Immobilized Enzyme Technology Research Articles

Functionalized Chitosan and Alginate Composite Hydrogel-Immobilized Laccase with Sustainable Biocatalysts for the Effective Removal of Organic Pollutant Bisphenol A

The immobilization of enzymes is an important strategy to improve their stability and reusability. Enzyme immobilization technology has broad application prospects in biotechnology, biochemistry, environmental remediation, and other fields. In this study, composites of chitosan (CS) and sodium alginate (SA) with Cu2+ forming a double-network crosslinked structure of hydrogels were prepared and used for the immobilization of laccase. Fourier infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy tests revealed that laccase molecules were immobilized on the composite hydrogel surface by a covalent bonding method. Compared to free laccase, the pH, temperature, and storage stability of the immobilized laccase were markedly improved. In addition, the immobilized laccase could be easily separated from the reaction system and reused, and it maintained 81.6% of its initial viability after six cycles of use. Bisphenol A (BPA) in polluted water was efficiently degraded using immobilized laccase, and the factors affecting the degradation efficiency were analyzed. Under the optimal conditions, the BPA removal was greater than 82%, and the addition of a small amount of ABTS had a significant effect on BPA degradation, with a removal rate of up to 99.1%. Experimental results indicated that immobilized laccases had enormous potential in actual industrial applications.

Establishment and application of a screening method for α-glucosidase inhibitors based on dual sensing and affinity chromatography

α-Glucosidase plays a direct role in the metabolic pathways of starch and glycogen, any dysfunction in its activity could result in metabolic disease. Concurrently, this enzyme serves as a target for diverse drugs and inhibitors, contributing to the regulation of glucose metabolism in the human body. Here, an integrated analytical method was established to screen inhibitors of α-glucosidase. This step-by-step screening model was accomplished through the biosensing and affinity chromatography techniques. The newly proposed sensing program had a good linear relationship within the enzyme activity range of 0.25 U mL−1 to 1.25 U mL−1, which can quickly identify active ingredients in complex samples. Then the potential active ingredients can be captured, separated, and identified by an affinity chromatography model. The combination of the two parts was achieved by an immobilized enzyme technology and a microdevice for reaction, and the combination not only ensured efficiency and accuracy for inhibitor screening but also eliminated the occurrence of false positive results in the past. The emodin, with a notable inhibitory effect on α-glucosidase, was successfully screened from five traditional Chinese medicines using this method. The molecular docking results also demonstrated that emodin was well embedded into the active pocket of α-glucosidase. In summary, the strategy provided an efficient method for developing new enzyme inhibitors from natural products.

Calcium-based MOFs as scaffolds for shielding immobilized lipase and enhancing its stability

The enzyme immobilization technology has become a key tool in the field of enzyme applications; however, improving the activity recovery and stability of the immobilized enzymes is still challenging. Herein, we employed a magnetic carboxymethyl cellulose (MCMC) nanocomposite modified with ionic liquids (ILs) for covalent immobilization of lipase, and used Ca-based metal–organic frameworks (MOFs) as the support skeleton and protective layer for immobilized enzymes. The ILs contained long side chains (eight CH2 units), which not only enhanced the hydrophobicity of the carrier and its hydrophobic interaction with the enzymes, but also provided a certain buffering effect when the enzyme molecules were subjected to compression. Compared to free lipase, the obtained CaBPDC@PPL–IL–MCMC exhibited higher specific activity and enhanced stability. In addition, the biocatalyst could be easily separated using a magnetic field, which is beneficial for its reusability. After 10 cycles, the residual activity of CaBPDC@PPL–IL–MCMC could reach up to 86.9%. These features highlight the good application prospects of the present immobilization method.

Enhanced High-Fructose Corn Syrup Production: Immobilizing Serratia marcescens Glucose Isomerase on MOF (Co)-525 Reduces Co2+ Dependency in Glucose Isomerization to Fructose.

