Showing papers on "Immobilized enzyme published in 2014"

Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization

Abstract: Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.

Enzyme immobilization by adsorption: a review

Abstract: Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.

Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms

Abstract: Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides).

Materials-Based Strategies for Multi-Enzyme Immobilization and Co-Localization: A Review

Abstract: Immobilized enzymes as biocatalysts have great potential both scientifically and industrially because of their technological and economic importance. Their highly efficient catalytic mechanisms and reusability have made them excellent candidates for green and sustainable applications. Previous studies have primarily focused on single enzyme immobilization. However, there are many situations where a single enzyme cannot completely catalyze reactions and multiple enzymes working together in a cascade are needed. It is very challenging to efficiently drive the multi-step reaction toward the desired direction, which is especially true when reactive intermediates are present. Nature overcomes this limitation through the use of multi-enzyme complexes (MECs) to promote the overall catalytic efficiency, which has inspired researchers to synthesize artificial MECs to controllably enhance the production of the desired compounds in multi-step reaction cascades in vitro. The most common approaches to synthesize artificial MECs are to use genetic engineering techniques to create fusion proteins or to co-localize multiple enzymes on suitable carriers. This review focuses on the latter with a particular emphasis on materials-based approaches to enzyme co-localization, which builds on techniques developed for single enzyme immobilization. The attachment techniques used in single enzyme immobilization are also effective in multiple enzyme co-localization, which has a direct impact on the overall enzyme orientation and activity. For carrier-based strategies, the platforms developed for single enzyme immobilization are also appropriate for attaching and co-localizing multiple enzymes. However, the involvement of multiple components in co-localization brings many challenges. The properties of different enzymes makes co-localization complicated when selecting attachment techniques and platforms to preserve enzymatic activity, because the structure and function of each component enzyme needs to be taken into consideration to preserve the overall enzyme activity. In addition, the relative position of the multiple enzymes in a confined space plays a significant role in the interactions between different enzymes, which makes spatial control important for co-localization. This review focuses on the potential of materials-based approaches for multiple enzyme co-localization for the design of sustainable multi-enzyme biocatalysts. A critical analysis of the attachment techniques and carriers platforms that have been used in enzyme immobilization and multi-enzyme co-localization in vitro is provided.

Preparation of Magnetic Chitosan Nanoparticles As Support for Cellulase Immobilization

Abstract: A simple preparation process was developed for magnetic nanoparticles, consisting of chitosan coated on Fe3O4 nanoparticles, to be used as support for enzyme immobilization. Cellulase was covalentl...

Immobilization of cellulase enzyme on functionalized multiwall carbon nanotubes

Abstract: For the past decades, the global trends in the demand of cellulase has been arisen due to its extensive range of applications in food and agriculture industry, and its potential use in the fermentation of biomass into biofuels. However, the instability, highly solubility in water, low catalytic efficiency and high cost of enzyme has become the main obstacles for the development of large scale operations and applications. In this study, cellulase enzyme was immobilized onto functionalized multiwalled carbon nanotubes (MWCNTs) via physical adsorption method to yield a stable and ease of separate enzyme. Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) are used to confirm the successful immobilization of cellulase enzyme. In this approach, the efficiency of enzyme immobilization reaches an optimal value when 4 mg/mL enzyme concentration is used in which approximately 97% enzyme loading can be attained. Based on the UV–visible spectroscopy analysis, the optimum reaction conditions for immobilized cellulase are at pH 5 and a temperature of 50 °C. Results have revealed that MWCNT–cellulase composite still retained 52% of its cellulase activity after six cycles of the CMC analysis. This feature is beneficial to the industrial applications because of its potential to be easily separated from the end product at the end of the reaction, reuse for multiple times and allow the development of multiple enzyme reaction system.

Recent progress in nanobiocatalysis for enzyme immobilization and its application

Abstract: Recent advances in nanotechnology have provided various nanoscale materials that can be used as support for enzyme immobilization. Nanobiocatalysis integrating the biocatalyst and nanoscale materials is drawing great attention as innovative technology. Nanobiocatalysis could achieve not only a much higher enzyme loading capacity and a significantly enhanced mass transfer efficiency, but also unbelievable stabilization. In this review, we will present and discuss the recent progress in nanobiocatalysis and its applications in the fields of bioelectronics, bioconversion, and proteomics.

