Enzyme Immobilization Strategies Research Articles

The importance of factorial design on the optimization of biosensor performance: immobilization of glucose oxidase as a case study.

Conventionally, the optimization of glucose biosensors is achieved by varying the concentrations of the individual reagents used to immobilize the enzyme. In this work, the effect and interaction between glucose oxidase enzyme (GOx), ferrocene methanol (Fc), and multi-walled carbon nanotubes (MWCNTs) at different concentrations were investigated by a design of experiments (DoE). For this analysis, a factorial design with three factors and two levels each was used with the software RStudio for statistical analysis. The data were obtained by electrochemical experiments on the immobilization of GOx-Fc/MWCNT at different concentrations. The results showed that the factorial DoE method was confirmed by the non-normality of the residuals and the outliers of the experiment. When examining the effects of the variables, analyzing the half-normal distribution and the effects and contrasts for GOx-Fc/MWCNT, the factors that showed the greatest influence on the electrochemical response were GOx, MWCNT, Fc, and MWCNT:Fc, and there is a high correlation between the factors GOx, MWCNT, Fc, and MWCNT:Fc, as shown by the analysis of homoscedasticity and multicollinearity. With these statistical analyses and experimental designs, it was possible to find the optimal conditions for different factors: 10mMmL-1 GOx, 2mgmL-1 Fc, and 15mgmL-1 MWCNT show a greater amperometric response in the glucose oxidation. This work contributes to advancing enzyme immobilization strategies for glucose biosensor applications. Systematic investigation of DoE leads to optimized immobilization for GOx, enables better performance as a glucose biosensor, and allows the prediction of some outcomes.

Evaluation of oil transesterification in a packed‐bed reactor containing lipase immobilized in starch–alginate jet cutting beads

AbstractThere has been a growing interest in ecofriendly enzymatic processes. However, enzyme solubility limits the application of many biocatalysts in continuous systems, requiring the development of cost‐effective strategies for enzyme immobilization. Based on this premise, this study investigated the application of lipase immobilized in starch–alginate beads for oil transesterification in a tubular reactor. An economical derivative was produced by immobilizing Eversa Transform 2.0 in 50:50 (w/w) starch–alginate beads using the jet‐cutting technique. The biocatalyst had a particle size of about 500 μm and activity of 138.67 ± 18.53 U g−1. X‐ray photoelectron spectroscopy showed nitrogen content ranging from 6.38% to 7.29%, with uniform distribution of lipase throughout the beads. Nitrogen isotherms were characteristic of mesoporous materials, with an average pore diameter of 48.09 Å and low surface area (0.69 m2 g−1). A face‐centered central composite design was used to study soybean oil transesterification. In the best four runs, the process achieved a mean triglyceride conversion of 45%. High ester productivity levels (2.05 × 10−2% ester g−1 biocatalyst min−1 or 1.5 × 10−4% ester U−1 min−1) were obtained. Biocatalyst reuse led to a twofold increase in ester concentration (14.57% vs 7.7%). These findings confirm the successful development of a low‐cost biocatalyst suitable for use in continuous reactions.

Flexible cellulose paper-based biosensor from inkjet printing for non-invasive glucose monitoring