The escalating demand for processed foods has led to the widespread industrial use of glucose isomerase (GI) for high-fructose corn syrup (HFCS) production. This reliance on GIs necessitates continual Co2+ supplementation to sustain high catalytic activity across multiple reaction cycles. In this study, Serratia marcescens GI (SmGI) was immobilized onto surfaces of the metal-organic framework (MOF) material MOF (Co)-525 to generate MOF (Co)-525-GI for use in catalyzing glucose isomerization to generate fructose. Examination of MOF (Co)-525-GI structural features using scanning electron microscopy-energy dispersive spectroscopy, Fourier-transform infrared spectroscopy, and ultraviolet spectroscopy revealed no structural changes after SmGI immobilization and the addition of Co2+. Notably, MOF (Co)-525-GI exhibited optimal catalytic activity at pH 7.5 and 70 °C, with a maximum reaction rate (Vmax) of 37.24 ± 1.91 μM/min and Km value of 46.25 ± 3.03 mM observed. Remarkably, immobilized SmGI exhibited sustained high catalytic activity over multiple cycles without continuous Co2+ infusion, retaining its molecular structure and 96.38% of its initial activity after six reaction cycles. These results underscore the potential of MOF (Co)-525-GI to serve as a safer and more efficient immobilized enzyme technology compared to traditional GI-based food-processing technologies.

Pros and Cons in Various Immobilization Techniques and Carriers for Enzymes.

In recent years, enzyme immobilization technology has been developed, and studies on immobilized enzyme materials have become very prominent. With the immobilization technique, enzymes and compatible carrier materials are combined or enzyme crystals/aggregates are used in a carrier-free fashion, by physical, chemical, or biochemical methods. As a kind of biocatalyst, immobilized enzymes can catalyze certain chemical reactions with high selectivity and high efficiency under relatively mild reaction conditions and eliminate pollution to the environment. Considering the current status and applications of immobilized enzyme technology and materials emerging in the last 5years, this mini-review introduces the advantages and disadvantages of various enzyme immobilization techniques with carriers as well as the pros and cons of different materials for immobilization. The future prospects of immobilization technology and carrier materials are outlined, aiming to provide a reference for further research and applications of sustainable technology.

The effect of commercial β-glucosidase and its immobilization on quality of red wine production

To find a feasible strategy for commercial β-glucosidase to enhance red wine quality, immobilized enzyme technology were considered. The preferred epoxy resin using the covalent binding method was ES-103B, and its optimal immobilization conditions were pH 4.9, 25 h immobilization time and 3 mg/g enzyme concentration. Meanwhile, this immobilized β-glucosidase still contained weak esterase activity just like commercial β-glucosidase. Free commercial β-glucosidase had different effects on the fermentation of 11 clones from three grape varieties (Syrah, Merlot and Cabernet Sauvignon) to produce wine. For example, the free enzyme increased the concentrations of β-damascenone, 1-hexanol, phenylethyl alcohol, and three ethyl esters (ethyl hexanoate, ethyl octanoate and ethyl decanoate) in a Cabernet Sauvignon (CS169) wine, but total anthocyanins level was reduced, affecting the color depth (a* value). Notably, during the alcoholic fermentation stage of CS169, immobilized β-glucosidase did not significantly decrease polyphenolic levels and color depth (a* value). What's more, using immobilized β-glucosidase under this treatment condition resulted in the highest levels of higher alcohols, ethyl esters, and acetate. Moreover, β-damascenone content increased by more than 25.35%, as well as phenethyl alcohol and benzaldehyde of benzenic compounds significantly increased by over 72.23% and two-fold, respectively. As a result, CS169 wine possessed more complex aroma and highest sensory score. Therefore, enologists can use immobilized β-glucosidase to develop various strategies that enhance wine quality and produce different wine styles.

