Showing papers on "Immobilized enzyme published in 2013"

Modifying enzyme activity and selectivity by immobilization.

Abstract: Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Enzyme immobilization: an overview on techniques and support materials.

Abstract: The current demands of the world’s biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. These requirements are inevitable to facilitate large-scale and economic formulation. Enzyme immobilization provides an excellent base for increasing availability of enzyme to the substrate with greater turnover over a considerable period of time. Several natural and synthetic supports have been assessed for their efficiency for enzyme immobilization. Nowadays, immobilized enzymes are preferred over their free counterpart due to their prolonged availability that curtails redundant downstream and purification processes. Future investigations should endeavor at adopting logistic and sensible entrapment techniques along with innovatively modified supports to improve the state of enzyme immobilization and provide new perspectives to the industrial sector.

Industrial use of immobilized enzymes

Abstract: Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization.

Enzyme immobilization: an update

Abstract: Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need for immobilization and different applications in industry, covering the last two decades. The most recent papers, patents, and reviews on immobilization strategies and application are reviewed.

Evaluation of immobilized enzymes for industrial applications

Abstract: In contrast to the application of soluble enzymes in industry, immobilized enzymes often offer advantages in view of stability, volume specific biocatalyst loading, recyclability as well as simplified downstream processing. In this tutorial review the focus is set on the evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.

Progress in enzyme immobilization in ordered mesoporous materials and related applications

Abstract: A short time after the discovery of ordered mesoporous materials, which possess unique features such as high specific surface area and pore volume, highly uniform pore distribution and tunable pore size, these materials have been prospected as promising carriers for enzyme immobilization. The immobilization of enzymes in ordered mesoporous materials has been studied for almost two decades. With the development of tailored ordered mesoporous materials and advances in enzyme technology, this field attracted increasing interest due to its quickly expanded functions and applications. This review focuses on the relation between the progress in ordered mesoporous materials and its corresponding contribution to enzyme immobilization as well as the applications of these materials in biocatalysis. The potential trends in the future development of this field are also pointed out.

Enzyme stabilization by nano/microsized hybrid materials

Abstract: Immobilization is a key technology for successful realization of enzyme-based industrial processes, particularly for production of green and sustainable energy or chemicals from biomass-derived catalytic conversion. Different methods to immobilize enzymes are critically reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross-linking (carrier free). As a result, immobilizing enzymes on certain supports can enhance storage and operational stability. In addition, recent breakthroughs in nano and hybrid technology have made various materials more affordable hosts for enzyme immobilization. This review discusses different approaches to improve enzyme stability in various materials such as nanoparticles, nanofibers, mesoporous materials, sol–gel silica, and alginate-based microspheres. The advantages of stabilized enzyme systems are from its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This review also considers the latest studies conducted on different enzymes immobilized on various support materials with immense potential for biosensor, antibiotic production, food industry, biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be environmental friendly, inexpensive, and easy to use for enzyme-based industrial applications.

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

Abstract: Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Immobilization of enzymes: a literature survey.

Abstract: The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.

Surface Modification of Halloysite Nanotubes with Dopamine for Enzyme Immobilization

Abstract: Halloysite nanotubes (HNTs) have been proposed as a potential support to immobilize enzymes. Improving enzyme loading on HNTs is critical to their practical applications. Herein, we reported a simple method on the preparation of high-enzyme-loading support by modification with dopamine on the surface of HNTs. The modified HNTs were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy analyses. The results showed that dopamine could self-polymerize to adhere to the surface of HNTs and form a thin active coating. While the prepared hybrid nanotubes were used to immobilize enzyme of laccase, they exhibited high loading ability of 168.8 mg/g support, which was greatly higher than that on the pristine HNTs (11.6 mg/g support). The immobilized laccase could retain more than 90% initial activity after 30 days of storage and the free laccase only 32%. The immobilized laccase could also maintain more than 90% initial activity after five re...

Enzyme immobilisation on amino-functionalised multi-walled carbon nanotubes: structural and biocatalytic characterisation.

Abstract: Background: The aim of this work is to investigate the structure and function of enzymes immobilised on nanomaterials. This work will allow better understanding of enzyme-nanomaterial interactions, as well as designing functional proteinnanomaterial conjugates. Methodology/Principal Findings: Multiwalled carbon nanotubes (MWNTs) were functionalised with amino groups to improve solubility and biocompatibility. The pristine and functionalised forms of MWNTs were characterised with Fouriertransform infrared spectroscopy. Thermogravimetric analysis was done to examine the degree of the functionalisation process. An immobilised biocatalyst was prepared on functionalised nanomaterial by covalent binding. Thermomyces lanuginosus lipase was used as a model enzyme. The structural change of the immobilised and free lipases were characterised with transmission electron Microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy and Circular dichroism spectroscopy. Biochemical characterisation of immobilised enzyme showed broader pH and thermal optima compared to soluble form. Reusability of the immobilised enzyme for hydrolysis of long chain esters was demonstrated up to ten cycles. Conclusion/Significance: Lipase immobilised on MWNTs has exhibited significantly improved thermal stability. The exploration of advanced nanomaterial for enzyme immobilisation support using sophisticated techniques makes nanobiocatalyst of potential interest for biosensor applications.

