Immobilized Enzyme Research Articles

Piezoionic High Performance Hydrogel Generator and Active Protein Absorber via Microscopic Porosity and Phase Blending.

Generating electricity in hydrogel is very important but remains difficult. Hydrogel with electricity generation capability is more capable in bio-relevant tasks such as tissue engineering, artificial skin, or medical treatment, because electricity is indispensable in regulating physiological activities. Here, a porous and phase blending hydrogel structure for effective piezoionic electricity generation is developed. Dynamic electric field is generated taking advantage of the difference in streaming speeds of sodium and chloride in the material. Microscopic porosity and hydrophilic-hydrophobic phase blending are the two key factors for prominent piezoionic performance. Voltages as high as 600mV are first realized in hydrogels in response to medical ultrasound stimulation. The hydrogel structure is also subjective to effective substance exchange and can actively enrich proteins from surroundings under mechanical stimuli. Preliminary applications in neural stimulation, constructing complex spatial-temporal chemical and electric field distribution patterns, mimetic tactile sensor, sample pretreatment in fast detection, and enzyme immobilization are demonstrated.

Urease immobilized poly(AAm-AGE) cryogel for depletion of urea from human serum

Polyacrylamide-allyl glycidyl ether [poly(AAm-AGE)] cryogel monolith was prepared for covalent immobilization of urease enzyme and aimed for usage of urea depletion from human serum by immobilized urease. Immobilization was succeeded via epoxy groups of allyl glycidyl ether with an immobilization yield of 71.9 ± 2.65%. Characterization was performed by Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX) analysis and swelling analysis to reveal functionalities and morphological, elemental and macroporous structure of the cryogel. Effects of pH, temperature and urea concentration on catalytic activity profiles of free and immobilized urease were investigated. Thermal and storage stabilities of free urease were enhanced upon immobilization. Additionally, urea removal from artificial human serum was achieved by 92% and immobilized urease was reusable up to ten reuses.

Recent Advances in Enzyme Immobilisation Strategies: An Overview of Techniques and Composite Carriers

For over a century, enzyme immobilisation has been proven to be a superior strategy to improve catalytic activity and reusability and ensure easy separation, easy operation, and reduced cost. Enzyme immobilisation allows for an easier separation of the enzyme from the reaction mixture, thus simplifying downstream processing. This technology protects the enzyme from degradation or inactivation by harsh reaction conditions, making it more robust and suitable to be used in various applications. Recent strategies of immobilisation methods, such as adsorption, cross-linking, entrapment or encapsulation, and covalent bonding, were critically reviewed. These strategies have shown promising results in improving enzyme stability, activity, and reusability in various applications. A recent development in enzyme immobilisation in nanomaterials and agrowaste renewable carriers is underlined in the current review. Furthermore, the use of nanomaterials and agrowaste carriers in enzyme immobilisation has gained significant attention due to their unique properties, such as high surface area, high mass transfer, biocompatibility, and sustainability. These materials offer promising outcomes for developing more efficient and sustainable immobilised enzymes. This state-of-the-art strategy allows for better control over enzyme reactions and enhances their reusability, leading to more cost-effective and environmentally friendly processes. The use of renewable materials also helps to reduce waste generation and promote the utilisation of renewable resources, further contributing to the development of a circular economy.

Suppression mechanism of soilborne diseases by biochar: Immobilization and deactivation of pathogenic enzymes and toxic metabolites

Greenhouse vegetable production aggravates soilborne diseases. Biochar has been recently considered as soil amendment to effectively prevent soilborne diseases. This study evaluates the immobilization and deactivation of pathogen-produced cell wall degrading enzymes (CWDEs) and toxic metabolites (TMs) by biochar to understand the mechanism involved in tomato Fusarium wilt, with activated carbon as the control. The results reveal that cellulase takes shorter time to reach the adsorption equilibrium than pectinase does. The adsorption isotherm of pectinase follows the Langmuir model, and the adsorption isotherm of cellulase follows the Freundlich model. The adsorption capacity of pectinase is greater than that of cellulase on biochar (on activated carbon). The adsorbed enzymes are completely immobilized and observably deactivated. The activity of immobilized pectinase decreases as 43.01 % of the original activity on biochar and 44.91 % on activated carbon. But the activity of immobilized cellulase decreases only as 1.88 % on biochar and 3.89 % on activated carbon. The tomato seedling bioassay analysis demonstrates that the adsorption of CWDEs and TMs is conducive to the reduction of disease severity. However, these established models (or laboratory simulation) cannot explain the enzyme adsorption process of biochar in the real soil, which requires the further exploration in the future.

