With new chapters on protocols for immobilization of enzymes and cells which may be useful to greatly improve the functional properties of enzymes and cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Immobilization of Enzymes and Cells, Third Edition demonstrates simple and efficient protocols for the preparation, characterization, and utilization of immobilized enzymes and cells.
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Buy eBook. Buy Hardcover. The reversibly immobilized enzymes can be removed from the support under gentle conditions, a method highly attractive as when the enzymatic activity has decayed, the support can be regenerated and reloaded with fresh enzyme. This is because of economic reasons as the cost of the support is often a primary factor in the overall cost of immobilized catalysts. Physical adsorption usually requires soaking of the support into a solution of the enzyme and incubating to allow time for the physical adsorption to occur. Another way is allowing a solution of the enzyme to dry on the electrode surfaces and then rinsing away enzymes that are not adsorbed.
As for enzyme immobilization through purely ionic forces between the enzyme and support, it is based on the protein—ligand interaction principles used in chromatography, namely the reversible immobilization of enzymes which was first used in ion exchangers. The use of immobilized polymeric-ionic ligands has allowed for modulation of protein—matrix interactions and optimization of the derivative properties. In some cases, affinity binding is also included as one of the physical methods for immobilization of enzymes.
The remarkable selectivity of the interaction, control orientation of immobilized enzyme and minimal conformational changes caused by this type of binding resulting in high retention of the immobilized molecule activity are key advantages of the method [ 17 , 48 ] for instance, the binding between antibodies and antigens or haptens, lectins and free saccharidic chains or glycosylated macromolecules, nucleic acids and nucleic acid-binding proteins, hormones and their receptors, avidin and biotin, polyhistidine tag and metal ions, etc.
Entropically driven hydrophobic interactions are also used to bind enzymes to the surfaces of the support. When one enzyme molecule displaces a large number of water molecules both from the support and its own surface during immobilization, it results in entropy gain to produce the hydrophobic interactions between both entities.
Further modulation of the hydrophobic interactions between the enzyme and support is achieved through adjustment of the pH, temperature and concentration of salt during enzyme immobilization. In general, enzyme immobilization through the technique of physical adsorption is quite simple and may have a higher commercial potential due to its simplicity, low cost and retaining high enzyme activity [ 64 ] as well as a relatively chemical-free enzyme binding. Entrapment is defined as an irreversible method of enzyme immobilization where enzymes are entrapped in a support or inside of fibres, either the lattice structure of a material or in polymer membranes [ 69—71 ] that allows the substrate and products to pass through but retains the enzyme.
The method permits the ability to modify the encapsulating material and hopefully create an optimal microenvironment for the enzyme i. The ideal microenvironment could be optimal pH, polarity or amphilicity. The relation between support material pore size and adsorption is that the adsorption can be done only externally if pores are too small and vice versa. It is possible to use the following polymers as a matrix: alginate, carrageenan, collagen, polyacrylamide, gelatin, silicon rubber, polyurethane and polyvinyl alcohol with styrylpyridium group.
Also, the ratio of immobilized particle size to the support material pore size is a significant factor to be considered for the usability of ready probes. Cross-linking is another irreversible method of enzyme immobilization that does not require a support to prevent enzyme loss into the substrate solution. Technically, cross-linking is performed by formation of intermolecular cross-linkages between the enzyme molecules by means of bi- or multifunctional reagents.
The most commonly used cross-linking reagent is glutaraldehyde as it is economical and easily obtainable in large quantities. Cross-linked enzyme aggregates CLEAs are first prepared by aggregating the enzymes in precipitants such as acetone, ammonium sulphate and ethanol followed by a cross-linker,[ 57 ] and the reactions for enzyme immobilization can be executed in three different manners; either by mixing the prepolymers with a photosensitizer e. This is followed by polymerization initiated by gamma radiation, or the enzymes are mixed in a buffered aqueous solution containing acrylamide monomer and a cross-link agent before a chemically initiated polymerization is performed.