Direct electrochemistry of glucose oxidase and a biosensor for glucose based on a glass carbon electrode modified with MoS2 nanosheets decorated with gold nanoparticles

Abstract: An electrochemical glucose biosensor was developed by immobilizing glucose oxidase (GOx) on a glass carbon electrode that was modified with molybdenum disulfide (MoS2) nanosheets that were decorated with gold nanoparticles (AuNPs). The electrochemical performance of the modified electrode was investigated by cyclic voltammetry, and it is found that use of the AuNPs-decorated MoS2 nanocomposite accelerates the electron transfer from electrode to the immobilized enzyme. This enables the direct electrochemistry of GOx without any electron mediator. The synergistic effect the MoS2 nanosheets and the AuNPs result in excellent electrocatalytic activity. Glucose can be detected in the concentration range from 10 to 300 μM, and down to levels as low as 2.8 μM. The biosensor also displays good reproducibility and long-term stability, suggesting that it represents a promising tool for biological assays.

Graphene-based field effect transistor enzymatic glucose biosensor using silk protein for enzyme immobilization and device substrate

Abstract: A silk fibroin-encapsulated graphene field effect transistor (FET) enzymatic biosensor that utilizes silk protein as both device substrate and enzyme immobilization material was developed for glucose detection. This biosensor detected glucose levels by measuring the differential drain-source current and the Dirac point shift of the graphene transistor as the glucose is oxidized by glucose oxidase that was immobilized in silk fibroin film on the graphene FET. The fabricated biosensors showed 0.1–10 mM large linear detection range, which covers the reference range of medical examination for diabetes diagnostics. The detection limit of the fabricated biosensors was approximately 0.1 mM ( S / N = 3) with excellent selectivity, and the average sensitivity was 2.5 μA/mM measured at V ds = 100 mV and V g = 0 V. Because this fibroin-encapsulated graphene FET enzymatic biosensor is biocompatible, flexible, and long-term stable, it holds a great promise for portable, wearable, and implantable continuous glucose monitoring applications.

Exceptionally High Glucose Current on a Hierarchically Structured Porous Carbon Electrode with “Wired” Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase

Abstract: This article introduces a carbon electrode designed to achieve efficient enzymatic electrolysis by exploiting a hierarchical pore structure based on macropores for efficient mass transfer and mesopores for high enzyme loading. Magnesium oxide-templated mesoporous carbon (MgOC, mean pore diameter 38 nm) was used to increase the effective specific surface area for enzyme immobilization. MgOC particles were deposited on a current collector by an electrophoretic deposition method to generate micrometer-scale macropores to improve the mass transfer of glucose and electrolyte (buffer) ions. To create a glucose bioanode, the porous-carbon-modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, deglycosylated flavin adenine dinucleotide-dependent glucose dehydrogenase (d-FAD-GDH), and a cross-linker. Carbohydrate chains on the peripheral surfaces of the FAD-GDH molecules were removed by periodate oxidation before cross-linking. The current density for the oxidati...

Immobilization of Horseradish Peroxidase on NH2-Modified Magnetic Fe3O4/SiO2 Particles and Its Application in Removal of 2,4-Dichlorophenol

Abstract: Fe3O4 nanoparticles were prepared by a co-precipitation method with the assistance of ultrasound irradiation, and then coated with silica generated by hydrolysis and condensation of tetraethoxysilane. The silica-coated Fe3O4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in anchoring of primary amine groups on the surface of the particles. Horseradish peroxidase (HRP) was then immobilized on the magnetic core-shell particles by using glutaraldehyde as a crosslinking agent. Immobilization conditions were optimized to obtain the highest relative activity of the immobilized enzyme. It was found the durability of the immobilized enzyme to heating and pH variation were improved in comparison with free HRP. The apparent Michaelis constants of the immobilized HRP and free HRP with substrate were compared, showing that the enzyme activity of the immobilized HRP was close to that of free HRP. The HRP immobilized particles, as an enzyme catalyst, were used to activate H2O2 for degrading 2,4-dichlorophenol. The rapid degradation of 2,4-dichlorophenol indicated that the immobilized enzyme has potential applications for removing organic pollutants.