Bienzyme co-immobilized on Zr-Based MOF with hierarchical porous structure for visual detection of glucose

Metal-organic frameworks (MOFs) are considered as promising carriers for immobilized enzymes. Whereas, some interrelated and restrictive problems exist in the construction of MOFs immobilized enzymes with excellent catalytic performance, resulting in reduced catalytic activity of the enzymes. In this work, a novel hierarchical porous structure of Zr-based metal-organic frameworks (Zr-MOF, HPU) was accurately constructed for co-immobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx). The prepared HPU carrier can enrich the substrate by using the abundant micropore structure around the mesopore, thus reducing the mass transfer resistance and making up for the loss of enzyme activity during enzyme immobilization. Notably, GOx-HRP@HPU demonstrated outstanding catalytic efficiency and excellent stability, as well as ideal reusability, maintaining nearly 95% initial activity at 60 ℃ after 1 h incubation and above 65% initial activity after 10 reuses. Both the immobilization process and interaction of substrates with co-immobilized bienzyme reactor were investigated by means of microcalorimetry, which demonstrated HPU was an excellent platform for co-immobilized HRP and GOx. Furthermore, GOx-HRP@HPU was used for the detection of glucose with excellent detection performance.

Papain Covalent Immobilization in Bacterial Cellulose Films as a Wound Dressing

Ideally, the dressings used in the clinic have characteristics that help the wound closure process. Among several factors that affect the success of this healing process, there is debridement. It manages the wound bed components and the re-epithelialization process. Still, the property of debridement is not generally associated with dressings. Here, we show a chemically modified bacterial cellulose film conjugated to a proteolytic enzyme, papain, as a dressing with debridement properties. Bacterial cellulose films were reacted with a spacer derived from succinic acid and finally had this enzyme covalently immobilized in its structure by an amide bond. FT-IR and UV–vis showed bands typically of bioconjugated polymer. Enzymatic immobilization was very effective under the conditions applied with high yield (33% w/w), and these remained activated after the coupling reaction. The bacterial cellulose film with the enzyme papain attached to it was also very compatible with fibroblast cells, suggesting that it could be a promising wound dressing material for future research.

Application of Immobilized Enzymes in Juice Clarification.

Immobilized enzymes are currently being rapidly developed and are widely used in juice clarification. Immobilized enzymes have many advantages, and they show great advantages in juice clarification. The commonly used methods for immobilizing enzymes include adsorption, entrapment, covalent bonding, and cross-linking. Different immobilization methods are adopted for different enzymes to accommodate their different characteristics. This article systematically reviews the methods of enzyme immobilization and the use of immobilized supports in juice clarification. In addition, the mechanisms and effects of clarification with immobilized pectinase, immobilized laccase, and immobilized xylanase in fruit juice are elaborated upon. Furthermore, suggestions and prospects are provided for future studies in this area.

Immobilizing white-rot fungi laccase: Towardbio-derived supports as a circular economy approach in organochlorine removal.

Despite their high persistence in the environment, organochlorines (OC) are widely used in the pharmaceutical industry, in plastics, and in the manufacture of pesticides, among other applications. These compounds and the byproducts of their decomposition deserve attention and efficient proposals for their treatment. Among sustainable alternatives, the use of ligninolytic enzymes (LEs) from fungi stands out, as these molecules can catalyze the transformation of a wide range of pollutants. Among LEs, laccases (Lac) are known for their efficiency as biocatalysts in the conversion of organic pollutants. Their application in biotechnological processes is possible, but the enzymes are often unstable and difficult to recover after use, driving up costs. Immobilization of enzymes on a matrix (support or solid carrier) allows recovery and stabilization of this catalytic capacity. Agricultural residual biomass is a passive environmental asset. Although underestimated and still treated as an undesirable component, residual biomass can be used as a low-cost adsorbent and as a support for the immobilization of enzymes. In this review, the adsorption capacity and immobilization of fungal Lacon supports made from residual biomass, including compounds such as biochar, for the removal of OC compounds are analyzed and compared with the use of synthetic supports. A qualitative and quantitative comparison of the reported results was made. In this context, the use of peanut shells is highlighted in view of the increasing peanut production worldwide. The linkage of methods with circular economy approaches that can be applied in practice is discussed.

