Immobilized Enzyme Research Articles

Activated carbon from sugarcane bagasse as support for lipase immobilization by physical adsorption technique

Lipases are recognized as the most important group of catalysts in biotechnology. However, utilization of free enzymes is often hampered by the need for more operational stability, high cost, and non-reusability. Most of these obstacles can be solved by lipase immobilization. This work's objective was to evaluate the performance of the activated carbon obtained from sugarcane straw (SAC) as a support for lipase immobilization. Two lipases were immobilized by physical adsorption on SAC: Aspergillus niger 11T53A14 lipase and CalB (lipase B from Candida antarctica, Novozymes). Results revealed that the lipase had been anchored on the activated carbon with the lipase binding efficiency of 89 % (A. niger lipase) and 100 % (CalB) at the optimum experimental conditions (initial protein concentration 0.1 mg mL-1, 0.15 g of SAC, 25 °C, and 120 min). Langmuir isotherm fitted the adsorption equilibrium data of the lipases on SAC. SAC presents a high surface area and protein adsorption capacity. These results show that activated carbon synthesized from the sugarcane straw is a promising support for enzyme immobilization.

Multi-point immobilization of GH 11 endo-β-1,4-xylanase on magnetic MOF composites for higher yield of xylo-oligosaccharides

GH 11 endo-β-1,4-xylanase (Xy) was a crucial enzyme for xylooligosaccharides (XOS) production. The lower reusability and higher cost of purification has limited the industrial application of Xy. Addressing these challenges, our study utilized various immobilization techniques, different supports and forces for Xy immobilization. This study presents a new method in the development of Fe3O4@PDA@MOF-Xy which is immobilized via multi-point interaction forces, demonstrating a significant advancement in protein loading capacity (80.67 mg/g), and exhibiting remarkable tolerance to acidic and alkaline conditions. This method significantly improved Xy reusability and efficiency for industrial applications, maintaining 60 % activity over 10 cycles. Approximately 23 % XOS production was achieved by Fe3O4@PDA@MOF-Xy. Moreover, the yield of XOS from cobcorn xylan using this system was 1.15 times higher than that of the free enzyme system. These results provide a theoretical and applicative basis for enzyme immobilization and XOS industrial production.

Immobilized cold-active enzymes onto magnetic chitosan microparticles as a highly stable and reusable carrier for p-xylene biodegradation

Stability and reusability properties are the two most important factors that determine an enzyme’s application in industry. To this end, cold-active crude enzymes from a psychrophile (xylene monooxygenase (XMO) and catechol 1,2-dioxygenase (C1,2D) were immobilized on magnetic chitosan microparticles for the first-time using glutaraldehyde as a linker. The potential application of enzyme-loaded magnetic particles to remove and detoxify dissolved p-xylene from water confirmed the synergistic mechanism of degradation for in-situ bioremediation in soil and water. Immobilization was optimized based on four variables, such as magnetic particle (MPs), chitosan, glutaraldehyde, and enzyme concentrations. The immobilized enzymes were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). The immobilized enzymes showed improved pH tolerance ranging from 4.0 to 9.0, better temperature stability ranging from 5 to 50, higher storage stability (∼70% activity after 30 days of storage), and more importantly, reusability (∼40% activity after 10 repetitive cycles of usage) compared to their free form. Also, the immobilization of enzymes increased the effectiveness of the enzymatic treatment of p-xylene in soil (10,000 mg/kg) and water (200 mg/L) samples. As a result of the superior catalytic properties of immobilized XMO and C1,2D, they offer great potential for in situ or ex-situ bioremediation of pollutants in soil or water.