Preparation and nanoencapsulation of l -asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

Abstract: This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene (ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.

Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads

Abstract: This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C.

Tandem Catalytic Conversion of Glucose to 5-Hydroxymethylfurfural with an Immobilized Enzyme and a Solid Acid

Abstract: Conversion of cellulosic biomass to renewable chemicals such as 5-hydroxymethylfurfural (HMF) is of high current interest. Herein, we report a rare example of one-pot synthesis of HMF from glucose by tandem catalysis. The system is composed of a thermophilic glucose isomerase enzyme for glucose isomerization to fructose and a solid acid catalyst for fructose dehydration to HMF. A base (−NH2) functionalized mesoporous silica (aminopropyl-FMS) with large pore size was deployed successfully to immobilize and protect the thermophilic glucose isomerase in organic solvents at high temperature. The combination of this catalyst with a Bronsted acid (−SO3H) functionalized mesoporous silica (propylsulfonic acid-FMS) allowed us to conduct a one-pot transformation of glucose to HMF directly in a monophasic solvent system composed of tetrahydrofuran (THF) and H2O (4:1 v/v) with 61% yield of fructose and 30% yield of HMF at temperatures >363 K in 24 h.

Poly(2-Vinyl-4,4-dimethylazlactone)-Functionalized Magnetic Nanoparticles as Carriers for Enzyme Immobilization and Its Application

Abstract: Fabrication of various efficient enzyme reactors has triggered increasing interests for its extensive applications in biological and clinical research. In this study, magnetic nanoparticles were functionalized by a biocompatible reactive polymer, poly(2-vinyl-4,4-dimethylazlactone), which was synthesized by reversible addition–fragmentation chain transfer polymerization. Then, the prepared polymer-modified magnetic nanoparticles were employed as favorable carriers for enzyme immobilization. l-Asparaginase was selected as the model enzyme to fabricate the enzyme reactor, and the prepared enzyme reactor exhibited high loading capacity of 318.0 μg mg–1 magnetic nanoparticle. Interestingly, it has been observed that the enzymolysis efficiency increased slightly with the lengthened polymer chain, resulting from the increased immobilization amount of enzyme. Meanwhile, the immobilized enzyme could retain more than 95.7% activity after 10 repeated uses and maintain more than 72.6% activity after 10 weeks storage...

Glutaraldehyde in Bio-Catalysts Design: A Useful Crosslinker and a Versatile Tool in Enzyme Immobilization

Abstract: Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.

Electrochemical biosensing of carbofuran based on acetylcholinesterase immobilized onto iron oxide–chitosan nanocomposite

Abstract: We report a highly sensitive square wave voltammetric biosensor for the determination of carbofuran using an acetylcholinesterase (AChE) enzyme immobilized iron oxide–chitosan nanocomposite film modified glassy carbon electrode (AChE/Fe3O4–CH/GCE). The Fe3O4–CH nanocomposite was prepared by a simple solution mixing process and its formation was confirmed by FT-IR spectroscopy. The effective enzyme immobilization onto the nanocomposite matrix was confirmed by electrochemical impedance, scanning electron and atomic force microscopy studies. Various experimental parameters such as effect of scan rate, inhibition time and substrate concentration were optimized. The nanocomposite-based biosensor could detect carbofuran as low as 3.6 × 10−9 M. The reproducibility of the AChE/Fe3O4–CH/GCE was ascertained by performing intra-assay and inter-assay experiments using cyclic voltammetry. The practical application of the biosensor was ascertained by the determination of carbofuran from cabbage samples and by comparing the results with those obtained by the standard high-performance liquid chromatography (HPLC) method.

