and on-site detection. In recent years, electrochemical enzymatic biosensors have attracted increasing attention for use in the recognition of phenolic compounds, which is considered an effective strategy for the product transfer of portable analytical devices. Although electrochemical enzymatic biosensors provide a fast, accurate on-site detection technique, the difficulties of enzyme deactivation, poor stability and low sensitivity remain to be solved. Thus, effective immobilization methods of enzymes and nanomaterials with excellent properties have been extensively researched to obtain a high-sensitivity and high-stability biosensing platform. Simultaneous detection of multiple phenols may become the focus of further research. In this review, we provide an overview of recent progress toward electrochemical enzymatic biosensors for the detection of phenolic compounds, including enzyme immobilization approaches and advanced nanomaterials, especially nanocomposites with attractive properties such as good conductivity, high specific surface area, and porous structure. We will comprehensively discuss the features and mechanisms of the main enzymes adopted in the construction of different phenolic biosensors, as well as traditional methods (e.g., adsorption, covalent bonding, entrapment, encapsulation, cross-linking) of enzyme immobilization. The most effective method is based on the properties of enzymes, supports and application objective because there is no one-size-fits-all method of enzymatic immobilization. The emphasis will be given to various advanced nanomaterials, including their special nanostructures, preparation methods and performance. Finally, the main challenges in future research on electrochemical phenolic biosensors will be discussed to provide further perspectives for practical applications in dynamic and on-site monitoring. We believe this review will deliver an important inspiration for the construction of novel and high-performance electrochemical biosensors from enzyme selection to nanomaterial design for the detection of various hazardous materials. We believe this review will deliver an important inspiration on the construction of novel and high-performance electrochemical biosensors from the enzyme selection to the nanomaterial design for detections of various hazardous materials.

Termofilik Anoxybacillus gonensis glukoz izomerazının DEAE-Sefaroz üzerine iyonik ve kovalent immobilizasyonu

High fructose corn syrup (HFCS), which is produced by the conversion of one sugar into another (glucose to fructose), has a marketing value. Hence, different glucose isomerases [(GI) (D-xylose ketol isomerase, EC 5.3.1.5)] isolated from different sources (macro-and microorganisms) were researched until today. In addition, the cost reduction of GI production for industrial applications has been investigated and applied with different techniques. Enzyme immobilization approaches have prominent features because they allow enzymes to be used repeatedly. In the current study, Anoxybacillus gonensis G2T glucose isomerase (AgoGI) (wild type) were immobilized with ionic and covalent binding on DEAE-sepharose matrix. Afterward, kinetic and biochemical parameters of the immobilized enzymes were evaluated. The pH and temperature parameters, in which the ionic and covalent immobilized enzymes showed the best activity, were determined as 6.50 and 85 °C, respectively. The kinetic data (Vmax and Km) of ionic bound AgoGI on DEAE-sepharose were 4.85±2.09 μmol/min/mg protein and 130,57±5,42 mM, as covalent immobilized AgoGI on the same matrix were 40.51± 0.81 μmol/min/mg protein µmol/min and 127,28±2,96 mM, respectively. Consequently, the usage of DEAE-sepharose for both covalent and ionic immobilization as immobilization matrix did not exhibit any negative effects on biochemical and kinetic parameters of glucose isomerase. Therefore, immobilized AgoGI on DEAE-sepharose was an excellent and promising tool for HFCS production.

Effect of process parameters on immobilization of cyclodextrin glucanotranferase (CGTase) from Bacillus licheniformis on pineapple waste

Cyclodextrin glucanotransferase (CGTase) is capable of degrading starch to produce cyclodextrin (CD). CD production has increased over the years due to its applications in numerous industries like environmental engineering, food industries, and pharmaceutical industries. The enzyme immobilization approach was commonly implemented to overcome low CD production during the reaction process due to the instability and denaturation of CGTase. The objective of this study is to immobilize CGTase produced by Bacillus licheniformis on pineapple peel. The influence of process parameters on the CGTase immobilization process such as pH (5, 6, 7, 8, and 9), contact time (4, 8, 12, 16, 20, 24, and 28 hr), and temperature (20, 25, 30, 35 and 40 °C) was studied. pH 7 was the best pH for CGTase immobilization, with a 75.97% of immobilization yield. In addition, the optimal temperature that produced the high immobilization yield (76.80%) was at 25 ℃. Meanwhile, the optimum contact time was detected at 24 hr, with 75.53% of immobilized yield. In conclusion, the immobilization process could increase the enzyme stability and increase CD production, valuable for application in industries.