Wearable glucose sensors have attracted significant attention for enabling non-invasive blood glucose measurement without discomfort and risk of infection. However, it is a challenge to simultaneously realize wearable adaptability, biodegradability, and excellent sensing performance. Herein, a cellulose paper-based non-invasive biosensor relying on reverse iontophoresis was designed to detect glucose in interstitial fluid (ISF), and two different enzyme immobilization strategies have been compared. The results showed inkjet-printed cellulose paper-based biosensor (IPB) performances better than the drop-coated cellulose paper-based biosensor (DPB). IPB has twice response current more than DPB in detection range (0–10 mM). In sensitivity, IPB is 1.170 μA/mM three times higher than the DPB (0.376 μA/mM). Besides, IPB's electron-transfer resistance (Rct) is 7.27 kΩ smaller than DPB (Rct = 10.51 kΩ) about 30 %. More importantly, IPB exhibited a good reproducibility (RSD, 4.82 %), which was much less than DPB (RSD, 18.35 %). Furthermore, the IPB realizes noninvasive continuous glucose monitoring over 6 h in volunteer experiments with great analytical performance comparable to commercial devices (Pearson correlation 0.732). Cellulose paper-based glucose sensors with inkjet printing provide non-invasive access to statistically significant diagnostic information, simple and cost-effective, which promotes the application of flexible, wearable, degradable bioelectrodes in continuous glucose monitoring at home, providing a concept for full integration in a compact and portable way in the future.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.

Horseradish Peroxidase Immobilized onto Mesoporous Magnetic Hybrid Nanoflowers for Enzymatic Decolorization of Textile Dyes: A Highly Robust Bioreactor and Boosted Enzyme Stability.

Recently, hybrid nanoflowers (hNFs), which are accepted as popular carrier supports in the development of enzyme immobilization strategies, have attracted much attention. In this study, the horseradish peroxidase (HRP) was immobilized to mesoporous magnetic Fe3O4-NH2 by forming Schiff base compounds and the HRP@Fe3O4-NH2/hNFs were then synthesized. Under optimal conditions, 95.0% of the available HRP was immobilized on the Fe3O4-NH2/hNFs. Structural morphology and characterization of synthesized HRP@Fe3O4-NH2/hNFs were investigated. The results demonstrated that the average size of HRP@Fe3O4-NH2/hNFs was determined to be around 220 nm. The ζ-potential and magnetic saturation values of HRP@Fe3O4-NH2/hNFs were -33.58 mV and ∼30 emu/g, respectively. Additionally, the optimum pH, optimum temperature, thermal stability, kinetic parameters, reusability, and storage stability were examined. It was observed that the optimum pH value shifted from 5.0 to pH 8.0 after immobilization, while the optimum temperature shifted from 30 to 80 °C. K m values were calculated to be 15.5502 and 7.6707 mM for free HRP and the HRP@Fe3O4-NH2/hNFs, respectively, and V max values were calculated to be 0.0701 and 0.0038 mM min-1. The low K m value observed after immobilization indicated that the affinity of HRP for its substrate increased. The HRP@Fe3O4-NH2/hNFs showed higher thermal stability than free HRP, and its residual activity after six usage cycles was approximately 45%. While free HRP lost all of its activity within 120 min at 65 °C, the HRP@Fe3O4-NH2/hNFs retained almost all of its activity during the 6 h incubation period at 80 °C. Most importantly, the HRP@Fe3O4-NH2/hNFs demonstrated good potential efficiency for the biodegradation of methyl orange, phenol red, and methylene blue dyes. The HRP@Fe3O4-NH2/hNFs were used for a total of 8 cycles to degrade methyl orange, phenol red, and methylene blue, and degradation of around 81, 96, and 56% was obtained in 8 h, respectively. Overall, we believe that the HRP@Fe3O4-NH2/hNFs reported in this work can be potentially used in various industrial and environmental applications, particularly for the biodegradation of recalcitrant compounds, such as textile dyes.

Wall-Immobilized Biocatalyst vs. Packed Bed in Miniaturized Continuous Reactors: Performances and Scale-Up.

