Immobilized enzyme explained

An immobilized enzyme is an enzyme, with restricted mobility, 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.[1] [2] Immobilized enzymes are easily to be handled, simply separated from their products, and can be reused.[3]

Enzymes are bio-catalysts which play an essential role in the enhancement of chemical reactions in cells without being persistently modified, wasted, nor resulting in the loss of equilibrium of chemical reactions. Although the characteristics of enzymes are extremely unique, their utility in the industry is limited due to the lack of re-usability, stability, and high-cost of production.[4]

History

The first synthetic immobilized enzyme was made in the 1950s, performed by the inclusion of enzyme into polymeric matrices or binding onto carrier substances. Also cross-linking procedure was applied by cross-linking of protein alone or along with the addition of inert materials. Over the last decade various immobilization methods have been developed. Binding the enzyme to previously synthesized carrier materials for example is the mostly preferred method so far. Newly, the procedure of cross-linking of crystals of enzyme is also considered as an exciting substitute.  Utilization rate of immobilized enzymes is growing constantly.[5]

Considerations

Before performing any kind of immobilization techniques, some factors should be in mind. It is necessary to understand the chemical and physical effects on an enzyme following immobilization. Enzyme stability and kinetic characteristics can be altered due to changes in the microenvironment conditions of the enzyme after entrapment, support material attachment, or products of enzymatic actions for instance. Additionally, it is important to consider maintaining the tertiary structure of an enzyme prior to immobilizing to have a functional enzyme. Similarly, another crucial site for the functionality of an enzyme is the active-site, which should also be maintained while enzyme is being attached to a surface for immobilization, it is a must to have a selective method for the attachment of surface/material to not end up with an immobilized, but dysfunctional enzyme. Consequently, there are three foundational factors to be thought of for the production of functional immobilized enzymes: immobilization supports selection, conditions and methods of immobilization.[6]

Support selection

For a support material to be ideal, it must be hydrophilic, inert towards enzymes, biocompatible, microbial attack and compression resistant, and must be affordable.[7] [8] Support materials can be organic or inorganic, synthetic or natural (depending on the composition), since they are biomaterial types at the end. There is no universal type of a support material to be used for the immobilization of all enzymes. However, there are some commonly used supports such as silica-based carriers, acrylic resins, synthetic polymers, active membranes and exchange resins. One of the hardest processes before the immobilization process itself, is the selection of support material since it relies on the enzyme type, reaction of media, safety policy of hydrodynamic and reaction conditions. As different types of support give different physical and chemical characteristics and properties, which would effect enzyme function, such as: Hydrophilicity/hydrophobicity, surface chemistry, and pore size.

Methods

Enzymes can be immobilized by physical, or chemical methods including:

Physical

Adsorption

Entrapment

Chemical

Cross-linking

Covalent bonding

Affinity-tag binding: is an immobilization method combining physical, and chemical methods where 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. This technique is the 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.

Random versus site-directed

Numerous enzymes of biotechnological importance have been immobilized on various supports (inorganic, organic, composite and nanomaterials) via random multipoint attachment. However, immobilization via random chemical modification results in a heterogeneous protein population where more than one side chains (amino, carboxyl, thiol etc) present in proteins are linked with the support with potential reduction in activity due to restriction of substrate access to the active site.[11]

In contrast, in site-directed enzyme immobilization, the support can be linked to a single specific amino acid (generally N- or C-termini) in a protein molecule away from the active-site. This way maximal enzyme activity is retained due to the free access of the substrate to the active-site. These strategies are mainly chemical but may additionally require genetic and enzymatic methods to generate functional groups (that are absent in protein) on the support and enzyme.

The choice of SDCM method depends on many factors, such as the type of enzyme (less stable psychrophilic, or more stable thermophilic homologue), pH stability of enzyme, the availability of N- or C-termini to the reagent, non-interference of the enzyme terminus with the enzyme activity, type of catalytic amino acid residue, the availability, price and the ease of preparation of reagents. For example, the generation of complementary clickable functionalities (alkyne and azide) on the support and enzyme is one of the most convenient way for immobilizing enzymes via site-directed chemical modification.[12]

Substrate immobilization

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.[13]

Commercial use

Immobilized enzymes have important application uses as they reduce costs and improve the outcome of the reaction they catalyze. Advantages include:

Convenience: Minuscule amounts of protein dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.
  • Economy: The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst. This is particularly useful in processes such as the production of Lactose Free Milk, as the milk can be drained from a container leaving the enzyme (Lactase) inside ready for the next batch.
  • Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme.[14] In the past, biological washing powders and detergents contained many proteases and lipases that broke down dirt. However, when the cleaning products contacted human skin, they created allergic reactions. This is why immobilization of enzymes is important, for many application fields.
  • Immobilized enzymes are used in various applications including: food, chemical, pharmaceutical, and medical industry. In the food industry for example, Immobilized enzymes are used for the manufacturing of several types of zero-calorie sweetners, Allulose for instance is an epimer of fructose, which is different structurally, resulting in it not being absorbable by human bodies when ingested. Another example of immobilized-enzyme-based sweetners include: Tagatose (immobilized β-galactosidase).

