INTRODUCTION:

An enzyme is a substance that catalyzesa metabolic reaction in living organisms andthe substrate in various chemical reactions. The enzymes can be consideredas biocatalyst that works in mild temperature, pressure, pH, substrate specificity under suitable reaction conditions leading to the production of desired products without any intermediate products as a contaminant. Because of these advantages, the enzymeis put under various application such as in cosmetics, paper industry, food industry, textile industry, the pharmaceutical industry, laundry and in detergents,etc.^1-5.

In biotechnology, a method of producing enzyme is expensive; hence novel methodologies and techniques have been implemented to bring down the productioncost. Some limitations of enzyme biotechnology are: low stability, high sensitivity to the process conditions and these problems can be overcome by the immobilization techniques ^{6, 7, 8}. Immobilized enzymes have been in use 1916, post the discovery of Nelson and Griffin that invertase, when absorbed to charcoal canhydrolyze the sucrose ⁹. The property and possibility of reuse and stability of the immobilized enzyme were identified by Grubhofer and Schelth, who reported the covalent immobilization of several enzymes¹⁰. The repeated assay with the immobilizedenzyme can be done, which in turn brings down the cost of the assay. The process of reuse of enzyme is very simple and can be attained through a technique like ultrafiltration. Enzyme immobilization is classified as Irreversible enzyme immobilization and Reversible enzyme immobilization methods.Irreversible enzyme immobilization methods are covalent binding and Entrapment. Reversible enzyme immobilization is adsorption, ionic binding, affinity binding and metal binding.

Enzymes are attached or fixedto a specific support material to make it immobilized (fig. 1). Support materials for immobilization are mostly inert polymers or inorganic compounds. An ideal support material should possess following properties: it should be affordable and must be inert, physically strong and regeneratable. In the present study, graduates have gathered the information from the available resources and compiled the article so that it can be available to them in published form. Although the subject is explored one, the intention for the publication of the present work is to seed the interest towards the publication¹¹.

Fig. 1: Enzyme Immobilization

TYPES OF IMMOBILIZATION

1. Support binding

In this method, a support/carrier materialis used to bound the enzyme. This is done by covalent, physical or ionic interactions. As it is difficult to keep the enzyme fixed to the carrier under industrial conditions of high reactant and product concentrations and high ionic strength that is why if these two are bound by physical interactions like van der Waals and hydrophobic interactions. Though ionic interaction is usually stronger than physical binding, covalent binding is the strongest. Covalent binding has an advantage that it prevents the leakage of the enzyme from the surface of support material¹².

2. Entrapment of enzyme by the inclusion

In this method, entrapment of enzyme in a gel lattice (polymer network) such as an organic polymer or sol-gelis done. Though the physical barriers are not too strong to prevent enzyme leakage entirely, covalent binding is also done in addition to entrapment¹³. Entrapment involves the synthesis of the polymeric meshwork catalyzed by the enzyme, whereas in support binding, the enzymeis attachedtothe prefabricated support.

3. Cross-linking of enzyme aggregates or crystals

Cross-linking is a relatively newer and advanced method of immobilizing an enzyme.In this method, the enzymeis immobilized free of any carrier material only bycross-linking enzyme crystals (CLECs), and cross-linking enzyme aggregates (CLEAs)¹⁴.The advantages of this method are increased activity of the enzyme, high stability,and the cost of production and processing is lowered as no carrier is required.

TECHNIQUES OF IMMOBILIZATION

1. Covalent Binding

Covalent binding is the most convenientway forimmobilization of enzyme. It is done by direct attachment of enzyme and the material through the covalent linkage¹⁵ .The covalent linkage is the strongest and most stable. The support material of enzymes includes polyacrylamide, porous glass, agarose and porous silica. This covalent binding includes two main steps, first- the activation of the support material bythe addition of the reactive compound and the second one is the modification of the polymer backbone to activate the matrix¹⁶. (fig. 2(a))

2. Adsorption

Enzyme adsorption is done by hydrophobic bonding or salt linkages between the enzyme and the support materials¹⁷. The support is dipped in enzyme solution to let it physically adsorbed or by drying it on the surface of an electrode to protect it against the aggregate formation, denaturation and hydrophobic interactions¹⁸.Coconut Fibre can be used as a support for this purpose. Production costs can be lowered by using environmental friendly support materials for immobilizing enzymes. For this purpose, biocompatible support material such as mesoporous silica nanoparticles (MSNs) can be used for immobilization results in long-term durability and efficiency of enzyme¹⁹.(fig. 2(b))