Silaffin-3-derived pentalysine cluster as a new fusion tag for one-step immobilization and purification of recombinant Bacillus subtilis catalase on bare silica particles

Bio-catalysis by enzymes on solid surfaces has been implemented in several practical applications. However, the current methods for efficient enzyme immobilization with retained activity need further development. Herein, a simple, rapid, and economical, bio-affinity-based approach was developed for the direct immobilization with high activity recovery of the Bacillus subtilis catalase (CAT), recombinantly expressed in Escherichia coli. Silaffin-3-derived pentalysine cluster (Sil3K) from Thalassiosira pseudonana and its mutant variant (penta-arginine peptide; Sil3R) were used for the first time in the non-covalent immobilization of the recombinant enzyme on silica particles. The fusion proteins CAT-Sil3K and CAT-Sil3R were selectively loaded from the cell lysates onto the silica surface. Unexpectedly, the Lys-based tag (Sil3K) was the superior to Arg-based tag (Sil3R) or tag-less system for the high recovery of CAT activity upon immobilization; an 8.4-fold and 1.5-fold increase in the catalytic activity was observed for CAT-Sil3K compared with the tag-less CAT and CAT-Sil3R, respectively. Furthermore, the CAT-Sil3K immobilized on silica particles exhibited improved thermal, pH and storage stabilities, and retained 72% of the initial activity after five reaction cycles. Moreover, CAT-Sil3K was released with approximately 85% recovery and 91% purity, in a biologically active form when free lysine solution was used as the eluent. Our data proved that Sil3K-tag, 12-mer peptide, can be a highly promising silica-affinity tag for effective enzyme immobilization with preserved activity. Additionally, the novel findings obtained here may open a new route not only for cost-effective enzyme immobilization approaches but also for high recovery of enzyme activity.

Functionalization of multiwalled carbon nanotubes for enzyme immobilization.

A simple strategy for enzyme immobilization onto homo- and hetero-functionalized multiwalled carbon nanotubes (MWCNTs) is described. Homo-functionalization of MWCNTs can be carried out using an amino-silane compound, i.e., 3-aminopropyl-triethoxysilane (APTES). It is an important silane coupling agent, which promotes interfacial behavior of nanomaterials. Whereas, hetero-functionalization of MWCNTs can be performed using APTES and cross-linking agent glutaraldehyde (GA). Key parameters for the immobilization of an enzyme on MWCNTs are selection of an efficient functionalizing agent, sonication of MWCNTs, enzyme load and enzyme coupling time. The optimal level of these factors is very important to obtain high specific activity and immobilization efficiency of the developed immobilized biocatalyst. Both homo- and hetero-functionalized MWCNTs can be used to develop an efficient immobilized biocatalyst having good operational stability. However, hetero-functionalized MWCNTs are more potent candidate for enzyme immobilization. The present methodology provides very efficient approach for enzyme immobilization on a versatile support to achieve high enzyme selectivity and operational stability for various industrial applications.

A Versatile Approach for Enzyme Immobilization Using Chemically Modified 3D-Printed Scaffolds

The tedious preparation procedures and difficulty in free structure formation and recycling have restricted the widespread application of existing enzyme immobilization strategies. Here, we report ...

Tackling the Challenges of Enzymatic (Bio)Fuel Cells.

The ever-increasing demands for clean and sustainable energy sources combined with rapid advances in biointegrated portable or implantable electronic devices have stimulated intensive research activities in enzymatic (bio)fuel cells (EFCs). The use of renewable biocatalysts, the utilization of abundant green, safe, and high energy density fuels, together with the capability of working at modest and biocompatible conditions make EFCs promising as next generation alternative power sources. However, the main challenges (low energy density, relatively low power density, poor operational stability, and limited voltage output) hinder future applications of EFCs. This review aims at exploring the underlying mechanism of EFCs and providing possible practical strategies, methodologies and insights to tackle these issues. First, this review summarizes approaches in achieving high energy densities in EFCs, particularly, employing enzyme cascades for the deep/complete oxidation of fuels. Second, strategies for increasing power densities in EFCs, including increasing enzyme activities, facilitating electron transfers, employing nanomaterials, and designing more efficient enzyme-electrode interfaces, are described. The potential of EFCs/(super)capacitor combination is discussed. Third, the review evaluates a range of strategies for improving the stability of EFCs, including the use of different enzyme immobilization approaches, tuning enzyme properties, designing protective matrixes, and using microbial surface displaying enzymes. Fourth, approaches for the improvement of the cell voltage of EFCs are highlighted. Finally, future developments and a prospective on EFCs are envisioned.

Facile immobilization of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function.

Gold nanoparticles provide a user-friendly and efficient surface for immobilization of enzymes and proteins. In this paper, we present a novel approach for enzyme bioconjugation to gold nanostars (AuNSs). AuNSs were modified with l-cysteine (Cys) and covalently bound to N-hydroxysulfosuccinimide (sulfo-NHS) activated intermediate glucose oxidase (GOx) to fabricate a stable and sensitive AuNSs–Cys–GOx bioconjugate complex. Such a strategy has the potential for increased attachment affinity without protein adsorption onto the AuNSs surface. Good dispersity in buffer suspension was observed, as well as stability in high ionic environments. Using the AuNSs–Cys–GOx bioconjugates showed greater sensitivity in the measuring of low concentrations of glucose based on plasmonic and colorimetric detection. Such a novel approach for enzyme immobilization can lead to AuNSs–Cys–GOx bioconjugate complexes that can be used as catalytic nanodevices in nanobiosensors based on oxidases in biomedical applications.