Lysozyme-immobilized electrospun Chitosan CS nanofibres via CLEAs have also been reported to be effective in continuous antibacterial applications. Covalent bonding is one of the most widely used methods for irreversible enzyme immobilization. For the covalent attachment between enzyme and support, the direction of the enzyme binding is a crucial factor that determines its stability. It has been reported that the highest enzyme activity level is achieved when the active centre amino acids is not involved in the binding with the support.
The coupling with the support can be done in two ways, depending on active groups present in the molecule that is to be immobilized. The reactive functional groups can be added to the support without modifications, or the support matrix is modified to generate activated groups. In both cases, it is anticipated that the electrophilic groups generated on the support will react with strong nucleophiles on the protein.
Matrices of choice for such interactions usually include agarose, cellulose, poly vinyl chloride , ion exchange resins and porous glass. Dandavate [ 90 ] and Yilmaz and co-workers [ 91 ] covalently immobilized Candida rugosa lipase onto the surface of silica nanoparticles and glutaraldehyde-activated aminopropyl glass beads which resulted in easy recovery and reuse of the enzyme for ester synthesis. Covalent bonds provide powerful link between the lipase and its carrier matrix, allow its reuse more often than with other available immobilization methods [ 5 , 93 ] and prevent enzyme release into the reaction environment.
Localization of the enzyme on the surface of the support further enhances enzyme attachment and enzyme loading binding method. The orientation and three-dimensional structure of immobilized enzymes are crucial to ensure high enzyme stability and activity. Most immobilization procedures do not actively control the orientation of the enzymes, resulting in the inevitable burying and inaccessibility of their active site.
Enzymes undergo substantial changes in the surface microenvironment, conformation and protein refolding following an immobilization process. This could explain the dramatically diminished activity and stability which is often observed when an enzyme is immobilized on a surface. TG may be used to monitor any reaction that involves a gaseous phase, such as oxidation or dehydration. The sample size varies from a few mg to 10 g depending on the equipment used.
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The method measures a sample's weight as it is heated or cooled in a furnace and is widely used to characterize and verify materials. In the thermograms weight versus temperature or time helps to generate information about thermal stability of the sample, quantitative determination of components, reaction rates, oxidation and kinetics of decomposition.
In previous studies, TGA is usually used to characterize the change in thermal stability of the support used in the immobilization process, either it has been modified or unmodified by measuring the decomposition or weight loss of the sample. Electron microscopy is the only technique available to obtain structural information on materials at nanometre scale resolution.
SEM is an analytical technique in which an image is formed on a cathode ray tube whose raster is synchronized with the raster of a point beam of electrons scanned over the surface of a specimen. The device bounces electrons off the surface of a sample to produce an image. The drive to improve SEM technology was the need for an FESEM capable of ultrahigh resolution over the entire accelerating voltage range by advances in secondary electron detector technology. It was also required for greater flexibility for a wider range of analytical applications.
Both SEM and FESEM are the most highly implemented techniques to characterize the morphological surface of the enzyme as well as the support for immobilization. SEM samples are commonly used to observe the morphology to confirm success of enzyme immobilization,[ , ] while FESEM is used to visualize very small topographic or morphology details on the surface or entire or fractioned objects. Structural information can also be obtained through the use of transmission electron microscopy TEM. The device allows for a much higher resolution than can be obtained with a light microscope, allowing for the visualization of even a single column of atoms.
The electrons are shot completely through the sample, using a tungsten filament to produce an electron beam in a vacuum chamber. The emitted electrons are accelerated through an electromagnetic field that also narrowly focuses the beam. The beam is then passed through the sample material, in which electrons that pass through the sample hit a phosphor screen, charge coupled device CCD or film and produce an image.
Sample of lower density allows more electrons to get through and the image is brighter. A staining method was adopted from the positive staining method for electron microscopy for biological samples whereby a darker image is produced in areas where the sample is denser and therefore, fewer electrons pass through.