Sustainable biocatalysis syntheses have gained considerable popularity over the years. However, further optimizations - notably to reduce costs - are required if the methods are to be successfully deployed in a range of areas. As part of this drive, various enzyme immobilization strategies have been studied, alongside process intensification from batch to continuous production. The flow bioreactor portfolio mainly ranges between packed bed reactors and wall-immobilized enzyme miniaturized reactors. Because of their simplicity, packed bed reactors are the most frequently encountered at lab-scale. However, at industrial scale, the growing pressure drop induced by the increase in equipment size hampers their implementation for some applications. Wall-immobilized miniaturized reactors require less pumping power, but a new problem arises due to their reduced enzyme-loading capacity. This review starts with a presentation of the current technology portfolio and a reminder of the metrics to be applied with flow bioreactors. Then, a benchmarking of the most recent relevant works is presented. The scale-up perspectives of the various options are presented in detail, highlighting key features of industrial requirements. One of the main objectives of this review is to clarify the strategies on which future study should center to maximize the performance of wall-immobilized enzyme reactors.

An Exploration of various Fructooligosaccharides production methods using novel engineered enzymes with innovative core-shell chitosan beads

This study investigates Fructooligosaccharides (FOSs) production as prebiotics to promote gut health and overall well-being. Various production methods for FOSs were explored, leveraging novel engineered inulosucrase (IN) and levansucrase (LV) enzymes. The methodologies included batch stirred reactors with free enzymes and fixed-bed reactors utilizing enzymes immobilized on hydrogel chitosan beads (HGBs) and core-shell chitosan beads (CSBs). The study found that free enzymes exhibited the highest sucrose consumption rates, with IN achieving 142.8 g/L∙h and LV achieving 82.9 g/L∙h. However, CSB-immobilized enzymes demonstrated substantial enzyme savings (55% and 41% reduction in IN and LV consumption, respectively) through reuse across multiple cycles, while delivering superior FOSs yields. CSB-immobilized enzymes outperformed HGB-immobilized enzymes in terms of immobilization efficiency, activity recovery, sucrose conversion, FOSs yields, and repeatability. A novel reactor setup involving interconnected fixed-bed reactors with different enzymes in series produced promising results, generating novel FOSs species alongside expected I-FOSs and L-FOSs. Consumable cost analysis indicated that while free enzyme usage is initially cost-effective (5.09–7.06 USD/kg FOSs), significant increases in LV prices could make CSB immobilization a cost-competitive alternative. This study highlights the potential of innovative enzyme immobilization strategies and reactor designs for efficient FOSs production, considering both performance and economic feasibility.

Recent Advances in the Strategies of Simultaneous Enzyme Immobilization Accompanied by Nanocarrier Synthesis

In recent years, with advancements in nanotechnology and materials science, new enzyme immobilization strategies based on nanomaterials have continuously emerged. These strategies have shown significant effects on enhancing enzyme catalytic performance and stability due to their high surface area, good chemical stability, and ease of enzyme binding, demonstrating tremendous potential for industrial applications. Those methods that can rapidly synthesize nanocarriers under mild conditions allow for the one-step synthesis of nanocarriers and enzyme complexes, thereby exhibiting advantages such as simplicity of process, minimal enzyme damage, short processing times, and environmental friendliness. This paper provides an overview of simultaneous enzyme immobilization strategies accompanied by nanocarrier synthesis, including organic–inorganic hybrid nano-flowers (HNFs), metal–organic frameworks (MOFs), and conductive polymers (CPs). It covers their preparation principles, post-immobilization performance, applications, and existing challenges.

Emerging Trends of Bilirubin Oxidases at the Bioelectrochemical Interface: Paving the Way for Self-Powered Electrochemical Devices and Biosensors.

Bilirubin oxidases (BODs) [EC 1.3.3.5 - bilirubin: oxygen oxido-reductase] are enzymes that belong to the multicopper oxidase family and can oxidize bilirubin, diphenols, and aryl amines and reduce the oxygen by direct four-electron transfer from the electrode with almost no electrochemical overpotential. Therefore, BOD is a promising bioelectrocatalyst for (self-powered) biosensors and/or enzymatic fuel cells. The advantages of electrochemically active BOD enzymes include selective biosensing, biocatalysis for efficient energy conversion, and electrosynthesis. Owing to the rise in publications and patents, as well as the expanding interest in BODs for a range of physiological conditions, this Review analyzes scientific literature reports on BOD enzymes and current hypotheses on their bioelectrocatalysis. This Review evaluates the specific research outcomes of the BOD in enzyme (protein) engineering, immobilization strategies, and challenges along with their bioelectrochemical properties, limitations, and applications in the fields of (i) biosensors, (ii) self-powered biosensors, and (iii) biofuel cells for powering bioelectronics.