Fabrication of flexible glucose sensor based on heterostructure ZnO nanosheets decorated PU/Chitosan-PANI hybrid nanofiber

Designing and fabrication of immensely accurate, affordable, sensitive, and portable glucose sensors is rapidly increasing in clinical applications. However, commercialization of wearable sensors has been impeded by inadequate conductivity, insufficient durability, poor flexibility, and enzyme immobilization. Herein, a scalable approach for fabricating a nanofiber-based highly electroactive, flexible, and disposable (PUC-PANI-ZnO@GOx) sensor that can assess glucose stimuli with sufficient liquid permeability by anchoring ZnO nanosheets over polyaniline (PANI) coated polyurethane-chitosan (PU-Chitosan; PUC) fibrous mat followed by glucose oxidase (GOx) immobilization is described. Regarding the electrocatalytic performance, PUC-PANI-ZnO@GOx sensor showed a high sensitivity (∼998.72 µA mM−1 cm−2) and a low detection limit (∼7.31 µM) for a broad linear range of 0.01 – 9.48 mM with acceptable stability and reproducibility. The fabricated sensor was highly selective towards glucose in the presence of exogenous and endogenous interfering species. To the best of our knowledge, this novel polymer-based nanocomposite adorned on nanofiber substrate was utilized for the first time for the fabrication of a flexible glucose sensor. It can be used for producing a large-scale, cost-effective, and high-performing sensor. Hence, this sustainable and eco-friendly approach can lead to new techniques for designing next-generation wearable and implantable glucose sensors.

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.

An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications.

Enzymes are commonly used as biocatalysts for various biological and chemical processes in industrial applications. However, their limited operational stability, catalytic efficiency, poor reusability, and high-cost hamper further industrial usage. Thus, crosslinked enzyme aggregates (CLEAs) are developed as a better enzyme immobilization tool to extend the enzymes' operational stability. This immobilization method is appealing because it is simpler due to the absence of ballast and permits the collective use of crude enzyme cocktails. CLEAs, so far, have been successfully developed using a variety of enzymes, viz., hydrolases, proteases, amidases, lipases, esterases, and oxidoreductase. Recent years have seen the emergence of novel strategies for preparing better CLEAs, which include the combi- and multi-CLEAs, magnetics CLEAs, and porous CLEAs for various industrial applications, viz., laundry detergents, organic synthesis, food industries, pharmaceutical applications, oils, and biodiesel production. To better understand the different strategies for CLEAs' development, this review explores these strategies and highlights the relevant concerns in designing innovative CLEAs. This article also details the challenges faced during CLEAs preparation and solutions for overcoming them. Finally, the trending strategies to improve the preparation of CLEAs alongside their industrial application trends are also discussed.

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.

Sustainable 3D-printed β-galactosidase immobilization coupled with continuous-flow reactor for efficient lactose-free milk production

The production of low-lactose and lactose-free milk remains a critical challenge. Novel enzyme immobilization integrated with the flow reactor that could be applied to hydrolyze milk with cost-effectiveness, facilitation, and safety is highly expected. Here, we present a novel continuous-flow bioreactor loaded with β-galactosidase (β-Gal) covalently immobilized 3D-printed scaffolds for the production of lactose-free milk. 3D-printed polylactic acid (PLA) scaffolds modified by self-assembly of polyethyleneimine and sodium hyaluronate layers are rapidly branched with functional groups, followed by covalent immobilization of β-Gal using genipin as the cross-linker. The use of genipin allows efficient and stable immobilization of the enzyme (92.87 % immobilization efficiency), and the immobilized enzyme retains 90.53 % activity after 6th repeated use. Moreover, the 3D-printed scaffolds integrated into the flow reactor perturbed the fluids in the different reactors through directional channels, which improved the internal mass transfer to achieve high catalytic activity (94.20 % lactose hydrolysis efficiency). The lactose residues of milk reacted in the β-Gal flow reactor for 6 and 12 h were 1.87 g dL-1 and 0.38 g dL-1, which fully met the requirements of the national food safety standards for low-lactose and lactose-free milk. Due to the high efficiency of enzyme immobilization and the stable catalytic activity of the reactor, it has a broad application prospect for the production of low-lactose and lactose-free milk and other biocatalytic fields.

