An immobilized enzyme, also referred to as sound, insolubilized, supported or matrix-linked enzymes is an enzyme whose movement is completely or severely restricted, usually resulting in a water-insoluble form of the enzyme. Key features of an enzyme-immobilized system include the enzyme, the matrix and the method by which the enzyme is arrached to the matrix. [1]
In biological systems, analogous structures are membrane bound proteins.
Artificially, enzymes can be 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.[2][3]
History edit
Enzyme immobilization is an old technique that evolved from fixing biocatalysts. Empirical development began as early as 1815 for the treatment of waste water and vinegar production. In the latter, the biocatalyst, typically bacteria, was fixed in wood shavings and alcohol would be passed over the shavings to produce vinegar. [4]
Modern technologies in enzyme immobilization were first characterised in Japan in 1967 for the isolation of L-amino acids with an aminoacylase from Aspergillus oryzae. Afterwards, a significant amount of research based interest was generated, which led to present day technologies. [5]
More complex systems were developed between 1985 to 1995, involving multiple enzymes, living cells and co-factor regeneration. An early example of this is the production of L-amino acids from keto-acids via reductive amination using L-aminoacid dehydrogenase. A second enzyme, formate dehydrogenase was fixed to regenerate the co-enzyme NADH, which is necessary for the reaction to proceed. [6]
Commercial use edit
For commercial purposes, immobilized enzymes primarily add value to processes by being reusable, a property without which its use would typically be economically unviable. The greatest disadvantage comes from mass transfer effects since the substrate must take a more convoluted path in order to access the active site. As enzymes towards the centre of the pore are less likely to participate in a reaction, typical enzyme kinetics no longer apply since the total amount of enzyme cannot be assumed to be active. The result is that reaction rates tend to be below that of the theoretical maximum.
Additional advantages and downsides are:
Advantages edit
- Re-usability of the enzyme, which if attached to a larger molecule may be recovered with centrifugation.
- Increased enzyme efficiency.
- Increased thermal and operational stability stability. [7]
- Easier to isolate a pure product; protein impurities such as allergens can be avoided.
- Choice of reactor: usable in continuous and batch reactors.
- Enables multiple use of a single batch of enzymes.
- Allows for complex multi-enzyme reaction systems, meaning that a greater number of reactions can be commercialised.
Disadvantages edit
- Additional costs towards enzyme immobilization process.
- Possible negative effects on enzyme stability and activity. Additional efforts to engineer the enzyme or reaction mixture may be necessary.
- Diffusion limitations, subject to mass transfer effects. It is possible to mitigate this through stirring of a reaction vessel.
Examples of commercially used enzymes edit
| Enzyme used | Product formed | Immobilisation methods |
|---|---|---|
| Glucose isomerase | High-fructose corn syrup | Covalently bonded to carrier, adsorption to ion exchange resin. [8] |
| Amino acid acylase | Amino acid production | Ionic, entrapment, polymer adsorption. |
| Penicillin acylase | Semi-synthetic penicillins | Adsorption, entrapment, microencapsulation, cross linking, covalent. [9] |
| Nitrile hydratase | Acrylamide | Cross linking with glutaryldehyde. [10] |
| β-Galactosidase | Hydrolyzed lactose | Adsorption, entrapment, covalent binding. [11] |
Techniques for Immobilization edit
There are various ways by which one can immobilize an enzyme. These techniques can be broadly catagorised as reversible and irreversible methods. Alternatively a distiction could also be made between coupling, where the enzyme is bonded to a support, and entrapment, where the enzyme is physically isolated with a barrier.
