Immobilization Of Enzymes And Cells Pdf

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Mateo, V. Pessela, T. Montes, J. Palomo, R.

Immobilization of Enzymes and Cells

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. Biocatalysis has been widely accepted in diverse sectors owing to their ease of production, substrate specificity and green chemistry. However, for large extent commercialization of these bio-derived catalysts, their reusability factor becomes mandatory, failing which they would no longer be economic.

Maintenance of their structural stability during any biochemical reaction is highly challenging. Consequently, immobilized enzymes with functional efficiency and enhanced reproducibility are used as alternatives in spite of their expensiveness. Immobilized biocatalysts can either be enzymes or whole cells Kawaguti et al. Inert polymers and inorganic materials are usually used as carrier matrices. Factors influencing performance of immobilized enzymes Cao This article reviews the existing techniques used for immobilization along with providing insights into the recent developments for each of them.

We have tried to throw light on significant modifications with respect to the techniques and innovative support materials employed for immobilization of biocatalysts that have potential implication on future enzyme market. Enzyme adsorption results from hydrophobic interactions and salt linkages where either the support is bathed in enzyme for physical adsorption or the enzyme is dried on electrode surfaces.

Adsorbed enzymes are shielded from aggregation, proteolysis and interaction with hydrophobic interfaces Spahn and Minteer Silanized molecular sieves have also been successfully used as supports for enzyme adsorption owing to the presence of silanols on pore walls that facilitate enzyme immobilization by hydrogen bonding Diaz and Balkus Various chemical modifications of the currently used supports would definitely help in better immobilization.

It would be important to note that Accurel with smaller particle sizes increases reaction rates and enantiomeric ratios during biocatalyzation Sabbani et al. For better process control and economic production, Yarrowia lipolytica lipase was immobilized on octyl-agarose and octadecyl-sepabeads supports by physical adsorption that resulted in higher yields and greater tenfold stability than that of free lipase.

This was accounted by the hydrophobicity of octadecyl-sepabeads that enhances affinity between the enzyme and support Cunha et al. These supports were preferred because they are less tough and crystalline than polyhydroxybutyrate. Eco-friendly supports of biological origin not only prevent cropping up of ethical issues, but also cut down the production costs.

Of late, biocompatible mesoporous silica nanoparticles MSNs supports have been used for biocatalysis in energy applications owing to their long-term durability and efficiency Popat et al. Covalent association of enzymes to supports occurs owing to their side chain amino acids like arginine, aspartic acid, histidine and degree of reactivity based on different functional groups like imidazole, indolyl, phenolic hydroxyl, etc.

Peptide-modified surfaces when used for enzyme linkage results in higher specific activity and stability with controlled protein orientation Fu et al. Cyanogen bromide CNBr -agarose and CNBr-activated-Sepharose containing carbohydrate moiety and glutaraldehyde as a spacer arm have imparted thermal stability to covalently bound enzymes Hsieh et al.

Highly stable and hyperactive biocatalysts have been reported by covalent binding of enzymes to silica gel carriers modified by silanization with elimination of unreacted aldehyde groups and to SBA supports containing cage-like pores lined by Si—F moieties Lee et al. Increase in half-life and thermal stability of enzymes has been achieved by covalent coupling with different supports like mesoporous silica, chitosan, etc.

Hsieh et al. Cross-linking of enzymes to electrospun nanofibers has shown greater residual activity due to increased surface area and porosity. Use of such nanodiametric supports have brought a turning point in the field of biocatalyst immobilization Wu et al. Covalent binding of alcohol dehydrogenase on attapulgite nanofibers hydrated magnesium silicate has been opted owing to its thermal endurance and variable nano sizes Zhao et al.

Biocatalytic membranes have been useful in unraveling effective covalent interactions with silicon-coated enzymes Hilal et al. Cross-linked enzyme aggregates produced by precipitation of enzyme from aqueous solution by addition of organic solvents or ionic polymers have been reported Sheldon Different orientations of immobilized enzyme on magnetic nanoclusters obtained by covalent binding have found their applications in pharmaceutical industries owing to their enhanced longevity, operational stability and reusability Yusdy et al.

Maintaining the structural and functional property of enzymes during immobilization is one of the major roles played by a cross-linking agent. One such agent is glutaraldehyde, popularly used as bifunctional cross-linker, because they are soluble in aqueous solvents and can form stable inter- and intra-subunit covalent bonds.

Affinity immobilization exploits specificity of enzyme to its support under different physiological conditions. It is achieved by two ways: either the matrix is precoupled to an affinity ligand for target enzyme or the enzyme is conjugated to an entity that develops affinity toward the matrix Sardar et al.

Affinity adsorbents have also been used for simultaneous purification of enzymes Ho et al. Complex affinity supports like alkali stable chitosan-coated porous silica beads and agarose-linked multilayered concanavalin A harbor higher amounts of enzymes which lead to increased stability and efficiency Shi et al.

