Showing papers on "Immobilized enzyme published in 2016"

Encapsulation of a Nerve Agent Detoxifying Enzyme by a Mesoporous Zirconium Metal-Organic Framework Engenders Thermal and Long-Term Stability.

Abstract: Immobilized enzymes typically have greater thermal and operational stability than their soluble form. Here we report that for the first time, a nerve agent detoxifying enzyme, organophosphorus acid anhydrolase (OPAA), has been successfully encapsulated into a water-stable zirconium metal–organic framework (MOF). This MOF features a hierarchical mesoporous channel structure and exhibits a 12 wt % loading capacity of OPAA. The thermal and long-term stabilities of OPAA are both significantly enhanced after immobilization.

Application of magnetic nanoparticles in smart enzyme immobilization.

Abstract: Immobilization of enzymes enhances their properties for efficient utilization in industrial processes. Magnetic nanoparticles, due to their high surface area, large surface-to-volume ratio and easy separation under external magnetic fields, are highly valued. Significant progress has been made to develop new catalytic systems that are immobilized onto magnetic nanocarriers. This review provides an overview of recent developments in enzyme immobilization and stabilization protocols using this technology. The current applications of immobilized enzymes based on magnetic nanoparticles are summarized and future growth prospects are discussed. Recommendations are also given for areas of future research.

Oriented immobilization of proteins on solid supports for use in biosensors and biochips: a review

Abstract: Immobilization of proteins on a solid support is critical with respect to the fabrication and performance of biosensors and biochips. Protein attachment with a preferable orientation can effectively avoid its denaturation and keeps its active sites fully exposed to solution, thus maximally preserving the bioaffinity or bioactivity. This review (with 140 refs.) summarises the recent advances in oriented immobilization of proteins with a particular focus on antibodies and enzymes. Following an introduction that describes reasons for oriented immobilization on (nano)surfaces, we summarize (a) methods for (bio)chemical affinity-mediated oriented immobilization (with sections on immunoglobulin G (IgG)-binding protein as the capture ligand, DNA-directed immobilization, aptamer- and peptide-mediated immobilization, affinity ligand and fusion tag-mediated immobilization, material-binding peptide-assisted immobilization); (b) methods for covalent oriented immobilization (with sections on immobilization via cysteine residues or cysteine tags, via carbohydrate moieties; via enzyme fusion or enzymatic catalysis, and via nucleotide binding sites of antibodies); (c) methods based on molecular imprinting techniques; (d) methods for characterization of oriented immobilized proteins; and then make conclusions and give perspectives.

Agarose and Its Derivatives as Supports for Enzyme Immobilization

Abstract: Agarose is a polysaccharide obtained from some seaweeds, with a quite particular structure that allows spontaneous gelation. Agarose-based beads are highly porous, mechanically resistant, chemically and physically inert, and sharply hydrophilic. These features—that could be further improved by means of covalent cross-linking—render them particularly suitable for enzyme immobilization with a wide range of derivatization methods taking advantage of chemical modification of a fraction of the polymer hydroxyls. The main properties of the polymer are described here, followed by a review of cross-linking and derivatization methods. Some recent, innovative procedures to optimize the catalytic activity and operational stability of the obtained preparations are also described, together with multi-enzyme immobilized systems and the main guidelines to exploit their performances.

Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

Abstract: Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies.

Construction of Thermophilic Lipase-Embedded Metal–Organic Frameworks via Biomimetic Mineralization: A Biocatalyst for Ester Hydrolysis and Kinetic Resolution

Abstract: Enhancing the activity and stability of enzymes and improving their reusability are critical challenges in the field of enzyme immobilization. Here we report a facile and efficient biomimetic mineralization to embed thermophilic lipase QLM in zeolite imidazolate framework-8 (ZIF-8). Systematic characterization indicated that the entrapment of lipase molecules was successfully achieved during the crystal growth of ZIF-8 with an enzyme loading of ∼72.2 ± 1.88 mg/g lipase@ZIF-8, and the enzymes could facilitate the construction of framework building blocks. Then the composite lipase@ZIF-8 was observed to possess favorable catalytic activity and stability in the ester hydrolysis, using the hydrolysis of p-nitrophenyl caprylate as a model. Finally, the composite was successfully applied in the kinetic resolution of (R,S)-2-octanol, with favorable catalytic activity and enantioselectivity during 10 cycle reactions. Thus, the biomimetic mineralization process can be potentially used as an effective technique for...