Polydopamine modified cellulose nanocrystals (CNC) for efficient cellulase immobilization towards advanced bamboo fiber flexibility and tissue softness

Herein, a green facile approach to improve the flexibility of unbleached bamboo kraft pulp (UBKP) via an immobilized enzyme technology is proposed. Polydopamine (PDA) acts as versatile modification and coating materials of cellulose nanocrystals (CNC) for assembling versatile bio-carriers (PDA@CNC). Cellulase biomacromolecules are efficiently immobilized on PDA@CNC to form cellulase@PDA@CNC nanocomposites. The relative enzyme activity, temperature/pH tolerance, and storage stability of cellulase were significantly improved after immobilization. The degree of polymerization treated UBKP decreased by 5.42 % (25 U/g pulp) compared to the control sample. The flexibility of treated fibers was 6.61 × 1014/(N·m2), which was 96.93 % higher (25 U/g) compared to the control and 3.88 times higher than that of the blank fibers. Cellulase@PDA@CNC performs excellent accessibility to fiber structure and induces high degree of fibrillation and hydrolysis of UBKP fibers, which contributes high softness of obtained tissue handsheets. The bio-carrier PDA@CNC within paper framework may further enhance tissue tensile strength. This study proposes a practical and environmentally friendly immobilization approach of cellulase@PDA@CNC for improving the hydrolysis efficiency and flexibility of UBKP fibers, which provides the possibility to maintain the strength of tissue paper while improving its softness, thus broadening the high-value application of immobilized enzyme technology in tissue production.

[Development of enzyme immobilization systems for CO2 bioconversion: advances and challenges].

Enzyme-catalyzed CO2 reduction to value-added commodities is important for alleviating the global environmental issues and energy crises due to high selectivity and mild conditions. Owing to high energy density, formic acid or methanol produced from CO2 using formate dehydrogenase (FDH) or multi-enzyme cascades are promising target chemicals for CO2 utilization. However, the low activity, poor stability and low reusability of key enzymes involved in such process hampered its large-scale application. Enzyme immobilization provides an effective solution to these problems and significant progress have been made in immobilization carriers. Moreover, integration of enzyme immobilization with other catalysis techniques have been explored extensively. This review summarized the recent advances in the immobilization of enzymes using membranes, inorganic materials, metal-organic frameworks, covalent organic frameworks and other carriers, and illustrated the characteristics and advantages of different immobilization materials and immobilization methods. The synergistic effects and applications of immobilized enzymes and electrocatalytic or photocatalytic coupling reaction systems for CO2 reduction were further summarized. Finally, the current challenges of enzyme immobilization technology and coupling reaction systems were pointed out and their development prospects were presented.

Enzyme immobilization technology as a tool to innovate in the production of biofuels: A special review of the Cross-Linked Enzyme Aggregates (CLEAs) strategy

This review emphasizes the crucial role of enzyme immobilization technology in advancing the production of two main biofuels, ethanol and biodiesel, with a specific focus on the Cross-linked Enzyme Aggregates (CLEAs) strategy. This method of immobilization has gained attention due to its simplicity and affordability, as it does not initially require a solid support. CLEAs synthesis protocol includes two steps: enzyme precipitation and cross-linking of aggregates using bifunctional agents. We conducted a thorough search for papers detailing the synthesis of CLEAs utilizing amylases, cellulases, and hemicellulases. These key enzymes are involved in breaking down starch or lignocellulosic materials to produce ethanol, both in first and second-generation processes. CLEAs of lipases were included as these enzymes play a crucial role in the enzymatic process of biodiesel production. However, when dealing with large or diverse substrates such as lignocellulosic materials for ethanol production and oils/fats for biodiesel production, the use of individual enzymes may not be the most efficient method. Instead, a system that utilizes a blend of enzymes may prove to be more effective. To innovate in the production of biofuels (ethanol and biodiesel), enzyme co-immobilization using different enzyme species to produce Combi-CLEAs is a promising trend.