A newly synthesized magnetic nanoparticle coated with glycidyl methacrylate monomer and 1,2,4-Triazole: Immobilization of α-Amylase from Bacillus licheniformis for more reuse, stability, and activity in the presence of H2O2

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1–3% H2O2.

CREATION AND PROPERTIES OF BIOCOMPOSITE NANOPARTICLES BASED ON FUCOIDAN AS CARRIERS OF FIBRINOLYTIC ENZYME

Magnetically guided biodegradable nanocomposites are a new class of functional materials that expand the possibilities of clinical therapy. The paper describes the chemistry of the formation of a nanobiocomposite material based on a natural polymer synthesized by electrostatic interaction of a polysaccharide and magnetite in an aqueous medium. The possibility of electrostatic immobilization of a model fibrinolytic enzyme was studied. An increase in the electrochemical potential of particles during immobilization of a fibrinolytic enzyme was established by the method of dynamic light scattering. The degree of enzyme inclusion was 9.95±0.05 mass%. The average particle size of the nanobiocomposite material after immobilization of the enzyme was 264.3 nm. The study of the magnetic properties showed the absence of residual magnetization, which makes it possible to exclude the possibility of magnetic aggregation when using the biocomposite in a biological application. The saturation magnetization of the formed material was 57.1 ± 2.3 kA/m, which is lower than that of pure magnetite sol (MS ∼ 73.8 ± 3.4 kA/m), but sufficient for manipulation under conditions of resistance to blood flow. The kinetics of the release of an enzyme preparation in a phosphate buffer from a nanocomposite carrier based on fucoidan was studied. To study the kinetics of enzyme release by mathematical modeling, zero and first order models, the Korsmeier-Peppas model, the Higuchi model, and the Baker-Lonsdale model were used. It has been established that the kinetics of fibrinolytic enzyme release is described with high accuracy (r2 = 0.98) by the Korsmeier–Peppas equation, where the process of release of the enzyme preparation is influenced by diffusion obeying Fick's law. It has been established that zero and first order models are applicable only to the initial (0-40 min, where r2 = 0.96) and final (80-320 min, r2 = 0.93) stage of release.

Advancements in enzyme immobilization on magnetic nanomaterials: toward sustainable industrial applications.

Enzymes are widely used in biofuels, food, and pharmaceuticals. The immobilization of enzymes on solid supports, particularly magnetic nanomaterials, enhances their stability and catalytic activity. Magnetic nanomaterials are chosen for their versatility, large surface area, and superparamagnetic properties, which allow for easy separation and reuse in industrial processes. Researchers focus on the synthesis of appropriate nanomaterials tailored for specific purposes. Immobilization protocols are predefined and adapted to both enzymes and support requirements for optimal efficiency. This review provides a detailed exploration of the application of magnetic nanomaterials in enzyme immobilization protocols. It covers methods, challenges, advantages, and future perspectives, starting with general aspects of magnetic nanomaterials, their synthesis, and applications as matrices for solid enzyme stabilization. The discussion then delves into existing enzymatic immobilization methods on magnetic nanomaterials, highlighting advantages, challenges, and potential applications. Further sections explore the industrial use of various enzymes immobilized on these materials, the development of enzyme-based bioreactors, and prospects for these biocatalysts. In summary, this review provides a concise comparison of the use of magnetic nanomaterials for enzyme stabilization, highlighting potential industrial applications and contributing to manufacturing optimization.

Preparation and Application of Complexes Based on Biopolymers of Animal Origin

Immobilization of enzymes (IoE) from animal origin on natural carriers increases the system stability; facilitates the separation and accelerates the recovery of the enzyme; makes reuse possible; provides a significant reduction in operating costs. There are numerous IoE methods and systems, including immobilization of various lipases on major carbohydrate biopolymers (chitin, chitosan, cellulose, etc.), discussed in this review. The key points of the most encouraging methods “for increasing the activity and stability” of such biopolymer systems are the “chitosan particle activation” by “ultra-sonication” and multiplicative “addition of glutaraldehyde” to these abovementioned systems. The design of such complex biopolymer preparations (in their various forms) is an important area of modern agrosciences, biomedicine, veterinary, zootechnology and bionanotechnology.