The method can produce images with resolution down to 0. Nowadays, TEM has become even more important as the structural dimensions in many materials science applications are rapidly approaching the nanometre length scale and are beyond the spatial resolution limits of other methods. In enzyme immobilization, the distribution of enzyme onto the support material needs to be visualized as it is an important parameter related to the accessibility of the enzyme to the substrate.
Another method of molecular analysis for immobilized enzymes is X-ray photoelectron spectroscopy XPS , also called electron spectroscopy for chemical analysis. The working of XPS is based on the photoelectric effect whereby each atom on the surface has core electron with the characteristic binding energy that is conceptually but not strictly equal to the ionization energy of that electron. The technique uses X-ray to probe the energy distribution of electrons ejected from the solid surface and the photoelectric effect: the electrons contain information regarding chemical oxidation state and electronic structure.
The surface of the sample is irradiated with a low-energy X-ray which excites the electrons of the sample atoms. If the binding energy is lower than the X-ray energy, the electrons are then emitted from the parent atom as a photoelectron. Only the photoelectrons at the outermost surface can escape from the sample surface, making this a surface analysis technique. XPS analysis provides valuable information about surface layers or thin film structures within the range of many industrial applications including catalysis, corrosion, adhesion, polymer surface modification,[ ] magnetic media, electronics, semiconductor, dielectric materials and thin film coatings packaging used in a number of industries.
Li and co-workers characterized the surface structure and composition of graft-modified and enzyme-functionalized polyaniline films using angle-resolved XPS. Subsequently, the binding energy and intensity of a photoelectron peak can be used to identify the elements contained in the sample surface. Plasmon resonance is described as a collective oscillation of conduction band electrons that occurs with peak wavelengths that depend on the size, shape and material composition of the nanoparticle.
The moving electrons generate an oscillating dipole that can couple with dipoles in other nearby nanoparticles. SPR has been used directly to measure the maximal binding and equilibrium fractional surface coverage as the concentration of enzyme in a recirculating solution is stepped up and down in a manner similar to that of the stepwise surface titration method previously described for the study of protein—protein and protein—DNA interactions. For instance, when specific cytochrome P enzymes are attached to the alternate gold surface, SPR was used to distinguish between inhibitors and enzyme substrates based on the shift in absorbance wavelength.
This is a feature particularly useful for any research studies that intend to ensure safety profile for potential drug candidates. Circular dichroism CD spectroscopy is a powerful method in structural biology that has been used to examine proteins, polypeptides and peptide structures since the s. The technique uses a source of circularly polarized light, in which the vector oscillates rotationally to the right or to the left, forming a helix around the axis of propagation.
To compare, when light is depolarized the electromagnetic vector oscillates in any direction perpendicular to the direction of propagation. When light is linearly polarized in a plane, the vector oscillates on a single plane in the direction of propagation. Overall, the contribution is very small but when the content of these residues is very high, the estimation of secondary structure becomes complicated.
Atomic force microscopy AFM is the only microscopic technique able to visualize biomolecules at the single-molecule level with sub-nanometre accuracy in liquid. The AFM probe consists of a microfabricated cantilever which tapers into a sharp nanotip that can be moved in three dimensions with sub-nanometre accuracy by means of several piezoelectric scanners. The tip is brought near the sample surface so that forces acting on the tip cause the cantilever to bend. A laser beam is aimed at the top of the cantilever and reflected onto a photodiode.
In the SMFS, the cantilever deflection is recorded as a function of the vertical displacement of the piezo scanner to quantitatively analyse ligand—receptor interactions to reveal the nature and magnitude of forces, and the related binding energy landscape.https://ipoketon.tk
Immobilization of Enzymes and Cells
The force-scan-based JM is able to obtain a simultaneous topography assessment and also quantify the unbinding [ ] between receptor molecules on a sample and a ligand using tip—sample adhesion maps obtained through the attached AFM tips. In another study, fractal dimension of the immobilization sensor surface was used as a parameter to evaluate the quality of the immobilized biosensors [ ] as well as to assess the controlled and oriented immobilization of ordered monolayers of enzymes prepared using a novel method.