Entrapment in HydrIL gels: Hydro-Ionic Liquid polymer gels for enzyme immobilization

Enzyme entrapment in hydro-ionic liquid gels (HydrIL gels) was explored as an enzyme immobilization strategy. The liquid phase of the gels was a mixture of buffer and tributyl methyl phosphonium alkanesulfonate [P4441][RSO3] ionic liquids. The ionic liquid modifies the liquid phase creating a more “organic” hydrophobic microenvironment around the enzyme. In the esterification of a secondary alcohol by Candida antarctica Lipase B, a 20-fold enhancement in final substrate conversion was achieved using an HydrIL gel compared to a standard poly(acrylamide) hydrogel. Co-polymerization of the poly(acrylamide) matrix with acrylate monomers led to further increases in reaction rate. A CalB HydrIL gel was reused over 10 reaction cycles, achieving a consistent performance from the fifth cycle. IL leaching became undetectable from the third reaction cycle.

Electrostatically Mediated In Situ Polymerization for Enzyme Immobilization and Activation.

Enzyme immobilization in nanoparticles is of interest for boosting their catalytic applications, yet rational approaches to designs achieving both high enzyme loading and activation remain a challenge. Herein, we report an electrostatically mediated in situ polymerization strategy that simultaneously realizes enzyme immobilization and activation. This was achieved by copolymerizing cationic monomers with a cross-linker in the presence of the enzyme lipase (anionic) as the template, which produces enzyme-loaded nanogels. The effects of different control factors such as pH, lipase dosage, and cross-linker fraction on nanogel formation are investigated systematically, and optimal conditions for enzyme loading and activation have been determined. A central finding is that the cationic polymer network of the nanogel creates a favorable environment that not only protects the enzyme but also boosts enzymatic activity nearly 2-fold as compared to free lipase. The nanogels improve the stability of the lipase to tolerate a broader working range of pH (5.5-8.5) and temperature (25-70 °C) and allow recycling such that after six cycles of reaction, 70% of the initial activity is conserved. The established fabrication strategy can be applied generally to different cationic monomers, and most of these nanogels exhibit adequate immobilization and activation of lipase. Our study confirms that in situ polymerization based on electrostatic interaction provides a facile and robust strategy for enzyme immobilization and activation. The wide variety of ionic monomers, therefore, features great potential for developing functional platforms toward satisfying enzyme immobilization and demanding applications.

Enzyme Immobilization on Stainless Steel Fleece and Its Mass Transfer Enhancement of Enzymatic Catalysis in a Rotating Packed Bed Reactor

Rotating packed beds (RPB) facilitate the mixing of heterogeneous substrates, and promote high mass transfer efficiency in heterogeneous reactions. For the enzymatic reactions, traditional porous particles with immobilized enzymes are sensitive to the strong sheer force of the RPB, thus limiting its application. This work offers a strategy for enzyme immobilization on the surface of stainless-steel fleece, to improve the shear strength resistance of immobilized enzymes. Lipase was applied to investigate and optimize the immobilization. Finally, a fatty acid hydratase (FAH) was applied for immobilization based on the optimized method, which was further applied for evaluating its performance in RPB. The results indicated that metal immobilized enzymes resist a higher shear force than their particle-immobilized alternatives. Operating at a centrifugal force factor (β) of 30, the hydration conversion rate of 96% is achieved after 8 h, which was from nearly 38% faster than in a stirrer tank reactor (hydration yield of 60%). The metal immobilization, moreover, efficiently improved the enzyme reusability, as demonstrated by a conversion rate remaining above 90% after 15 batches. These results indicated that a metal immobilization method combined with an RPB reactor significantly increases the efficiency of enzymatic reactions.