Irreversible edit
Irreversible immobilization results in either an irrecoverable support or biocatalyst. The most common approaches are:
- Covalent bond: Among one of the most widely used methods. The enzyme is bound covalentely to an insoluble support (such as silica gel). An advantage is that this approach provides the strongest enzyme/support interaction, and so the lowest protein leakage during catalysis. However, due to the nature of these bonds, the amino acid residues involved in the covalent bonds must not be integral to the catalytic function of the enzyme itself. This issue is typically resolved by carrying out the binding process in the prescence of chemicals analagous to the intended substrate. [12]
- Cross-linkage: Enzyme molecules are covalently bonded to each other to create a matrix consisting of almost only enzyme. The reaction ensures that the binding site does not cover the enzyme's active site, the activity of the enzyme is only affected by immobility. However, the inflexibility of the covalent bonds precludes the self-healing properties exhibited by chemoadsorbed self-assembled monolayers. Use of a spacer molecule like poly(ethylene glycol) helps reduce the steric hindrance by the substrate in this case.
- Entrapment: The enzyme is trapped in a polymer network that allows both substrate and product to flow through but not the enzyme. This is distinct from cross-linkage and covalent binding because the enzyme is not bound to the matrix containing it. However, mass transfer effects are most pronounced in this method.
- Microencapsulation is a form of entraptment where the enzyme is occluded from the rest of the reaction mixture by encapsulation in insoluble spherical beads or microspheres, such as calcium alginate. [13]
Reversible edit
Reversible methods of immobilization allow for detachment of the enzyme from its support or matrix under mild conditions. Commercially, this is attractive in enhancing the re-usability of the materials involved; if the enzyme degrades, it can be easily replaced.
- Adsorption is a catch-all term for non-covalent interactions but refers to physical adsorption in its narrowest sense. It can be done on glass, alginate beads or a matrix. In the simplest cases the enzyme is mixed with glass beads and will naturally attach to the outside of an inert material. In general, this method is the slowest among those listed here. As adsorption is not a chemical reaction, the active site of the immobilized enzyme may be blocked by the matrix or bead, greatly reducing the activity of the enzyme. In addition, due to the weak bonds involved, leakage could be problematic.
- Affinity-tag binding: Enzymes may be immobilized to a surface, e.g. in a porous material, using non-covalent or covalent Protein tags. This technology has been established for protein purification purposes, and has recently been applied for biocatalysis applications by EziG™ with the His-tag. This technique is the only one generally applicable, and can be performed without prior enzyme purification with a pure preparation as the result. Porous glass and derivatives thereof are used, where the porous surface can be adapted in terms of hydrophobicity to suit the enzyme in question. A significant advantage of this method is the high degree of selectivity between bound molecules. [14]
- Ionic binding is analagous to the interactions seen in chromatography and uses ion-exchangers. A difficulty in this method is finding the correct balance between enzyme functionality and binding strength to prevent leakage. Further challenges arise when support with a high electrostatic charge is used due to the distortions in enzyme kinetics although it has been possible to use this property for engineering purposes.
- Chelation/metal binding uses salts or hydroxides of transition metals on organic molecules (acting as carriers) to coordinate and bind to nucleophilic regions of molecules on the matrix. The metal salt is heated until it precipitates onto the matrix and certain positions of the metal salt are free to interact with the enzyme. The enzyme can be detached through competition (with an analagous ligand) or through decreased pH. Issues in this method relate to the reproducability of results, high variations in enzyme stability and also metal ion leakage.
- Disulfide bonds constitute a unique category because, while being covalent bonds, are reversible by addition of dithiothreitol. The degree of reactivity of the thiol group is also dependant on pH, meaning that the formation process of disulfide bonds is potentially very high.
Immobilization of a Substrate for Enzymatic Reactions edit
Another widely used application of the immobilization approach together with enzymes has been the enzymatic reactions on immobilized substrates. This approach facilitates the analysis of enzyme activities and mimics the performance of enzymes on e.g. cell walls.[15]
References edit
- ^ Engineers, NIIR Board of Consultants & (2004-01-01). Enzymes Biotechnology Handbook. ASIA PACIFIC BUSINESS PRESS Inc. ISBN 9788178330785.
- ^ Zaushitsyna, O.; Berillo, D.; Kirsebom, H.; Mattiasson, B. (2013). "Cryostructured and Crosslinked Viable Cells Forming Monoliths Suitable for Bioreactor Applications". Topics in Catalysis. 57 (5): 339. doi:10.1007/s11244-013-0189-9.