Parameters Affecting the Performance of Immobilized Enzyme

Abstract: Enzyme immobilization has been investigated to improve lipase properties over the past few decades. Different methods and various carriers have been employed to immobilize enzyme. However, the application of enzymatic technology in large scale is rarely seen during the industrial process. The main obstacles are a high cost of the immobilization and the poor performance of immobilized lipase. This review focuses on the current status of enzyme immobilization, which aims to summarize the latest research on the parameters affecting the performance of immobilized enzyme. Particularly, the effect of immobilization methods, immobilization carriers, and enzyme loading has been discussed.

Modifying Enzyme Activity and Selectivity by Immobilization

Abstract: Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Rapid Detection of Phenol Using a Membrane Containing Laccase Nanoflowers

Abstract: With the rapid development of nanoscience and nanotechnology, nanostructured biocatalysts that take the advantage of nanomaterials in terms of both functional and structural availability have offered new opportunities for improving biological functions of enzymes and expanding applications in areas such as biosensors, bioanalytical devices, and industrial biocatalysis. Recently, we reported a method of preparing protein–inorganic hybrid nanostructures with flower-like shapes, which have shown much greater activities than free enzymes and most of the reported immobilized enzymes. To bring this appealing catalyst into practical use, however, an effective accommodation of these high-performance enzyme catalysts is required. One way is to weakly attach these enzyme nanoflowers to porous materials by physical adsorption. Recently, Krieg et al. reported the fabrication of a supramolecular membrane by noncovalent modification of a commercial membrane, which suggests the possibility of fabricating functional filtration membranes by a simple post-modification procedure, thus enabling many new and interesting applications. It thus came to our mind to fabricate a membrane incorporating enzyme nanoflowers for the rapid detection of hazardous compounds through visualization of the catalyzed product. Owing to their high toxicity even at a low concentration, phenols are listed as major toxic pollutants by the Environmental Protection Agency of the USA and other countries. Sensitive detection of phenolic compounds has been well established using instrumental analysis such as liquid chromatography. However, these methods usually require sophisticated instrumentation and a multistep procedure, making them less convenient for rapid and on-site detection. The present study started by the fabrication of an enzyme nanoflower incorporated into a membrane. As shown in Figure 1, a suspension of laccase–inorganic hybrid nanoflowers, which have a high activity (ca. 200% that of free laccase) for phenol oxidization, as we observed previously, was injected into a commercial disposable syringe filter equipped with a cellulose acetate membrane (pore size 0.2 mm). This procedure thus deposited enzyme nanoflowers with an average size of 4 mm onto the membrane. Then the aqueous sample containing phenol was mixed with an aqueous solution of 4-aminoantipyrine and was passed through the membrane with incorporated laccase nanoflowers, causing oxidative coupling of phenol with 4-aminoantipyrine to form an antipyrine dye that has an absorption maximum at 495 nm. This procedure allowed rapid analysis by a UV/ Vis spectrophotometer or by the naked eye. Finally, pure water was injected into the filter to remove unreacted reagents and the reaction products, followed by drying the membrane in air for the next use. For the preparation of laccase–copper phosphate nanoflowers, typically, 0.8 mm aqueous CuSO4 was added to phosphate buffered saline (PBS) containing 0.1 mgmL 1 laccase at pH 7.4 and 25 8C. After three days, the precipitate of laccase nanoflowers appeared with porous, flower-like structures. Scanning electron microscopy (SEM) images of the nanoflowers are presented in Figure 2a,b, from which the average diameter of the laccase nanoflowers was determined [a] L. Zhu, L. Gong, Y. Zhang, R. Wang, Prof. J. Ge, Prof. Z. Liu Department of Chemical Engineering, Tsinghua University Beijing 100084 (China) E-mail : junge@mail.tsinghua.edu.cn liuzheng@mail.tsinghua.edu.cn [b] Prof. R. N. Zare Department of Chemistry, Stanford University Stanford, CA 94305-5080 (USA) E-mail : zare@stanford.edu [] These authors contributed equally to this work. Figure 1. Fabrication, use, washing, and reuse of the membrane with incorporated laccase nanoflowers. Phenol and ortho-, meta-, and para-substituted phenols carrying carboxy, halogen, methoxy, or sulfonic acid groups react with 4-aminoantipyrine to form colored compounds, which can then be readily detected.

Preparation of Well-Dispersed Gold/Magnetite Nanoparticles Embedded on Cellulose Nanocrystals for Efficient Immobilization of Papain Enzyme

Abstract: A nanocomposite consisting of magnetite nanoparticles (Fe3O4NPs) and Au nanoparticles (AuNPs) embedded on cellulose nanocrystals (CNCs) was used as a magnetic support for the covalent conjugation of papain and facilitated recovery of this immobilized enzyme. Fe3O4NPs (10-20 nm in diameter) and AuNPs (3-7 nm in diameter) were stable and well-dispersed on the CNC surface. Energy-dispersive spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy were used to evaluate the surface composition and structure of CNC/Fe3O4NPs/AuNPs. The nanocomposite was successfully used for the immobilization and separation of papain from the reaction mixture. The optimal enzyme loading was 186 mg protein/g CNC/Fe3O4NPs/AuNPs, significantly higher than the value reported in the literature. The activity of immobilized papain was studied by electrochemical detection of its specific binding to the Thc-Fca-Gly-Gly-Tyr-Arg inhibitory sequence bound to an Au electrode. The immobilized enzyme retained 95% of its initial activity after 35 days of storage at 4 °C, compared to 41% for its free form counterpart.