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

Abstract: Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.

Large-scale aerosol-assisted synthesis of biofriendly Fe2O3 yolk–shell particles: a promising support for enzyme immobilization

Abstract: Multiple-shelled Fe2O3 yolk–shell particles were synthesized using the spray drying method and intended as a suitable support for the immobilization of commercial enzymes such as glucose oxidase (GOx), horseradish peroxidase (HRP), and laccase as model enzymes. Yolk–shell particles have an average diameter of 1–3 μm with pore diameters in the range of 16 to 28 nm. The maximum immobilization of GOx, HRP, and laccase resulted in the enzyme loading of 292, 307 and 398 mg per g of support, respectively. After cross-linking of immobilized laccase by glutaraldehyde, immobilization efficiency was improved from 83.5% to 90.2%. Km and Vmax values were 41.5 μM and 1722 μmol min−1 per mg protein for cross-linked laccase and those for free laccase were 29.3 μM and 1890 μmol min−1 per mg protein, respectively. The thermal stability of the enzyme was enhanced up to 18-fold upon cross-linking, and the enzyme retained 93.1% of residual activity after ten cycles of reuse. The immobilized enzyme has shown up to 32-fold higher stability than the free enzyme towards different solvents and it showed higher efficiency than free laccase in the decolorization of dyes and degradation of bisphenol A. The synthesized yolk–shell particles have 3-fold higher enzyme loading efficiency and lower acute toxicity than the commercial Fe2O3 spherical particles. Therefore, the use of unique yolk–shell structure Fe2O3 particles with multiple-shells will be promising for the immobilization of various enzymes in biotechnological applications with improved electrochemical properties. To the best of our knowledge, this is the first report on the use of one pot synthesized Fe2O3 yolk–shell structure particles for the immobilization of enzymes.

A Novel Acetylcholinesterase Biosensor: Core-Shell Magnetic Nanoparticles Incorporating a Conjugated Polymer for the Detection of Organophosphorus Pesticides.

Abstract: To construct a sensing interface, in the present work, a conjugated polymer and core-shell magnetic nanoparticle containing biosensor was constructed for the pesticide analysis. The monomer 4,7-di(furan-2-yl)benzo[c][1,2,5]thiadiazole (FBThF) and core-shell magnetic nanoparticles were designed and synthesized for fabrication of the biosensing device. The magnetic nanoparticles were first treated with silica and then modified using carboxyl groups, which enabled binding of the biomolecules covalently. For the construction of the proposed sensor a two-step procedure was performed. First, the poly(FBThF) was electrochemically generated on the electrode surface. Then, carboxyl group modified magnetic nanoparticles (f-MNPs) and acetylcholinesterase (AChE), the model enzyme, were co-immobilized on the polymer-coated surface. Thereby, a robust and novel surface, conjugated polymer bearing magnetic nanoparticles with pendant carboxyl groups, was constructed, which was characterized using Fourier transform infrared spectrometer, cyclic voltammetry, scanning electron microscopy, and contact angle measurements. This novel architecture was then applied as an immobilization platform to detect pesticides. To the best of our knowledge, a sensor design that combines both conjugated polymer and magnetic nanoparticles was attempted for the first time, and this approach resulted in improved biosensor characteristics. Hence, this approach opens a new perspective in the field of enzyme immobilization and sensing applications. Paraoxon and trichlorfon were selected as the model toxicants. To obtain best biosensor performance, optimization studies were performed. Under optimized conditions, the biosensor in concern revealed a rapid response (5 s), a low detection limit (6.66 × 10(-3) mM), and high sensitivity (45.01 μA mM(-1) cm(-2)). The KM(app) value of poly(FBThF)/f-MNPs/AChE were determined as 0.73 mM. Furthermore, there was no considerable activity loss for 10 d for poly(FBThF)/f-MNPs/AChE biofilm.

Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance.

Abstract: Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel.

Surface functionalized halloysite nanotubes decorated with silver nanoparticles for enzyme immobilization and biosensing

Abstract: Improving enzyme immobilization with high loading capacity and achieving direct electron transfer (DET) between the enzyme and the electrode surface is key to designing highly sensitive enzymatic electrochemical biosensors. Herein, we report a novel approach based on the selective modification of the outer surface of halloysite nanotubes (HNTs) that supports silver nanoparticles (AgNPs) to obtain a hybrid nanocomposite. AgNPs of about 10 nm average size could be uniformly supported on silane-modified HNTs through in situ reduction of Ag+ ions. The resultant nanocomposite shows an excellent support capability for the effective immobilization and electrical wiring of redox enzyme glucose oxidase (GOx). The GOx immobilized HNT/AgNPs were deposited on the glassy carbon electrode (GCE) and utilized for the bioelectrocatalyzed electrochemical detection of glucose. The GOx modified composite electrodes show glucose sensitivity as high as 5.1 μA mM−1 cm−2, which is higher than for the electrodes prepared without surface functionalization.

Development of simple protocols to solve the problems of enzyme coimmobilization. Application to coimmobilize a lipase and a β-galactosidase

Abstract: This paper shows the coimmobilization of β-galactosidase from Aspergillus oryzae (β-gal) and lipase B from Candida antarctica (CALB). The combi-biocatalyst was designed in a way that permits an optimal immobilization of CALB on octyl-agarose (OC) and the reuse of this enzyme after β-gal (an enzyme with lower stability and altogether not very stabilized by multipoint covalent attachment) inactivation, both of them serious problems in enzyme co-immobilization. With this aim, OC-CALB was coated with polyethylenimine (PEI) (this treatment did not affect the enzyme activity and even improved enzyme stability, mainly in organic medium). Then, β-gal was immobilized by ion exchange on the PEI coated support. We found that PEI can become weakly adsorbed on an OC support, but the adsorption of PEI to CALB was quite strong. The immobilized β-gal can be desorbed by incubation in 300 mM NaCl. Fresh β-gal could be adsorbed afterwards, and this could be repeated for several cycles, but the amount of PEI showed a small decrease that made reincubation of the OC-CALB–PEI composite in PEI preferable in order to retain the amount of polymer. CALB activity remained unaltered under all these treatments. The combi-catalyst was submitted to inactivation at 60 °C and pH 7, conditions where β-gal was rapidly inactivated while CALB maintained its activity unaltered. All β-gal activity could be removed by incubation in 300 mM NaCl, however, SDS analysis showed that part of the enzyme β-gal molecules remained immobilized on the OC-CALC–PEI composite, as the inactivated enzyme may become more strongly adsorbed on the ion exchanger. Full release of the β-gal after inactivation was achieved using 1 M NaCl and 40 °C, conditions where CALB remained fully stable. This way, the proposed protocol permitted the reuse of the most stable enzyme after inactivation of the least stable one. It is compatible with any immobilization protocol of the first enzyme that does not involve ion exchange as only reason for enzyme immobilization.

Printable enzyme-embedded materials for methane to methanol conversion.

Abstract: An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas-liquid reactions.

Enzyme immobilization on cellulose matrixes

Abstract: Enzymes are excellent catalysts in many applications due to their biocompatibility, low energy consumption, unique selectivity, and mild reaction condition. However, some disadvantages limit the usage of enzymes in end uses, such as low stabilities and difficult recovery. In order to overcome these disadvantages, enzyme immobilization was developed. Among various kinds of substrates for attaching enzyme, cellulose and its derivatives are one of the ideal matrixes because they are low cost, nontoxic, renewable, biodegradable, and biocompatible. In this review, we summarize recent progress in the research of enzyme immobilization on cellulose matrixes.