Multi-Parameter Detection of Urine Based on Electropolymerized PANI: PSS/AuNPs/SPCE

Urine analysis is widely used in clinical practice to indicate human heathy status and is important for diagnosing chronic kidney disease (CKD). Ammonium ions (NH4+), urea, and creatinine metabolites are main clinical indicators in urine analysis of CKD patients. In this paper, NH4+ selective electrodes were prepared using electropolymerized polyaniline-polystyrene sulfonate (PANI: PSS), and urea- and creatinine-sensing electrodes were prepared by modifying urease and creatinine deiminase, respectively. First, PANI: PSS was modified on the surface of an AuNPs-modified screen-printed electrode, as a NH4+-sensitive film. The experimental results showed that the detection range of the NH4+ selective electrode was 0.5~40 mM, and the sensitivity reached 192.6 mA M-1 cm-2 with good selectivity, consistency, and stability. Based on the NH4+-sensitive film, urease and creatinine deaminase were modified by enzyme immobilization technology to achieve urea and creatinine detection, respectively. Finally, we further integrated NH4+, urea, and creatinine electrodes into a paper-based device and tested real human urine samples. In summary, this multi-parameter urine testing device offers the potential for point-of-care testing of urine and benefits the efficient chronic kidney disease management.

Enzyme Immobilization Technologies and Industrial Applications.

Enzymes play vital roles in diverse industrial sectors and are essential components of many industrial products. Immobilized enzymes possess higher resistance to environmental changes and can be recovered/recycled easily when compared to the free forms. The primary benefit of immobilization is protecting the enzymes from the harsh environmental conditions (e.g., elevated temperatures, extreme pH values, etc.). The immobilized enzymes can be utilized in various large-scale industries, e.g., medical, food, detergent, textile, and pharmaceutical industries, besides being used in water treatment plants. According to the required application, a suitable enzyme immobilization technique and suitable carrier materials are chosen. Enzyme immobilization techniques involve covalent binding, encapsulation, entrapment, adsorption, etc. This review mainly covers enzyme immobilization by various techniques and their usage in different industrial applications starting from 1992 until 2022. It also focuses on the multiscale operation of immobilized enzymes to maximize yields of certain products. Lastly, the severe consequence of the COVID-19 pandemic on global enzyme production is briefly discussed.

Recent advances in enzyme immobilization based on nanoflowers

In general, enzyme immobilization technologies were applied for seeking to create reusable enzymes with improved enzyme properties for industrial applications. However, to traditional immobilization technologies, such as adsorption, covalent binding, cross-linking, and entrapment, the reusability and stability are improved, the activity is often obviously reduced compared with the free enzyme. To resolve this challenge, the recently appeared hierarchical Nanoflowers (NFs) technology opened a new door for enzyme immobilization, which possesses the inherent merits such as enhanced loading capacity and high specific surface area. Recent researches have clearly stated that it is an inexpensive and environmentally benign technology for enzyme immobilization. In this review, we focus on the recent advances in the immobilization of enzymes with the environmentally benign hierarchical NFs, as well as their application.

Nanosurface-immobilized lipase and its degradation of phthalate wastewater

Phthalic acid esters (PAEs) are hard-to-degrade organic pollutants of current concern, and enzyme degradation is a green method for effective degradation of PAEs. To improve the recognition of PAEs, the combination of molecular blotting and immobilized enzyme technology is a novel and efficient method for the degradation of PAEs. In this study, molecularly imprinted carrier (MIP-HNTs) was prepared from halloysite nanotubes (HNTs), using di(2-ethylhexyl) phthalate (DEHP) as the template molecule, methyl methacrylate (MMA) as the functional monomer and (ethylene methacrylate) EDGMA as the cross-linking agent. A novel immobilized enzyme (MIP-HNTs@lipase) was prepared successfully via glutaraldehyde cross-linking, subsequently. The lipase loading efficiency was 76%. The optimal enzymatic activity conditions for MIP-HNTs@lipase were determined to be pH=9 and 50 °C. The MIP-HNTs@lipase exhibited good stability and efficient degradation of DEHP with a degradation rate of 94.758% when the DEHP concentration was at 5 mg·L–1. This research provided a new strategy for industrial application of enzymes and degradation of persistent pollutants.