Immobilization of cellulase on magnetoresponsive graphene nano-supports

Abstract: In this study, we report the preparation of pH tunable, temperature sensitive magnetoresponsive graphene-based nano-bio carriers for cellulase immobilization. We discuss a simple route to overcome the geometric disadvantage imposed by most 2D immobilization supports and make them capable of closely mimicking free enzymes (FE) operating under similar reaction conditions. The supramolecular assembly of oppositely charged quenched polyelectrolytes and maghemite–magnetite nanoparticles on 2D graphene supports followed by covalent immobilization of cellulase shows a marked improvement in the bio-receptivity of graphene supports. The incorporation of magnetic nanoparticles opens up the possibility of recovery and reuse of the enzyme over multiple cycles. The immobilized enzymes retained about 55% of the original specific activity even after four cycles of reuse. Cellulase immobilization is achieved by a combination of annealed polyelectrolyte brushes and zero-length spacer molecules. The swelling behavior of annealed polyelectrolyte brushes is a strong function of the environmental conditions. The degree of polyelectrolyte swelling can be easily tweaked by manipulating the pH and temperature, providing us an effective tool to control the activity of immobilized enzymes. At a pH of 5.1 and a temperature of 50 °C, the immobilized enzymes with the annealed polyelectrolyte brushes displayed close to 1.5-fold improvement in the activity as compared to immobilized enzymes without the brushes. Activity of immobilized cellulase is evaluated using both soluble as well as insoluble substrates like 2% (w/v) CMC and avicel respectively.

Industrial Use of Immobilized Enzymes

Abstract: Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization.

Uniform Magnetic Core/Shell Microspheres Functionalized with Ni2+–Iminodiacetic Acid for One Step Purification and Immobilization of His-Tagged Enzymes

Abstract: A facile approach has been developed to synthesize Fe3O4/PMG (poly (N,N′-methylenebisacrylamide-co-glycidyl methacrylate)) core/shell microspheres using distillation–precipitation polymerization. Treating PMG shell with iminodiacetic acid (IDA) and Ni2+ yields composite microspheres of Fe3O4/PMG/IDA–Ni2+. The Ni2+ ions loaded on the surface of microspheres provide abundant docking sites for immobilization of histidine-tagged proteins. The high saturation magnetization of Fe3O4/PMG (23 emu/g), determined by vibrating sample magnetometer (VSM), allows an easy separation of the microspheres from solution under an external magnetic field. The composite microspheres were used to purify two His-tagged cellulolytic enzymes (Cel48F and Cel9G) directly from crude cell lysates with high binding affinity, capacity, and specificity. The microspheres can be recycled for many times without significant loss of binding capacity to enzymes. The immobilized enzymes on the surface of microspheres well retain their biologica...

Hierachically Structured Hollow Silica Spheres for High Efficiency Immobilization of Enzymes

Abstract: In this work, the first example of a hierarchically structured hollow silica system is reported without any chemical modification to the enzyme involved in the process. The leaching of the physically adsorbed enzyme is substantially restrained in comparison to pure hollow silica supports. The hierarchical architecture is composed of the ordered hollow silica spheres with a shell-in-shell structure. This rationally integrated architecture, which serves as the host for glucose oxidase immobilization, displays many significant advantages, including increases in mechanical stability, enzyme loading, and bioactivity, and a decrease in enzyme leaching compared to existing pure hollow silica matrices. This facilitates further multifarious applications for enhanced enzyme immobilization, biosensors, and biocatalysis.

Nanocomposites of nanocrystalline cellulose for enzyme immobilization

Abstract: We describe the synthesis, characterization and use of a composite material made of a renewable source and metallic nanoparticles for biosensing applications. Nanocrystalline cellulose (NCC) is a product isolated from natural cellulose fibers, which is of approximately 100 nm long and 10 nm wide in size. We augmented the surface area and tailored the chemical affinity of NCC by optimally dressing it with gold nanoparticles (AuNPs). The deposition of AuNPs on NCC was controlled by using cationic polyethylenimine (PEI) at different pHs. AuNPs were thiol-functionalized using different linkers prior to enzyme immobilization. The enzyme (glucose oxidase or GOx) was conjugated on the composite by carbodiimide coupling, and subsequent activation of linker-carboxylic acid group. Our results showed that GOx was attached to the surface of the NCC nanocomposite. Moreover, the amount of GOx loaded onto the support depended on the length of the thiol-linker used. The lower value (20.3 mg/mg of support) was obtained with the longer thiol-linker (11 carbon chain) compared to 25.2 mg/mg of support for the smaller thiol-linker (3 carbon chain).