- ^ Aragão Börner, R.; Zaushitsyna, O.; Berillo, D.; Scaccia, N.; Mattiasson, B.; Kirsebom, H. (2014). "Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production". Process Biochemistry. 49: 10. doi:10.1016/j.procbio.2013.09.027.
- ^ Brena, Beatriz; González-Pombo, Paula; Batista-Viera, Francisco (2013-01-01). Guisan, Jose M. (ed.). Immobilization of Enzymes and Cells. Methods in Molecular Biology. Humana Press. pp. 15–31. doi:10.1007/978-1-62703-550-7_2. ISBN 9781627035491.
- ^ Tosa, Mori , Fuse , Chibata, T, T, N, I (1966). "Studies on continuous enzyme reactions. II. Preparation of DEAE-cellulose-aminoacylase column and continuous optical resolution of acetyl-DL-methionine". Enzymologia – via PubMed.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Brodelius, P.; Mosbach, K. [14] Overview. pp. 173–175. doi:10.1016/0076-6879(87)35075-x.
- ^ Wu, Hong; Liang, Yanpeng; Shi, Jiafu; Wang, Xiaoli; Yang, Dong; Jiang, Zhongyi (April 2013). "Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres". Materials Science and Engineering: C. 33 (3): 1438–1445. doi:10.1016/j.msec.2012.12.048.
- ^ Suekane, M. (1982-01-01). "Immobilization of glucose isomerase". Zeitschrift für allgemeine Mikrobiologie. 22 (8): 565–576. doi:10.1002/jobm.19820220808. ISSN 1521-4028.
- ^ Shah, Pallavi; Sridevi, N.; Prabhune, Asmita; Ramaswamy, Veda (2008-12-01). "Structural features of Penicillin acylase adsorption on APTES functionalized SBA-15". Microporous and Mesoporous Materials. 116 (1–3): 157–165. doi:10.1016/j.micromeso.2008.03.030.
- ^ van Pelt, Sander; Quignard, Sandrine; Kubáč, David; Sorokin, Dimitry Y.; Rantwijk, Fred van; Sheldon, Roger A. (2008-04-01). "Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme". Green Chem. 10 (4): 395–400. doi:10.1039/b714258g. ISSN 1463-9270.
- ^ Panesar, Parmjit S.; Kumari, Shweta; Panesar, Reeba (2010-12-27). "Potential Applications of Immobilizedβ-Galactosidase in Food Processing Industries". Enzyme Research. 2010: 1–16. doi:10.4061/2010/473137. PMC 3014700. PMID 21234407.
{{cite journal}}: CS1 maint: PMC format (link) CS1 maint: unflagged free DOI (link) - ^ Zucca, Paolo; Sanjust, Enrico (9 September 2014). "Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms". Molecules. 19 (9): 14139–14194. doi:10.3390/molecules190914139.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ Wadiak, D. T.; Carbonell, R. G. (1975-08-01). "Kinetic Behavior of Microencapsulated β-Galactosidase". Biotechnology and Bioengineering. 17 (8): 1157–1181. doi:10.1002/bit.260170806. ISSN 1097-0290.
- ^ Engelmark Cassimjee, K.; Kadow, M.; Wikmark, Y.; Svedendahl Humble, M.; Rothstein, M. L.; Rothstein, D. M.; Bäckvall, J. -E. (2014). "A general protein purification and immobilization method on controlled porosity glass: Biocatalytic applications". Chemical Communications. 50 (65): 9134. doi:10.1039/C4CC02605E.
- ^ Gray, C. J.; Weissenborn, M. J.; Eyers, C. E.; Flitsch, S. L. (2013). "Enzymatic reactions on immobilised substrates". Chemical Society Reviews. 42 (15): 6378. doi:10.1039/C3CS60018A. PMID 23579870.
Category:Enzymes Category:Organic chemistry Category:Stereochemistry