Surface modification with highly-homogeneous porous silica layer for enzyme immobilization in capillary enzyme microreactors

Immobilized enzyme micro-reactors (IMERs) are of vital importance in developing miniaturized bioanalytical systems and have promising applications in various biomanufacturing. An inherent limitation in designing IMERs is the one-dimensional cylindrical geometry of micro-channels that offers limited exposed surface area for molecular reorganization and enzyme immobilization. In this study, we report a robust capillary-IMER based on a three dimensional porous layer open tubular (3D-PLOT) column which is prepared by an easy-to-control surface modification strategy via single-step in situ biphasic reaction. The 3D-PLOT column with highly uniform porous geometry and narrow distribution of porosity can greatly enhance the surface-area-to-volume ratio of the micro-channels, showing the beneficial effects for enzyme immobilization to enhance reaction efficiency and shorten analysis time. Taking trypsin as a model enzyme, enzymatic activities of immobilized enzyme are analyzed. We compare enzyme assays using the proposed 3D-PLOT-IMER with those using normal capillary-IEMR without surface modification as well as free trypsin. The 3D-PLOT-IMER exhibits excellent stability and inter/intra-day reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate enzyme assay. The feasibility of the proposed method for potential application in biological analysis is demonstrated by coupling the 3D-PLOT-IMER with a nano-LC-MS/MS system for online digestion of standard proteins, cell extraction and living Hela cells. Our study show that the surface modification with the proposed 3D-porous layer is a simple and efficient approach for enzyme immobilization, and could be widely suitable for different kinds of IMERs.

Characterization of Magnetic Nanoparticles Coated with Chitosan: A Potential Approach for Enzyme Immobilization

Cross-linking of magnetic nanoparticles with proteins plays a significant role in the preparation of new materials for biotechnological applications. The aim was the maximization of the magnetic mass attracted and protein loading of magnetic iron oxide nanoparticles coated with chitosan, synthesized in a single step by alkaline precipitation. Chitosan-coated magnetite particles (Fe3O4@Chitosan) were cross-linked to a xylanase and a cellulase (Fe3O4@Chitosan@Proteins), showing a 93% of the magnetic saturation of the magnetite. X-ray diffraction pattern in composites corresponds to magnetite. Thermogravimetry and differential scanning calorimetry showed that 162 mg of chitosan was coating one gram of composite and 12 mg of protein was cross-linked to each gram of magnetic support. Cross-linking between enzymes and Fe3O4@Chitosan was confirmed by infrared spectroscopy with Fourier transform, X-ray energy, and X-ray photoelectron spectroscopy dispersion analysis. From dynamic light scattering, transmission and electron microscopy the average particle size distribution was 230 nm and 430 nm for Fe3O4@Chitosan and Fe3O4@Chitosan@Proteins, showing agglomerates of individual spherical particles, with an average diameter of 8.5 nm and 10.8 nm, respectively. The preparation method plays a key role in determining the particle size and shape, size distribution, surface chemistry, and, therefore, the applications of the superparamagnetic nanoparticles.

Nano-in-Nano Approach for Enzyme Immobilization Based on Block Copolymers.

We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.

A hierarchical assembly of flower-like hybrid Turkish black radish peroxidase-Cu2+ nanobiocatalyst and its effective use in dye decolorization

Effective dye decolorization in wastewater still shows a big challenge. Although the biological methods, especially using enzymes, offer alternative and effective process for dye degradation and overcome the limitations of chemical and physical methods such as the instability, lack of reusability and high cost of free enzymes strictly, which limit their use in many scientific and technical applications. Enzymes rapidly lose their activities in aqueous solutions and against environmental changes due to their very susceptibility and unfavorable conformations. Herein, we report preparation of the enzyme-inorganic hybrid nanostructures with flower-like shape consisting of Turkish black radish peroxidase and Cu2+ metal ions using an encouraging enzyme immobilization approach. The peroxidase-Cu2+ hybrid nanoflowers (NFs) exhibited enhanced stability and activity towards various pH values and provided excellent dye decolorization efficiency for Victoria blue (VB) dye with more than 90% within 1 h. The NFs were also repeatedly used in efficient and caused 77% VB decolorization efficiency even at tenth cycles. However, to the best of our knowledge, for the first time, we prepared peroxidase enzyme isolated from Turkish black radish incorporated NFs and used them for dye decolorization. We believe that the NFs can be promising materials for dye decolorization in real wastewater treatment.