Polymer-Based Strategies for Enzyme Immobilization: A Comprehensive Review

Enzyme immobilization refers to the process of attaching or confining enzymes onto a solid support or within a matrix, often made of polymers or other materials. This immobilization creates a stable and controlled environment for the enzyme to interact with substrates and perform catalysis. The primary goal of enzyme immobilization is to enhance enzyme stability, reusability, and activity under specific conditions, making them more practical and efficient for various biotechnological, industrial, and medical applications.
 Immobilization methods can vary widely, including physical adsorption, covalent bonding, entrapment within matrices, encapsulation, crosslinking, and more. These methods provide a means to control the interactions between the enzyme and the surrounding environment, affecting factors such as substrate accessibility, enzyme orientation, and stability.
 Due to their ease of fabrication and superior structural adaptability, polymer compounds in a variety of physical forms, including beads, films, fibers,and coatings,have become popular as supportive materials for enzyme immobilization. For enzyme immobilization, a number of natural polymers, including agar, agarose, alginate, dextran, chitosan,and carrageenan, as well as synthetic polymers, such as polyamides, polystyrene, and polyacrylamide, are often employed as a carrier system. The immobilization offers a cost-effective system for various applications in biotechnology, industry, and research.

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

AbstractThe natural micro‐ and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.

Nucleic Acid-based Enzyme Cascades-Current Trends and Future Perspectives.

The natural micro- and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.

Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts: A promising technology for SUMO fusion proteins

The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.

Sustainable Immobilization of β-Glucosidase onto Silver Ions and AgNPs-Loaded Acrylic Fabric with Enhanced Stability and Reusability.

Modified polymer design has attracted significant attention for enzyme immobilization, offering promising applications. In this study, amine-terminated polymers were synthesized by incorporating functional groups into polyacrylonitrile using hexamethylenediamine. This work highlights the successful enzyme immobilization strategy using modified polymers, offering improved stability and expanded operational conditions for potential biotechnological applications. The resulting amino groups were utilized to capture silver ions, which were subsequently converted to silver nanoparticles (AgNPs). The obtained materials, AgNPs@TA-HMDA (acrylic textiles coated silver nanoparticles AgNPs) and Ag(I)@TA-HMDA (acrylic textiles coated with Ag ion) were employed as supports for β-glucosidase enzyme immobilization. The highest immobilization yields (IY%) were achieved with AgNPs@TA-HMDA at 92%, followed by Ag(I)@TA-HMDA at 79.8%, resulting in activity yields (AY%) of 81% and 73%, respectively. Characterization techniques such as FTIR, FE-SEM, EDX, TG/DTG, DSC, and zeta potential were employed to investigate the structural composition, surface morphologies, elemental composition, thermal properties, and surface charge of the support materials. After 15 reuses, the preservation percentages decreased to 76% for AgNPs@TA-HMDA/β-Glu and 65% for Ag(I)@TA-HMDA/β-Glu. Storage stability revealed that the decrease in activity for the immobilized enzymes was smaller than the free enzyme. The optimal pH for the immobilized enzymes was broader (pH 5.5 to 6.5) compared to the free enzyme (pH 5.0), and the optimal temperature for the immobilized enzymes was 60 °C, slightly higher than the free enzyme's optimal temperature of 50 °C. The kinetic analysis showed a slight increase in Michaelis constant (Km) values for the immobilized enzymes and a decrease in maximum velocity (Vmax), turnover number (Kcat), and specificity constant (Kcat/Km) values compared to the free enzyme. Through extensive characterization, we gained valuable insights into the structural composition and properties of the modified polymer supports. This research significantly contributes to the development of efficient biotechnological processes by advancing the field of enzyme immobilization and offering valuable knowledge for its potential applications.