The term calorimetry is defined as a measurement of the relationship of the change of temperature according to time during the process of program-controlled temperature. Microcalorimetry is a versatile technique for studying thermal activities in terms of heat, heat flow and heat capacity. Samples in the form of solids, liquids and gases can all be investigated. It works on the principle that all physical and chemical processes are accompanied by a heat exchange with their surroundings.
When a reaction occurs a temperature gradient is formed between the sample and its surroundings to result in heat flow between the sample and the surrounding that is measured as a function of time. Different calorimetric techniques can be adapted to investigate the variable aspects of the protein chemistry, depending on the physical environment and the type of confinement. In differential scanning calorimetry experiments, the thermodynamic parameters such as the middle point temperature and enthalpy change of the unfolding transition of either the immobilized or free protein can be obtained.
The technique is fairly flexible to provide insight on the thermodynamic effects of the immobilization, as such from multipoint covalent attachment to simple absorption [ ] testing of protein stability, DNA—drug binding studies, protein formulation stability, stability of sutant and wild-type proteins, membrane and lipid stability and polynucleotide stability. Both techniques are suitable to establish reaction enthalpy changes and equilibrium constants from full protein—ligand titration curves. Forster resonance energy transfer FRET is a quantum mechanical phenomenon that occurs between two fluorescent molecules.
It is a distance-dependent physical process, by which non-radiative energy is transferred from an excited molecular fluorophore the donor to another fluorophore the acceptor through intermolecular long-range dipole—dipole coupling. FRET only needs one of the two molecules to be fluorescent and the distance between the donor and acceptor is kept to minimum to ensure high probability of energy transfer.
The donor and acceptor fluorophores must be adequately aligned for proper induction of the acceptor dipole by the donor. The use of FRET between fluorophores has been enhanced [ , ] by the introduction of new generation fluorophores which include small and photostable organic fluorophores as well as activated nanoparticles that were brought in as a substitute to the native fluorophores, such as tryptophan and green fluorescent protein.
The method requires covalent attachment of a FRET pair, the donor fluorophore and acceptor dye at specific sites of the biomolecules [ , ] to produce the overlapping emission peak of the donor and the excitation peak of the acceptor. The sample is irradiated at the absorption wavelength of the donor which is temporarily excited into a higher energetic electronic state. The donor fluorophore then gives up its energy non-radiatively to the acceptor fluorophore by dipole-induced dipole interaction and decay at its characteristic fluorescence emission wavelength.
The emission wavelengths of both donor and acceptor are monitored to quantify the efficiency of energy transfer between the donor and acceptor. Measurements of FRET efficiency have long been used at the ensemble level to monitor conformational changes of molecules in solution [ ] such as examining structural and dynamic properties of individual molecules on an atomic level, analysis of molecular interactions at the level of single cells, cell organelles[ , ] and single molecules including time trajectories of folding pathways and transient intermediates of enzymes when immobilized.
FRET offers the benefit of a practical and simple measurement for cases in which the main objective is to distinguish between two molecular states with different donor—acceptor distances. Hence, these factors account for biases due to 1 bleed-through in excitation, such as when a donor is excited by the acceptor's excitation wavelength and vice versa; and 2 crosstalk in emission detection, such as when the emission of a donor also contributes to the signal measured in a set-up for acceptor detection, and vice versa. It is often difficult to separate the contribution of direct crosstalk from the contribution of bleed-through signals.
Scanning electrochemical microscopy SECM was invented by Bard and co-workers in and it is an instrument which basically consists of a combination of electrochemical components, positioners and computer control. Current is allowed to flow through a microelectrode immersed in an electrolytic solution and situated close to a substrate.