Fabrication of lignin-based sub-micro hybrid particle as a novel support for adenylate cyclase immobilization

This study developed a surface functionalized lignin-based sub-microsphere as an innovative support for enzyme immobilization. Lignin was first modified with a silane reagent leading to lignin/SiO2 (LS) organic/inorganic hybrid particles, displayed as regular sub-micro spheres with a SiO2 shell as demonstrated in SEM images. The LS particles were further modified to introduce nickel ions, as evidenced in XPS spectra, facilitating affinity adsorption with a his-tagged enzyme. The immobilization of adenylate cyclase from Haloactinopolyspora alba (HaAC), expressed in Escherichia coli, was conducted on the surface functionalized LS (LS-G-NTA-Ni). The immobilization conditions were optimized to achieve the highest relative activity, which were determined to be using a Ni2+ concentration of 62.5 mM, at pH=9.5 and 25 °C, with an enzyme-to-support ratio of 4.0 for a duration of 2 h. The immobilized HaAC shows maximum relative activity at pH=9.5 and 40 °C, and exhibits significantly improved thermal stability compared to the free enzyme. After undergoing five reusing cycles, the immobilized HaAC maintains a satisfactory activity (54.15%), which is due to the surface chemistry and the structural stability of the functionalized LS. This work provides a valuable exploration for high-value application of industrial lignin.

WaveFlex Biosensor: MXene-Immobilized W-shaped Fiber-Based LSPR sensor for highly selective tyramine detection

Tyramine is generally produced via decarboxylation of tryptophan, and its long-term exposure can induce severe symptoms in humans like brain stroke and hypertensive crisis. It also served as an important factor to check the food quality, as its increased level is frequently found in rotten food products. Thus, more precise, practical, quick, and easy methods have been required for the detection of tyramine in various samples. In the present study, a localized surface plasmon resonance (LSPR) optical fiber-based, highly sensitive, and selective method for tyramine detection has been proposed. The fiber surface has been functionalized with MXene and gold nanoparticles to provide increased surface area for enzyme immobilization as well as enhance the performance of the sensor. The results showed that the developed sensor efficiently detects tyramine with high specificity due to the presence of the tyrosinase enzyme. Further studies revealed the stability, repeatability, and reusability of the proposed biosensor with a 6.96 µM detection limit and a sensitivity of 0.0385 nm/µM. As a result, the proposed optical fiber-based sensor has significant potential for application in the detection of the presence of tyramine in a wide variety of agricultural and food samples.

Magnetic macroporous chitin microsphere as a support for covalent enzyme immobilization

In this study, a novel magnetic macroporous chitin microsphere (MMCM) was developed for enzyme immobilization. Chitin nanofibers were prepared and subsequently subjected to self-assembly with magnetic nanoparticles and PMMA (polymethyl methacrylate). Following this, microspheres were formed through spray drying, achieving a porous structure through etching. The MMCM serves as an effective support for immobilizing enzymes, allowing for their covalent immobilization both on the microsphere's surface and within its pores. The substantial surface area resulting from the porous structure leads to a 2.1-fold increase in enzyme loading capacity compared to non-porous microspheres. The MMCM enhances stability of the immobilized enzymes under various pH and temperature conditions. Furthermore, after 20 days of storage at 4 °C, the residual activity of the immobilized enzyme was 2.93 times that of the free enzyme. Even after being recycled 10 times, the immobilized enzyme retained 56.7 % of its initial activity. It's noteworthy that the active sites of the enzymes remained unchanged after immobilization using the MMCM, and kinetic analysis revealed that the affinity of the immobilized enzymes rivals that of the free enzymes.

Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme Knockout for Androgenic Disorders

Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme Knockout for Androgenic Disorders

Synergistic treatment of textile wastewaters using spent diatomaceous earth loaded with laccases: A cost-effective and eco-friendly approach

Biosorption and enzymatic biodegradation are well-known methods for the removal/elimination of several pollutants from the environment. These techniques are usually evaluated individually; however, biosorption coupled with enzymatic biodegradation may be a cheap and promising technology for treating industrial wastewater. In this study, laccases from Trametes versicolor were immobilized on spent diatomaceous earth from brewing filtration (SDE) as a biosorbent previously functionalized with 3-aminopropyltriethoxysilane (APTES). A three-level factorial design was used to optimize the immobilization process. The biosorbent coupled with enzymes (SDE-LA) and without enzymes (SDE-S), as well as free laccases (LA), were applied for the treatment of real textile effluent. Enzyme immobilization on SDE increased pH and thermal and storage stability. SDE-LA was more effective in the removal of chemical oxygen demand from the raw textile effluent (66.2 %) than SDE-S and LA (59.0 % and 14.0 %, respectively). Effluent color and toxicity were also significantly reduced by the treatment with SDE-LA. Overall, laccase immobilized on SDE offers a cost-effective and sustainable alternative for treating/polishing textile wastewaters. This combined process of enzymatic action and biosorption can decrease pollutant loading and toxicity of industrial effluents, allowing their safer disposal in water bodies.

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.

