Immobilization Techniques

 

IMMOBILIZATION OF ENZYMES:

The word "immobilized enzymes" applies to "enzymes that are physically bound or placed in a given area of space with preservation of their catalytic activity and can be used repeatedly and continuously." Because of their superiority over soluble enzymes, immobilized enzymes are currently the topic of great concern. The immobilization techniques are the foundation for producing a variety of biotechnology items with use in diagnostics, bio-affinity chromatography, and biosensors, besides their usage in industrial processes. Initially, only immobilized single enzymes were used, and more complicated mechanisms were formed after the 1970s including two-enzyme reactions with cofactor regeneration and living cells.

Enzymes

Enzymes are considered to be extremely powerful and reliable catalysts in a broad spectrum in processes distinguished by strong selectivity and action. In addition, enzymes may minimize the number of reaction steps and the number of volatile solvents needed, thereby rendering a method more cost-effective and environmentally sustainable. For these purposes, enzymes have been highly effective catalysts that show tremendous potential in many functional applications of food to pharmaceutical industries.

The usage of enzymes in several catalytic processes have culminated in experiments which have resulted in substantial enzyme properties development. One of the most important and commonly employed methods are enzyme immobilization, where catalysts are bound to a strong base that is insoluble in the mixture of reactions.

Why is enzyme Immobilization important?

The main benefit of immobilization being that it greatly increases biomolecules 'stability under diverse reaction conditions and strengthens biomolecules' reusability over consecutive catalytic cycles. In addition, the catalysts move from a homogeneous to a heterogeneous shape after joining the enzyme molecules, which allows the easy isolation of the bio-catalytic mechanism from the reaction mixture and results in products with higher purity.

Besides this, immobilized enzymes are very relevant for commercial uses as they have many advantages to the reaction costs and processes which include:

Convenience: The reaction dissolves tiny quantities of protein, and science will be even simpler. Upon completion, there are usually only solvent and catalyst components in the process mixtures.

Economy: Simply extract the immobilized enzyme from the reaction allowing it possible to recover the biocatalyst. This is especially helpful in processes such as Lactose-Free Milk processing, since the milk may be removed from a jar that keeps the enzyme (Lactase) ready for the next sample.

Flexibility: Usually, immobilized enzymes have greater thermal and operating flexibility than the enzyme's soluble form.

Biological cleaning powders and detergents in the past included tons of proteases and lipases that break up water. However, they developed allergic reactions when the washing agents touched human skin. This is why not just economically, immobilization of enzymes is important.

TECHNIQUES TO IMMOBILIZED AN ENZYME

Different methods and supports for the immobilization have been developed to improve enzyme activity. Because it depends on the type of enzyme, reaction media, safety policy in the field of hydrodynamic conditions and reaction conditions, the choice of support the material can be a rather complex matter.

Some enzyme immobilization techniques are;

Adsorption: Enzyme adsorption results from hydrophobic interactions and salt connections where either the support for physical adsorption is bathed in enzyme or the enzyme is dried on electrode surfaces. Adsorbed enzymes are shielded against aggregation, proteolysis and hydrophobic interactions.

Researchers have used eco-friendly supports such as coconut fibers with good water holding capacity and high cation exchange properties; microcrystalline cellulose with irreversible binding capacity; kaolin with high enzyme retains chemical acetylation capability; and micro/mesoporous materials with thiol functionalized, large surface area ideally suited for reducing and oxidizing reactions.

Covalent bonding: Due to their side chain amino acids such as arginine, aspartic acid, histidine, and degree of reactivity dependent on various functional classes such as imidazole, indolyl, phenolic hydroxyl, etc., covalent interaction of enzymes to supports exist. When used for the enzyme linkage, peptide-modified surfaces result in higher specific activity and stability with protein orientation controlled. One such agent is glutaraldehyde, which is popularly used as a bi-functional cross-linker since it is soluble in aqueous solvents and can form stable covalent inter- and intra-subunit bonds.

Entrapment: Entrapment is the caging of enzymes within gels or fibers by covalent or noncovalent bonds. Efficient encapsulation was achieved with alginate – gelatin – calcium hybrid carriers that prevented enzyme leakage and gave mechanical stability increased. With its wide-ranging applications in the field of fine chemistry, biomedicine biosensors and biofuels, nanostructured supports such as electrospun nanofibers and pristine materials have revolutionized the world of enzyme immobilization.

MATRIX FOR IMMOBILIZATION OF ENZYMES

Materials used for immobilization supports are;

Alginate: Alginate obtained from brown algae cell walls are alginic acid phosphate, magnesium, and sodium salts which have been commonly used for immobilization such as xanthan – alginate beads, alginate – polyacrylamide gels, which phosphate alginate beads with improved enzyme activity and reusability. Cross-linking of alginate with divalent ions and glutaraldehyde enhance enzyme stability.

Chitosan and chitin: Solid polymers such as chitin and chitosan were used as immobilization aids. The enzyme moieties of proteins or carbohydrates are used for attaching them to chitosan. Chitosan can contain twice as many of the enzymes in the shape of beads. Bacillus circulans chitin-binding domain of chitinase A1 has a strong affinity to chitin; hence, this ability has been exploited for D-hydantoinase retention.

Gelatin: Gelatin is a hydrocolloid substance that is rich in amino acids that can adsorb to water up to ten times its weight. Its limitless shelf life gained publicity for immobilization of the enzyme. Gelatin has been used in a polyacrylamide mixed carrier method where chromium (III) acetate cross-linking proved stronger than chromium (III) sulfate and potassium (III) sulfate. Calcium alginate with gelatin provides a strong base for the deposition of calcium phosphate for the immobilization of enzymes.

APPLICATIONS AND SCOPE

Biocatalysts are the major competitors of the separate manufacturing processes. Constant attempts are ongoing to enhance the operation, performance, reproducibility, and stabilization of the enzyme during industrial processes. Currently, there are significant attempts being made to improve biosensor stability. The immobilization into nanocavities of biosensing enzymes has produced important effects. The development of nanotechnology has contributed to the use of silica nanoparticles with immobilized laccase to remove micropollutants from wastewater. Growing environmental issues have culminated in the usage of immobilized biocatalysts for the manufacture of biodiesel.

By: Dua'a Mughal

 

Reference:

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