The microelectrode and the substrate form part of an electrochemical cell which is also constituted by reference and auxiliary electrodes, and sometimes by a second working electrode. An ultramicroelectrode called a tip is used to scan a surface of interest in close proximity. The electrochemical response of the tip or of the substrate in response to the tip provides quantitative information about the interfacial region.
SECM has been used for the quantitative investigation and surface analysis of a wide range of processes that occur at interfaces [ ] and for probing a great variety of electrochemical processes in fundamental and applied electrochemistry,[ ] energy storage,[ ] materials science,[ ] corrosion science, biosensors research [ ] and biophysics.
The time-of-flight secondary ion mass spectroscopy TOF-SIMS is a surface-sensitive analytical tool that permits the submicron-scale mapping of complex sample surfaces such as protein-adsorbed materials, and chemical mapping information, such as the imaging of the distribution pattern of a particular protein. The components of TOF-SIMS consist of an ultrahigh vacuum system, a particle gun that uses Ga or Cs source, a circular designed flight path equipped with electrostatic analysers and a mass spectrometer that utilizes TOF analyser to enhance its sensitivity and increase its range of application.
The ions are allowed to travel through a path of a given length for a certain time span before reaching the detector. Since the velocity of each secondary ion is dependent on its weight, the mass of the ions is determined by measuring the exact time at which the ions reach the detector. Depending on the mass to charge ratio, generally ions that are lighter will reach the detector quicker.
The TOF-SIMS is available in three modes operating, namely, surface imaging and surface spectroscopy, for which both are used for visualization of distribution of individual species on a surface and analysis of elemental and molecular species on a surface, and depth profiling for determination of different chemical species as a function of depth from the surface. TOF-SIMS has been already used in the steric analysis of proteins,[ 16 ] lipid—lipid and lipid—protein interactions,[ , ] biomarkers [ ] as well as enzyme immobilization.
The dynamic mode is preferred when extreme high sensitivity analysis is required in which ions of high current densities are used to bombard the sample surface, which in tern leads to damaging the surface. This measurement method is more appropriate for analysis of inorganic materials such as trace elements.
Immobilized Enzymes and Cells, Part B, Volume 135 (Methods in Enzymology)
Hence, this method provides the chemical structure of the surface side of an immobilized enzyme representing the enzyme orientation. The method is appropriate for highly sensitive detection as it provides extremely high transmission in combination with parallel detection of all masses. However, TOF-SIMS has till now not come into widespread use in the field of enzyme immobilization because of its complicated spectrum interpretation. Enzyme-based strategies are increasingly favoured over the conventional chemical methods in industrial processes.
Utilization of immobilized enzymes as biocatalysts will continue to attract significant attention from industries as the technique is highly efficient, environmentally friendly and can potentially be cost saving when further research is done to seek out or manufacture new matrices that are cheaper and more robust, which can be used as supports for enzyme immobilization.
Also, more studies should be centred to overcome the current drawbacks in immobilization techniques, as well as development of simple and stable enzyme immobilization methods that could bring down the cost of immobilized enzymes. The present surface analytical technologies are useful for monitoring efficacy of an immobilization process as well as to keep track of post-immobilization changes in the enzyme.
These techniques provide vital insights into the effects of immobilization of enzyme on the stability and activity following treatment with different immobilization methods. In time, these modern techniques which combine tools from chemistry and molecular biology can be further developed to help improve enzyme immobilization strategies and expand the catalytic repertoire of immobilized enzymes for diverse application in various fields. We would like to acknowledge valuable help and suggestions provided by our colleagues. National Center for Biotechnology Information , U.
Biotechnology, Biotechnological Equipment. Biotechnol Biotechnol Equip. Published online Feb Author information Article notes Copyright and License information Disclaimer. Email: ym. Received Aug 27; Accepted Oct 7. This article has been cited by other articles in PMC. Abstract The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes.