Amorphous metal-organic frameworks on PtCu hydrogels: Enzyme immobilization platform with boosted activity and stability for sensitive biosensing

Cell-free enzymatic catalysis (CFEC) is emerging biotechnology that simulates biological transformations without living cells. However, the high cost of separation and preparation of the enzyme has hindered the practical application of the CFEC. Enzyme immobilization technologies using solid supports to stabilize enzymes have been regarded as an efficient strategy to address this issue. Nevertheless, the activity and stability of the immobilized enzymes are still crucial challenges for working in vitro. Herein, an enzyme immobilization platform is developed by using PtCu hydrogels coated with amorphous metallic-organic frameworks (MOFs) as multifunctional carriers to encapsulate horseradish peroxidase (HRP). Specifically, PtCu hydrogels acting as a "reservoir of metal ions" can interact with the immobilized enzyme and facilitate electron transfer, leading to the boosted enzyme catalytic performances. Furthermore, amorphous MOFs on the surface of PtCu hydrogels serve as an "armor" to protect the internal enzymes from various perturbation environments. The resultant enzyme immobilization platform (PtCu@HRP@ZIF-8) not only shows an approximately 2.4-fold enhanced activity compared with free enzyme but also exhibits improved stability against harsh conditions. The PtCu@HRP@ZIF-8-based biosensor is constructed for sensitive sensing of organophosphorus pesticides (OPs). The proposed biosensor exhibits a favorable linear relationship with the concentration of paraoxon-ethyl from 6 to 800 ng/mL, with a low detection limit of 1.8 ng/mL. This work reveals the promising potential of our proposed enzyme immobilization platform in practical applications.

An enzyme response-regulated colorimetric assay for pattern recognition sensing application using biomimetic inorganic-protein hybrid nanoflowers

Numerous detection strategies have been undertaken for quantitative analysis of single metal ion in the field of water-quality monitoring, whereas the complexity in the types and concentrations of potential contaminant leads us to should focus on groups of contaminants than to individual contaminants. To the best of our knowledge, there are few strategies to explore cross response-based semiselective sensor array in the area of water-quality monitoring. In this study, a colorimetric sensor array based on enzyme response analysis was innovatively developed for pattern recognition of various metal ions. Three types of metal phosphates-acetylcholinesterase nanoflowers (MP-AChE NFs) were prepared by using a green, facile, cost-efficient enzyme immobilization technology to construct the sensor array. With the help of a multivariate statistical analysis that can concentrate the most significant characteristics (variance) of the data into a lower dimensional space, principal component analysis (PCA) successfully identifies and distinguishes 11 species of metal ions, and gives unique fingerprint information for each analyte. Moreover, the sensor array can distinguish different concentrations of single model analyte, as well as a mixture of different contaminants in tap water.

Molecularly Imprinted Polymers with Enzymatic Properties Reduce Cytokine Release Syndrome.

A core-shell molecularly imprinted polymer nanoparticle with biological enzyme functional characteristics was developed by oxidative polymerization of template protein and polydopamine on the surface of protease-copper phosphate hybrid nanoflowers by molecular imprinting technology and enzyme immobilization technology. The obtained molecularly imprinted polymer showed specific binding characteristics with the template protein. It recognized and enriched the target molecules through the surface molecularly imprinted sites of the shell structure. In addition, the bound target molecules were further degraded into fragments by nanozymes with biological enzyme characteristics in the core. In this study, molecular imprinting technology and biotechnology were combined to obtain bifunctional molecularly imprinted polymer nanoparticles that can not only enrich template molecules but also degrade them into fragments. Herein, we selected interleukin 6 (IL-6), the target molecule of cytokine release syndrome (CRS), as a template molecule, and reported a molecularly imprinted polymer with degrading enzyme properties that can rapidly reduce IL-6 levels in vivo, including a molecularly imprinted layer that can recognize and bind IL-6 and nanozymes that can degrade IL-6 and deactivate it. It is used to clear the excessive secretion of IL-6 in CRS and reduce the level of IL-6 in the body to achieve the purpose of adjuvant treatment of CRS.