Immobilization of enzymes on polymers with upper critical solution temperature: promising engineering of enzymes for biocatalysis

A review of the developments in biocatalysis using upper critical solution temperature (UCST)-type polymer-immobilized enzymes.

Immobilization of L-asparaginase on Oxidized Bacterial Cellulose to Improve the Thermal Stability of the Enzyme

Bacterial cellulose (BC) membranes can be modified for covalent immobilization of macromolecules. One type of modification is oxidation, after which the oxidized BC membrane (OBC) could be used as a matrix for covalent immobilization of enzymes. In this work, the BC membrane was chemically oxidized with sodium periodate (NaIO4) to increase the stability of immobilized mesophilic L-asparaginase (L-ASNase) from Erwinia carotovora (EwA). IR spectroscopy confirmed the immobilization of L-ASNase EwA on OBC membranes. Immobilization of the enzyme increased its temperature optimum for its activity by 15°C and raised the inactivation temperature to 60°C. The OBC membrane could be used as a potential carrier for covalent immobilization of enzymes to improve their pharmacological properties by increasing their thermostability.

A photo-enzyme coupled system for carbon dioxide conversion to solar fuel: The rate-matching and compatibility between photocatalysis and enzyme catalysis

Using reduced nicotinamide adenine dinucleotide (NADH) as cofactor, formate dehydrogenase (FDH) can reversely catalyze CO2 reduction to formic acid. However, the industrialized application of FDH is fairly limited due to its high cost and stoichiometric cofactor consumption. Enzyme immobilization and cofactor regeneration are vital solutions to these problems. Ascribed to the benefits of utilizing a cheap, environmentally friendly, and renewable energy source, photocatalytic NADH regeneration is naturally sustainable and promising. Nonetheless, the rate-matching and compatibility between photocatalysis and enzyme catalysis have not been well revealed. In this study, polyethyleneimine (PEI) modified hollow fiber membrane (HFM) as the enzyme support was in-situ integrated with a visible-light-driven NADH regeneration system using thiophene-modified macroporous graphitic carbon nitride (ATCN-CN) as the catalyst. First, NADH regeneration conditions were regulated to accommodate enzyme catalysis. Then, the photo-enzyme coupled system (PECS) for the sustainable synthesis of formic acid with triethanolamine (TEOA) or H2O as electron donor was constructed, and the influencing factors were investigated. At last, the PECS was compared with other methods as well as a system that used FDH immobilized on silica nanoparticles. The results show that the reaction pH, electron donor, and catalyst concentration have a significant impact on NADH regeneration. For the PECS with TEOA as electron donor, the optimal pH value and photocatalyst concentration for formic acid synthesis are 7.0 and 1.2 mg mL−1, respectively. With the initial addition of 1 mmol L−1 NAD+, a formic acid yield of 35.3% was obtained after 5 h, standing out among the reported values. H2O as a green and low-cost electron donor meets the desire for low-cost photocatalysis. Although the production of formic acid after 5 h was lower in the PECS with H2O as electron donor than in the TEOA system, it was still 14.6 times that of the immobilized enzyme system without cofactor regeneration, and the yield of formic acid could reach 14.8% and 98.3% with 1 or 0.1 mmol L−1 NAD+, validating that current PECS is feasible to a sustainable and green synthesis of solar fuel from CO2.

Production and characterization of a collagen-blended electrospun nanofiber matrix for arginase immobilization

Enzyme immobilization onto nanofiber matrices presents a promising avenue for various biotechnological applications.

Stereoselective reduction of diarylmethanones via a ketoreductase@metal-organic framework.