Keywords: enzymes, immobilization, entrapment, surface analysis, nanoscale, atomic force spectroscopy, circular dichroism. Introduction One of the most important roles of enzymes as natural biocatalysts is their capacity to enhance the rate of virtually all chemical reactions within a cell. Factors to consider prior to enzyme immobilization It is important to recognize that an enzyme would undergo changes in the chemical and physical properties upon immobilization, depending on the choice of immobilization method. Choice of supports The characteristics of the matrix are paramount in determining the effectiveness of the immobilized enzyme system.
Techniques of enzyme immobilization Selection of the appropriate immobilization method is a very crucial part of the immobilization process as it plays the biggest role in determining the enzyme activity and characteristics in a particular reaction. Open in a separate window.
Figure 1. Physical adsorption The physical adsorption method can be defined as one of the straightforward methods of reversible immobilization that involve the enzymes being physically adsorbed or attached onto the support material. Entrapment Entrapment is defined as an irreversible method of enzyme immobilization where enzymes are entrapped in a support or inside of fibres, either the lattice structure of a material or in polymer membranes [ 69—71 ] that allows the substrate and products to pass through but retains the enzyme.
Cross-linking Cross-linking is another irreversible method of enzyme immobilization that does not require a support to prevent enzyme loss into the substrate solution. Covalent bonding Covalent bonding is one of the most widely used methods for irreversible enzyme immobilization. Surface analysis technology for enzyme immobilization The orientation and three-dimensional structure of immobilized enzymes are crucial to ensure high enzyme stability and activity.
X-ray photoelectron spectroscopy XPS Another method of molecular analysis for immobilized enzymes is X-ray photoelectron spectroscopy XPS , also called electron spectroscopy for chemical analysis. Surface plasmon resonance SPR by ultraviolet detection Plasmon resonance is described as a collective oscillation of conduction band electrons that occurs with peak wavelengths that depend on the size, shape and material composition of the nanoparticle.
Circular dichroism CD spectroscopy Circular dichroism CD spectroscopy is a powerful method in structural biology that has been used to examine proteins, polypeptides and peptide structures since the s. Atomic force microscopy AFM Atomic force microscopy AFM is the only microscopic technique able to visualize biomolecules at the single-molecule level with sub-nanometre accuracy in liquid.
Microcalorimetry The term calorimetry is defined as a measurement of the relationship of the change of temperature according to time during the process of program-controlled temperature. Scanning electrochemical microscopy SECM Scanning electrochemical microscopy SECM was invented by Bard and co-workers in and it is an instrument which basically consists of a combination of electrochemical components, positioners and computer control. Time-of-flight secondary ion mass spectroscopy TOF-SIMS The time-of-flight secondary ion mass spectroscopy TOF-SIMS is a surface-sensitive analytical tool that permits the submicron-scale mapping of complex sample surfaces such as protein-adsorbed materials, and chemical mapping information, such as the imaging of the distribution pattern of a particular protein.
Conclusion Enzyme-based strategies are increasingly favoured over the conventional chemical methods in industrial processes. Acknowledgements We would like to acknowledge valuable help and suggestions provided by our colleagues. Disclosure statement The authors declare no conflict of interest. References Cooper GM. The chemistry of cells: the central role of enzymes as biological catalysts.
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Immobilized enzymes and cells part B
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Immobilized Enzymes and Cells, Part B, Volume - 1st Edition
Nanostructures for enzyme stabilization. Chem Eng Sci. Direct immobilization of polyphenol oxidases on celite from ammonium sulphate fractionated proteins of potato Solanum tuberosum J Mol Catal B. Food Bioprod Process. Enzyme immobilization: an overview on techniques and support materials. Materials for enzyme engineering. In: Gemeiner P, editor. Enzyme engineering. Immobilization techniques and biopolymer carriers. Biotechnol Food Sci. Mesoporous materials for encapsulating enzymes.
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