Preparation of Robust Biocatalyst Based on Cross-Linked Enzyme Aggregates Entrapped in Three-Dimensionally Ordered Macroporous Silica

Abstract: With the aim to provide a highly stable and active biocatalyst, cross-linked enzyme aggregates (CLEAs) of lipase Candida sp. 99-125 were prepared in three-dimensionally ordered macroporous silica materials (CLEAs-LP@3DOM-SiO2). Lipase Candida sp. 99-125 was first precipitated in the pores of 3DOM SiO2 (named EAs-LP@3DOM-SiO2), and further cross-linked by glutaraldehyde to form CLEAs-LP@3DOM-SiO2. Saturated ammonium sulfate was used as a precipitant and glutaraldehyde with a concentration of 0.25% (w/w) was employed as a cross-linker. Compared with EAs-LP@3DOM-SiO2 and native lipase, CLEAs-LP@3DOM-SiO2 exhibited excellent thermal and mechanical stability, and could maintain more than 85% of initial activity after 16 days of shaking in organic and aqueous phase. When CLEAs-LP@3DOM-SiO2 was applied in esterification and transesterification reactions, improved activity and reusability were achieved. This method can be used for the immobilization of other enzymes of interest.

Kinetic Characterization and Effect of Immobilized Thermostable β-Glucosidase in Alginate Gel Beads on Sugarcane Juice.

Abstract: A thermostable β-glucosidase was effectively immobilized on alginate by the method of gel entrapment. After optimization of immobilized conditions, recovered enzyme activity was 60%. Optimum pH, temperature, kinetic parameters, thermal and pH stability, reusability, and storage stability were investigated. The K m and V max for immobilized β-glucosidase were estimated to be 5.0 mM and 0.64 U/ml, respectively. When comparing, free and immobilized enzyme, change was observed in optimum pH and temperature from 5.0 to 6.0 and 60°C to 80°C, respectively. Immobilized enzyme showed an increase in pH stability over the studied pH range (3.0-10.0) and stability at temperature up to 80°C. The storage stability and reusability of the immobilized β-glucosidase were improved significantly, with 12.09% activity retention at 30°C after being stored for 25 d and 17.85% residual activity after being repeatedly used for 4 times. The effect of both free and immobilized β-glucosidase enzyme on physicochemical properties of sugarcane juice was also analyzed.

Enhanced Activity and Stability of Lysozyme by Immobilization in the Matching Nanochannels of Mesoporous Silica Nanoparticles

Abstract: It is highly desirable to study the kinetics and spectroscopy of enzymes in a crowded and controllable microenvironment. In this work, we employ mesoporous silica of matching pore sizes to confine lysozyme in order to mimic enzyme in a crowded environment. The stability and activity of lysozyme immobilized in mesoporous silica nanoparticle (MSN) of various pore sizes were studied and correlated to spectroscopic data of the immobilized enzyme. By site-selective surface functionalization, we were able to avoid protein adsorbing on the external surfaces of MSNs and study specifically the protein immobilized in the nanochannels. Solution spectroscopic methods, CD and fluorescence, were used to study the secondary and tertiary structures of the immobilized enzyme because MSNs could be suspended very well in solution. To study the catalytic activity of lysozyme, we employed 4-methylumbelliferyl β-d-N,N′,N″-triacetylchitotrioside as a substrate that was hydrolyzed and detected by fluorescence spectroscopy. 8-Ani...

Bioinspired Silica Nanocomposite with Autoencapsulated Carbonic Anhydrase as a Robust Biocatalyst for CO2 Sequestration

Abstract: Here, we report on the development and characterization of a carbonic anhydrase (CA)-based biocatalyst encapsulated in a biosilica matrix for use in environmental CO2 sequestration. Encapsulation occurred simultaneously with autonomous silica synthesis by silica-condensing R5 peptide that was fused to recombinant CA. The encapsulation efficiency was greater than 95%, and the encapsulated CA was not leached from the silica matrix, demonstrating the highly efficient R5-mediated autoencapsulation process. The catalytic efficiencies for both esterase and CO2 hydratase activities tended to increase with increasing pH; however, the catalytic efficiency for CO2 hydration was much more pH dependent, suggesting that proton transfer from silica to water is a rate limiting step, especially for CO2 hydration. In addition to good reusability, the encapsulated CA exhibited outstanding thermostability, even retaining 80% activity after 5 days at 50 °C. The thermoactivity was also remarkable, showing ∼10-fold higher acti...