Immobilization on magnetic PVA/SA@Fe3O4 hydrogel beads enhances the activity and stability of neutral protease

Enzyme immobilization technology has a key role in improving the stability of enzyme reaction systems and biocatalyst utilization rates. In this study, polyvinyl alcohol/sodium alginate@Fe3O4 (PVA/SA@Fe3O4) magnetic immobilized-enzyme hydrogel beads were prepared. Their structure and morphology were characterized by scanning electron microscopy, surface area and porosity analyses, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and a vibrating sample magnetometer. The capability of PVA/SA@Fe3O4 to adsorb neutral protease was investigated with variations in composition, temperature, pH, stirring speed, enzyme concentration, and crosslinking concentration. The optimal parameters of the immobilization process were determined by response surface methodology (3% neutral protease, 4% crosslinking at 200 rpm, 45 °C and pH 7.2), under which an immobilization rate of 41.98 mg/g was obtained. The thermal stability, acid-base stability, and reusability of the immobilized enzyme were improved significantly. After seven cycles, the immobilized enzyme activity remained at 30.8% that of the initial enzyme activity. The results indicate that the immobilization of NP onto magnetic PVA/SA@Fe3O4 hydrogel beads improves enzyme efficiency, giving this process potential industrial applications.

Developing a novel strategy for light-triggered reversible enzyme immobilization and reuse of support

The reuse of support material still hamper the practical application of enzyme immobilization technology in wastewater treatment. Here, a novel strategy based on light-triggered reversible β-cyclodextrin (β-CD)-Azobenzene(Azo) host-guest interaction was developed. First, magnetic Fe3O4 nanoparticles were coated with porous β-CD-containing polymers(PCDP), and then the Azo-modified enzyme was immobilized in normal condition. The immobilization would enhance the stability and catalytic performance of enzymes, and the easy separation from reaction medium could be available with external magnetic field. Over broad ranges of temperature and pH, the immobilized enzyme showed higher activity than free enzyme, and the phenol removal efficiency retained about 58.63% even after five cycles. Under UV light, the photoisomerization of Azo would lead to disassemble of β-CD-Azo interaction, resulting in the detachment of the enzyme, and phenol removal efficiency decreased to only 16%. The support materials could be recovered for continue enzyme immobilization, results based on cyclic process of enzyme immobilization and detachment strongly demonstrated the detachment of enzyme and excellent reusability of Fe3O4@PCDP. And this strategy was proved to be a practical method based on batch experiments on the removal of phenol by tyrosinase (Tyr)-based remediation.

Advanced Enzyme Immobilization Technologies: An Eco-friendly Support, a Polymer-Stabilizing Immobilization Strategy, and an Improved Cofactor Co-immobilization Technique.

There is a wide variety of protocols for enzyme immobilization, allowing for the reuse of the enzyme, integration in flow bioreactors, and easy separation from the final product. However, none of them have reached a generalized implementation and new immobilization technologies are continuously being developed to improve the properties of the immobilized biocatalysts. In this chapter, we describe three advanced strategies looking at the key points of enzyme immobilization: the sustainability of the support, the recovered activity of the immobilized enzyme, and the reuse of the cofactors. Lignin is presented as a suitable and versatile support for enzyme immobilization, offering a more cost-effective and biodegradable strategy. A cationic polymer is used during the enzyme immobilization procedure to prevent the subunit dissociation of multimeric enzymes as well as to avoid excessive rigidification of the covalently immobilized enzyme. Finally, the reversible co-immobilization of cofactors has been improved by increasing the reactive groups of the support.

Recent Trends in Enzyme Immobilization-Concepts for Expanding the Biocatalysis Toolbox.

Enzymes have been exploited by humans for thousands of years in brewing and baking, but it is only recently that biocatalysis has become a mainstream technology for synthesis. Today, enzymes are used extensively in the manufacturing of pharmaceuticals, food, fine chemicals, flavors, fragrances and other products. Enzyme immobilization technology has also developed in parallel as a means of increasing enzyme performance and reducing process costs. The aim of this review is to present and discuss some of the more recent promising technical developments in enzyme immobilization, including the supports used, methods of fabrication, and their application in synthesis. The review highlights new support technologies such as the use of well-established polysaccharides in novel ways, the use of magnetic particles, DNA, renewable materials and hybrid organic–inorganic supports. The review also addresses how immobilization is being integrated into developing biocatalytic technology, for example in flow biocatalysis, the use of 3D printing and multi-enzymatic cascade reactions.