Mainly owing to their well-defined pore structures and high surface areas, metal-organic frameworks (MOFs) have recently become a versatile class of materials for enzyme immobilization. Nevertheless, most previous studies were focused on model enzymes such as cytochrome c, catalase, and glucose oxidase, with the application of MOF-derived biocomposites for (asymmetric) organic synthesis being rare. In the present work, the immobilization of the ketoreductase KmCR2 onto the zeolitic imidazolate framework (ZIF), a prominent type of MOF, was pursued using the controlled co-precipitation strategy, with a low 2-methylimidazole (2-mIM)/Zn molar ratio of 8 : 1 being employed. Such fabricated biocomposites denoted as KmCR2@ZIF were found to exist mainly in an amorphous phase, as suggested by the scanning electron microscopy (SEM) and powder X-ray diffraction (PXRD) data. Improved thermal and storage stabilities were observed for KmCR2@ZIF compared with the free enzyme. Stereoselective reduction of nine diarylmethanones 1 catalyzed by KmCR2@ZIF was performed, and the corresponding enantioenriched diarylmethanols 2 were afforded in 40-92% conversions with good to excellent optical purities (up to >99% ee). Critically, the current work demonstrated that the unique characteristic of KmCR2, namely the substituent position-controlled stereospecificity (meta versus para or ortho), was not altered upon the enzyme immobilization onto the ZIF.

Enzyme immobilization in Ru(N^N)3-modified covalent organic framework for photoenzymatic cascade catalysis

The co-immobilization of enzymes and metal catalysts addresses key challenges in chemo-enzymatic catalysis, leading to a more efficient and environmentally sustainable process. Central to this approach is the development of...

A green approach to encapsulate proteins and enzymes within crystalline lanthanide-based Tb and Gd MOFs.

In this work, bovine serum albumin (BSA) and Aspergillus sp. laccase (LC) were encapsulated in situ within two lanthanide-based MOFs (TbBTC and GdBTC) through a green one-pot synthesis (almost neutral aqueous solution, T = 25 °C, and atmospheric pressure) in about 1 h. Pristine MOFs and protein-encapsulated MOFs were characterized through wide angle X-ray scattering, scanning electron microscopy, thermogravimetric analysis, Fourier transform infrared and Raman spectroscopies. The location of immobilized BSA molecules, used as a model protein, was investigated through small angle X-ray scattering. BSA occurs both on the inner and on the outer surface of the MOFs. LC@TbBTC, and LC@GdBTC samples were also characterized in terms of specific activity, kinetic parameters, and storage stability both in water and acetate buffer. The specific activity of LC@TbBTC was almost twice that of LC@GdBTC (10.8 μmol min-1 mg-1vs. 6.6 μmol min-1 mg-1). Both biocatalysts showed similar storage stabilities retaining ∼60% of their initial activity after 7 days and ∼20% after 21 days. LC@TbBTC dispersed in acetate buffer exhibited a higher storage stability than LC@GdBTC. Additionally, terbium-based MOFs showed interesting luminescent properties. Together, these findings suggest that TbBTC and GdBTC are promising supports for the in situ immobilization of proteins and enzymes.

Laccase-based catalytic microreactor for BPA biotransformation

A laccase-based catalytic reactor was developed into a polydimethylsiloxane (PDMS) microfluidic device, allowing the degradation of different concentrations of the emergent pollutant, Bisphenol-A (BPA), at a rate similar to free enzyme. Among the immobilizing agents used, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was capable of immobilizing a more significant amount of the laccase enzyme in comparison to glutaraldehyde (GA), and the passive method (2989, 1537, and 1905 U/mL, respectively). The immobilized enzyme inside the microfluidic device could degrade 55 ppm of BPA at a reaction rate of 0.5309 U/mL*min with a contaminant initial concentration of 100 ppm at room temperature. In conclusion, the design of a microfluidic device and the immobilization of the laccase enzyme successfully allowed a high capacity of BPA degradation.