Design of a novel multi-layer enzyme membrane reactor for low-fouling, tailored production of oligodextran

Enzymatic conversion processes face challenges in controlling oligosaccharide molecular weight (Mw). Enzymatic membrane reactors (EMRs) with immobilized enzymes address this, but direct enzyme immobilization on the membrane surface can lead to deactivation and reduced hydrolysis efficiency. This study proposes a novel EMR configuration: a three-layer structure. An electrospun porous fibrous layer, modified with PDA, TA, and APTES, serves as a mechanical support layer. A commercial separation membrane is positioned below. This configuration enhances enzyme activity and selectivity. Using a “fouling-induced” technique, immobilized activity of the enzyme (i.e. dextranase) significantly increased to 11.5 µmol-isomaltose/min, surpassing incubation-immobilized dextranase (0.075 µmol-isomaltose/min). The additional layer preserves catalytic patterns, reducing fouling and ensuring high selectivity. The EMR configuration excels in producing low Mw oligosaccharides. The catalytic layer achieves 11.3 µmol-isomaltose/min, while the membrane exhibits exceptional selectivity and stability. The hydrophilic RC10 membrane with small pores performs best. This study highlights the potential of the EMR system for efficient production of stable low Mw oligosaccharides. Insights into optimizing enzyme immobilization strategies and membrane selection benefit enzymatic conversion processes.

Development of a novel ultrasound- and biocrosslinking-enhanced immobilization strategy with application to food enzymes.

The increasing demand for greener food production makes biocatalysts more desirable than traditional production approaches. One limiting factor for biocatalyst efficiency is the immobilization strategy. In this work, a novel immobilization method was developed with the tyrosine-tag crosslinking mechanism. The immobilization efficiency was further enhanced with ultrasound treatment. Such a strategy was proven to be efficient with food enzyme lipase, d-amino acid oxidase and glucose dehydrogenase when they were immobilized on macroporous resins, amino resins, epoxy resins, and multiwalled carbon nanotubes. For lipase, glucose dehydrogenase and d-amino acid oxidase, the immobilization yield on macroporous resins increased by 20.4%, 21.1% and 24.1%, respectively. In addition, the immobilized enzymes had enhanced reusability, with a high degree of activity (more than 85%) detected after six cycles. Furthermore, the enzyme electrochemical sensors constructed by enzyme crosslinking have higher sensitivity, with peak currents 4-8 times those of sensors with uncrosslinked enzymes. The enzyme immobilization strategy developed in this study paves the way for better application of biocatalysts in the food industry.

Peptide self‐assembly as a strategy for facile immobilization of redox enzymes on carbon electrodes

AbstractRedox–enzyme‐mediated electrochemical processes such as hydrogen production, nitrogen fixation, and CO2 reduction are at the forefront of the green chemistry revolution. To scale up, the inefficient two‐dimensional (2D) immobilization of redox enzymes on working electrodes must be replaced by an efficient dense 3D system. Fabrication of 3D electrodes was demonstrated by embedding enzymes in polymer matrices. However, several requirements, such as simple immobilization, prolonged stability, and resistance to enzyme leakage, still need to be addressed. The study presented here aims to overcome these gaps by immobilizing enzymes in a supramolecular hydrogel formed by the self‐assembly of the peptide hydrogelator fluorenylmethyloxycarbonyl‐diphenylalanine. Harnessing the self‐assembly process avoids the need for tedious and potentially harmful chemistry, allowing the rapid loading of enzymes on a 3D electrode under mild conditions. Using the [FeFe] hydrogenase enzyme, high enzyme loads, prolonged resistance against electrophoresis, and highly efficient hydrogen production are demonstrated. Further, this enzyme retention is shown to arise from its interaction with the peptide nanofibrils. Finally, this method is successfully used to retain other redox enzymes, paving the way for a variety of enzyme‐mediated electrochemical applications.