Green Synthesis of Biocatalysts Based on Nanocarriers Promises an Effective Role in Pharmaceutical and Biomedical Fields

Nanobiocatalysts (NBCs) are a promising new class of biocatalysts that combine the advantages of enzymes and nanomaterials. Enzymes are biological catalysts that are highly selective and efficient, but they can be unstable in harsh environments. Nanomaterials, on the other hand, are small particles with unique properties that can improve the stability, activity, and selectivity of enzymes. The development of NBCs has been driven by the need for more sustainable and environmentally friendly bioprocessing methods. Enzymes are inherently green catalysts, but they can be expensive and difficult to recover and reuse. NBCs can address these challenges by providing a stable and reusable platform for enzymes. One of the key challenges in the development of NBCs is the immobilization of enzymes on nanomaterials. Enzyme immobilization is a process that attaches enzymes to a solid support, which can protect the enzymes from harsh environments and make them easier to recover and reuse. There are many different methods for immobilizing enzymes, and the choice of method depends on the specific enzyme and nanomaterial being used. This review explores the effective role of NBCs in pharmaceutical and biomedical fields.

Phospholipase D Immobilization on Lignin Nanoparticles for Enzymatic Transformation of Phospholipids.

Lignin nanoparticles (LNPs) are promising components for various materials, given their controllable particle size and spherical shape. However, their origin from supramolecular aggregation has limited the applicability of LNPs as recoverable templates for immobilization of enzymes. In this study, we show that stabilized LNPs are highly promising for the immobilization of phospholipase D (PLD), the enzyme involved in the biocatalytic production of high-value polar head modified phospholipids of commercial interest, phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine. Starting from hydroxymethylated lignin, LNPs were prepared and successively hydrothermally treated to obtain c-HLNPs with high resistance to organic solvents and a wide range of pH values, covering the conditions for enzymatic reactions and enzyme recovery. The immobilization of PLD on c-HLNPs (PLD-c-HLNPs) was achieved through direct adsorption. We then successfully exploited this new enzymatic preparation in the preparation of pure polar head modified phospholipids with high yields (60-90 %). Furthermore, the high stability of PLD-c-HLNPs allows recycling for a number of reactions with appreciable maintenance of its catalytic activity. Thus, PLD-c-HLNPs can be regarded as a new, chemically stable, recyclable and user-friendly biocatalyst, based on a biobased inexpensive scaffold, to be employed in sustainable chemical processes for synthesis of value-added phospholipids.

Synergistic co-catalytic nanozyme system for highly efficient one-pot colorimetric sensing at neutral pH: Combining molybdenum trioxide and Fe(III)-Modified covalent triazine framework

This study investigates the co-catalytic capabilities of MoO3 nanosheets in enhancing the enzyme-like catalytic activity of a two-dimensional ultrathin Fe(III)-modified covalent triazine framework (Fe-CTF) under neutral pH conditions. The unique physicochemical surface properties and two-dimensional structures of Fe-CTF enable the direct immobilization of native enzymes (glucose oxidase (GOD) and xanthine oxidase (XOD)) through adsorption, eliminating the need for chemical processes. Efficient immobilization of the native enzymes within the Fe-CTF/GOD(XOD) hybrid is achieved through multipoint attachment involving various interactions. The Fe-CTF/MoO3 co-catalytic system exhibits enzyme-mimicking activity at neutral pH and, when combined with the high catalytic activity of the immobilized native enzymes, enables the development of a colorimetric method for glucose detection. This method demonstrates excellent facilitation, rapidity, sensitivity, and selectivity, with a linear detection range of 50–1000 μM and a limit of detection of 8.8 μM for glucose. Furthermore, a straightforward one-pot colorimetric method is established for screening XOD inhibitors. The inhibitory potential of a crude extract derived from Chinese water chestnut peel on XOD activity is evaluated using this method. The findings of this study pave the way for the utilization of nanozyme/native enzyme hybrids in pH-neutral conditions for one-pot colorimetric sensing. This work contributes to the advancement of enzyme-based sensing technologies and holds promise for various applications in biosensing and biomedical research.

Enterobacter aerogenes Lipase immobilized on ion exchange resin D152 as a supportive carrier

Enterobacter aerogenes Lipase used in this study was heterologously expressed by Pichia pastoris. D152, D152H, D151H, D113, 724), three types of anion resins ( D380, D 301R, D311) and two types of chelating r activities of the immobilized ( EAL). According to the results, D152 was selected for haydrolysis activity as the immobilized enzyme (EAL) . The D152 was selected for hydrolysis activity since the immobilized lipase exhibited the highest specific hydrolysis activity. Immobilization conditions (enzyme loading, immobilization time, temperature, and pH value) . The best results were enzyme loading 4mg/g, time 80min, temperature 30°C, and buffer pH 8; and under the optimized conditions the immobilization efficiency was 95% and the specific activity was 532841.34 U/g.esins (D401, D418) were sieved as support matrix. Hydrolysis assay was employed to evaluate the specific.