    In the chemical (cosmetics) industry as well, immobilized enzymes are used for the production of emollient esters by utilizing immobilized CalB enzyme. The first company to use such method is Evonik company in 2000. The enzyme Lipase-CalB in its immobilized state is actually used in other pharmaceutical applications for the production of Odanacatib, and Sofosbuvir.

    Notes and References

    1. Zaushitsyna O, Berillo D, Kirsebom H, Mattiasson B . Cryostructured and Crosslinked Viable Cells Forming Monoliths Suitable for Bioreactor Applications. Topics in Catalysis. 57. 5. 339–348. 2013. 10.1007/s11244-013-0189-9 . 94773366.
    2. Börner RA, Zaushitsyna O, Berillo D, Scaccia N, Mattiasson B, Kirsebom H . Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production. Process Biochemistry. 49. 10–18. 2014 . 10.1016/j.procbio.2013.09.027 .
    3. Book: Biocatalysis : from discovery to application . 1999 . Springer . Fessner WD . 3-540-64942-5 . Berlin . 40551838.
    4. Zhang DH, Yuwen LX, Peng LJ . 2013 . Parameters Affecting the Performance of Immobilized Enzyme . Journal of Chemistry . en . 2013 . 1–7 . 10.1155/2013/946248 . 2090-9063. free .
    5. Zelinski T, Waldmann H . 1997-04-04 . Quervernetzte Enzymkristalle (CLECs) — effiziente und stabile Biokatalysatoren für die präparative organische Chemie . Angewandte Chemie . de . 109 . 7 . 746–748 . 10.1002/ange.19971090709. 1997AngCh.109..746Z .
    6. Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA . An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes . Biotechnology, Biotechnological Equipment . 29 . 2 . 205–220 . March 2015 . 26019635 . 4434042 . 10.1080/13102818.2015.1008192 .
    7. Book: Brodelius P, Mosbach K . [14] Overview . 1987 . Methods in Enzymology . 135 . 173–175 . Elsevier . 10.1016/0076-6879(87)35075-x . 9780121820350 . 2022-12-28 .
    8. Book: Buchholz K, Klein J . Characterization of immobilized biocatalysts . [1] Characterization of immobilized biocatalysts . Methods in Enzymology . 135 . 3–30 . 1987 . 3600301 . 10.1016/0076-6879(87)35062-1 . Elsevier . 9780121820350 .
    9. Engelmark Cassimjee K, Kadow M, Wikmark Y, Svedendahl Humble M, Rothstein ML, Rothstein DM, Bäckvall JE . A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications . Chemical Communications . 50 . 65 . 9134–9137 . August 2014 . 24989793 . 10.1039/C4CC02605E .
    10. Zucca P, Sanjust E . Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms . Molecules . 19 . 9 . 14139–14194 . September 2014 . 25207718 . 6272024 . 10.3390/molecules190914139 . free .
    11. Book: Psychrophiles: From biodiversity to biotechnology . 2017-06-22 . Margesin R . 978-3-319-57057-0 . Second . Springer . Cham, Switzerland . 991854426.
    12. Shemsi AM, Khanday FA, Qurashi A, Khalil A, Guerriero G, Siddiqui KS . Site-directed chemically-modified magnetic enzymes: fabrication, improvements, biotechnological applications and future prospects . Biotechnology Advances . 37 . 3 . 357–381 . May 2019 . 30768953 . 10.1016/j.biotechadv.2019.02.002 . 73473772 .
    13. Gray CJ, Weissenborn MJ, Eyers CE, Flitsch SL . Enzymatic reactions on immobilised substrates . Chemical Society Reviews . 42 . 15 . 6378–6405 . August 2013 . 23579870 . 10.1039/C3CS60018A .
    14. Wu H, Liang Y, Shi J, Wang X, Yang D, Jiang Z . Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres . Materials Science & Engineering. C, Materials for Biological Applications . 33 . 3 . 1438–1445 . April 2013 . 23827593 . 10.1016/j.msec.2012.12.048 . free .