Bioaffinity layering is an improvisation of this technique that exponentially increases enzyme-binding capacity and reusability due to the presence of non-covalent forces such as coulombic, hydrogen bonding, van der Waals forces, etc. Sardar and Gupta ; Haider and Husain Entrapment is caging of enzymes by covalent or non-covalent bonds within gels or fibers Singh Efficient encapsulation has been achieved with alginate—gelatin—calcium hybrid carriers that prevented enzyme leakage and provided increased mechanical stability Shen et al.

Entrapment by nanostructured supports like electrospun nanofibers and pristine materials have revolutionalized the world of enzyme immobilization with their wide-ranging applications in the field of fine chemistry, biomedicine biosensors and biofuels Dai and Xia ; Kim et al.

Prevention of friability and leaching and augmentation of entrapment efficiency and enzyme activity by Candida rugosa lipase entrapped in chitosan have been reported.

This support has also been reported to be non-toxic, biocompatible and amenable to chemical modification and highly affinitive to protein due to its hydrophilic nature Betigeri and Neau Entrapment by mesoporous silica is attributed to its high surface area, uniform pore distribution, tunable pore size and high adsorption capacity Ispas et al. Simultaneous entrapment of lipase and magnetite nanoparticles with biomimetic silica enhanced its activity in varying silane additives Chen et al.

Sol—gel matrices with supramolecular calixarene polymers have been used for entrapment of C. Alginate derived from cell walls of brown algae are calcium, magnesium and sodium salts of alginic acid and have been extensively used for immobilization as xanthan—alginate beads, alginate—polyacrylamide gels and calcium alginate beads with enhanced enzyme activity and reusability.

Natural polymers like chitin and chitosan have been used as supports for immobilization Vaillant et al. The protein or carbohydrate moieties of enzymes are used for binding them to chitosan Hsieh et al. Chitosan has been used in combination with alginate where chitosan-coated enzymes had less leaching effect compared to alginate owing to the physical and ionic interactions between the enzyme and support Betigeri and Neau Similarly, a wet composite of chitosan and clay proved to be more reliable for enzyme trapping, because it has hydroxyl and amino groups, which easily link with enzymes, together with good hydrophilicity and high porosity.

Chitosan in the form of beads can entrap twice as much of the enzymes Chang and Juang According to Chern and Chao , the chitin-binding domain of chitinase A1 from Bacillus circulans has a high affinity to chitin; so, this property has been exploited to retain D-hydantoinase. Being a natural polymer, collagen has been used for immobilization of tannase employing glutaraldehyde as cross-linking agent Katwa et al.

This support is pseudoplastic in nature, which helps it to thin under shear stress and recover its viscosity once the stress is removed. Jegannathan et al. Gelatin is a hydrocolloid material, high in amino acids, and can adsorb up to ten times its weight in water.

Its indefinite shelf life has attracted attention for enzyme immobilization. Gelatin has been utilized in mixed carrier system with polyacrylamide where cross-linking with chromium III acetate proved better than chromium III sulfate and potassium chromium III sulfate Emregul et al. Immobilization with ionic liquid-cellulose film activated by glutaraldehyde gave better formability and flexibility Klein et al.

Made of linear amylase and branched amylopectin units, starch has been used as enzyme immobilizer. Calcium alginate—starch hybrid supports were applied for surface immobilization and entrapment of bitter gourd peroxidase. Entrapped enzyme was more stable in the presence of denaturants like urea due to internal carbohydrate moieties, while surface-immobilized enzyme had superior activity Matto and Husain Radiation grafting of substances like acrylamide and dimethylaminoethyl methacrylate onto starch are among the widely used industrial techniques for a high product yield Dung et al.

This structural heteropolysaccharide along with 0. CNBr-activated Sepharose-4B has been used to immobilize amylase and glucoamylase owing to its porosity and easy adsorption of macromolecules. Further matrix modifications like alkyl substituted Sepharose with multipoint attachment between hydrophobic clusters of the enzyme and alkyl residues of the support play a major role in retaining the catalytic properties at extremes of pH, high salt concentrations and elevated temperatures Hosseinkhani et al.

Another example of modified Sepharose matrix is concanavalin A Con A —Sepharose 4B where biospecific interaction between the glycosyl chains of the enzyme and Con A plays a pivotal role in fabrication of various biosensors Mirouliaei et al.

During white radish peroxidase immobilization, glutaraldehyde and polyethylene glycol act as an additive and protective layer around the active center of the enzyme to prevent the attack of free radicals Ashraf and Husain Likewise, Na Y zeolite was used to immobilize lysozyme because it had higher activity compared to other supports as reported by Chang and Chu The heterogeneous surface of zeolites with multiple adsorption sites are considered to be suitable for modulating the enzyme and support interactions Serralha et al.

Immobilization of Candida antarctica lipase on ceramic membrane showed that this inert support could be exploited for carrying out hydrolytic and synthetic reactions by limiting feedback inhibition Magnan et al. Another example of ceramics is toyonite whose variable pore structure can be modified using different organic coatings Kamori et al.

It provides resistance against high pH or temperature, urea, detergents and organic solvents Khan et al. It has been preferred due to its chemical inertness and interconnected pore structure Koszelewski et al. Enzymes like lignin peroxidase and horseradish peroxidase HRP immobilized on activated silica have been effectively used for the removal of chlorolignins from eucalyptus kraft effluent Dezott et al.

They have been used because of their nano-sized structures with high surface area, ordered arrangement and high stability to chemical and mechanical forces Soleimani et al. Surface modifications of silica by amination of hydroxyl and reactive siloxane groups and addition of methyl or polyvinyl alcohol groups strengthen enzyme and support bonds Rao et al.

Another enzyme nitrite reductase was immobilized on controlled pore glass beads, which served as a biosensing device for continuous monitoring Rosa et al. Both natural and hydrochloric acid-modified activated carbon has provided valuable support for enzyme adsorption Alkan et al.

Lately, mesoporous-activated carbon particles containing large contact sites for enzyme immobilization have been used for immobilizing acid protease and acidic lipases where catalytic efficiency has been significantly maintained after 21 cycles of reuse Kumar et al.

Chemical modification of charcoal by adsorbing papain with sulfhydryl groups increased the number of active sites and has been utilized for recovery of mercury from aqueous solution and efficiently employed for industrial wastewater treatment Dutta et al. As reported earlier by Kibarer and Akovali , charcoal is an excellent adsorbent with high adsorptive capacity and minimum fine particulate matter release. Biocatalysts are the key players in various industrial processes.

Production of regioselective and enantioselective compounds for biomedical application has been possible by immobilized enzymes Ren et al.

Glucose biosensors have been developed using electrospun PVA and surface-modified carbon nanotubes Wen et al. Agarose—guar has been successfully utilized for designing phenol biosensors Bagal and Karve Currently, keen efforts are being taken for increasing the stability of biosensors. Immobilization of biosensing enzymes into nanocavities showed significant results Vamvakaki and Chaniotakis Biosynthesis of polyester has been facilitated by immobilized C. With the advent of nanotechnology, silica nanoparticles with immobilized laccase have been applied for elimination of micropollutants from wastewater Zimmermann et al.

Increasing environmental concerns have led to the use of immobilized biocatalysts for biodiesel production Jegannathan et al. The different factors influencing enzyme immobilization and the possible modifications for their enhancement in activity have been chalked out in Fig.

Immobilization of Enzymes and Cells

An immobilized enzyme is an enzyme attached to an inert, insoluble material—such as calcium alginate produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride. This can provide increased resistance to changes in conditions such as pH or temperature. It also lets enzymes be held in place throughout the reaction, following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for enzyme catalysed reactions. An alternative to enzyme immobilization is whole cell immobilization. Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include:. In the past, biological washing powders and detergents contained many proteases and lipases that broke down dirt.

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From this point of view, immobilization, simplicity and stabilization have to be strongly related concepts. The third edition of Immobilization of Enzymes and Cells expands upon and updates the previous editions with current, detailed protocols for immobilization. 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. Skip to main content Skip to table of contents.

Immobilization of Enzymes and Cells: Methods, Effects and Applications

Yeast flocculation Saccharomyces cerevisiae is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes.

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.

Immobilized enzyme

Enzyme immobilization is a process where the movement of the enzyme is severely restricted in space in such a way that its catalytic activity is still preserved. Enzyme immobilization allows continuous use and reuse of the catalyst. Some advantages of the immobilized enzymes over their soluble forms are: increased enzyme stability reduced enzyme costs greater ease of enzyme separation and recovery for reutilization possibility of operating continuously easy product separation reduced effluent problems and , in some cases, increased activity. Despite this advantages industrial application is still limited by: the comparatively low cost of soluble enzymes traditional attitudes the investment needed for introducing new equipment to already implanted processes the nature and cost of the immobilizing support and the immobilizing process including losses of activity the performance of the system The choice of the matrix is very important for the good performance of an immobilized enzyme system. It is then desirable that an enzyme carrier possesses: large surface area permeability hydrophilic character insolubility chemical, mechanical and thermal stability high rigidity suitable shape and particle size resistance to microbial attack regenerability Alternatively it is also possible to immobilize, either viable or non-viable, whole cells. However, side reactions are possible and thus less pure products are obtained.

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Read this article to learn about the methods, effects and applications of immobilization of enzymes and cells. Traditionally, enzymes in free solutions i. Such use of enzymes is wasteful, particularly for industrial purposes, since enzymes are not stable, and they cannot be recovered for reuse. By employing this technique, enzymes are made more efficient and cost-effective for their industrial use. Some workers regard immobilization as a goose with a golden egg in enzyme technology. Immobilized enzymes retain their structural conformation necessary for catalysis. The possibility of loss of biological activity of an enzyme during immobilization or while it is in use.

Cesar Mateo, Benevides C. C. Pessela, Valeria Grazu, Rodrigo Torres, Fernando López-Gallego, Jose M. Guisan et al. Pages PDF.

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