3. Entrapment

Enzymes are boundedbya synthetic or natural polymeric network which are a permeable membrane that permits the substrates and the products to pass, but it retains the enzyme inside the network²⁰. The entrapment can be done by the gel, fiber entrapping and microencapsulation).The advantage of this method is that it is fast, cheap and mild conditions required for reaction process. The support matrix protects the enzymes from microbial contamination. This method provides a greater surface area for reaction²¹. (fig. 2(c))

4. Affinity immobilization

Affinity immobilization can be done by two methods: firstly, by coupling the template to a ligand to which it has an affinity for or the enzyme to a substance that develops an affinity for the template. In affinity immobilization purification of enzymes is also done. Stable chitosan- coated porous silica beads and agarose-linked multi-layered concanavalin can be used asthe supportthey can hold larger amounts of enzymes and hence increase stability and efficiency ²².(fig. 2(d))

5. Ionic Bonding

The bonding between the enzyme and the support material is doneby salt linkages²³. The nature of this noncovalent immobilization the process will be reversed by changing the temperature polarity and ionic strength conditions. This principle is similar to protein-ligand interactions principles used in chromatography²⁴.(fig. 2(e))

6. Metal Linked immobilization:

Metal salts are precipitated over the surface of the carriers,and it is bound with the nucleophilic groups on thematrix²⁵. The precipitation can be achieved by heating. This method is relativelysimple,and the activity is high. The carrier and the enzyme are separated by decreasing the pH,and this makes it a reversibleprocess²⁶.(fig. 2(f))

DIFFERENT SUPPORTS MATERIALS USED FOR ENZYME IMMOBILIZATION

Enzymes and its supportive material both are essential for the preparation of supported enzymes properties,andthe immobilized enzyme can be prepared by these properties²⁷.Immobilized enzymes have their own specific biochemical, chemical, Kinetic and mechanical properties. There are different types of polymeric materials are used for this purpose such as synthetic organic polymers, biopolymers, smart polymers and some kinds of other supports such as hydrogels and inorganic supports²⁸.

1. Synthetic organic support materials

One of the types of acrylic resins such as Eupergit C is used as a support materialas it does not shrink and swell under extreme conditions and is porous The components posses N,N-methylene-bi (methacrylamide), glycidyl methacrylate, allylglycidyl ether,and methacrylamide‖. It shows covalent interaction with the amino group of the enzyme through its oxirane component

2. Biopolymers

Biopolymers are polysaccharides that includecellulose, starch, chitosan agarose and some natural protein such as albumin; gelatinis mainly used as supportive material for enzyme immobilization²⁹. Cellulose modification is done by DEAE-Sephadex with diethylaminoethyl functionalities. This modification is carried out in a continuous process in fixed bed reactor,and the process has a wide range ofapplication³⁰.

3. Hydrogels

The hydrogel is a material by which enzyme can be immobilized in non-aqueous media. Hydrogels and cryogelsare used for this purpose which can be natural and synthetic polyvinyl alcohol (PVA) cryogels³¹. PVA can be prepared through a freeze-drying method onanother handa good and mechanically stable quality of PVA can be prepared by partial drying of afforded lens-shaped hydrogels and utilized in immobilization.

4. Inorganic supports

Silica granulation is most cost-friendly processes to immobilize the enzyme. This method is employed for washing purpose because of its strong detergent effects during washing³². Mesoporous silica‘s that also known as nanosilica are also used as support material.

The advantage of this support material is that this supporthas larger pore size so that they can uptake smaller size enzymes into them easily³³.

5. Smart polymers

Smart polymers are polymers that are stimulus responsive,and this makes them different from other supports. Smart polymer helps in enzyme immobilization through the technique of covalent attachment. Smart polymers are very responsive to any changes in the environment and react by changing their confirmation³⁴.

APPLICATION OF THE IMMOBILIZED ENZYMES:

1. Biomedical Application

Immobilized enzymes have been used in medicine for long. Immobilized enzymes are utilizedin diagnosis

and treatment of various diseases.The enzyme encapsulation done through the electroporation is a convenient way of immobilization in the biomedical field.The biomaterials are used in tissue engineering also.In biomedical application, in the cancer therapy enzyme is delivered to theoncogenic sites can be improved with new methods.

2. Use in Antibiotic production

For antibiotic production, scientists are constantly searching for a mostfine-tuned process for antibiotic production such as Beta-lactam. Beta lactumacrylase are the enzymes used for the hydrolysis of Penicillin G and cephalosporins C. Penicillin G . Acrylate is an immobilized enzyme used in aqueous medium,and mutatedacrylatesare used in fermentation and semi-syntheticbeta-lactam antibiotics synthesis.

Other than this bio-medical and pharmaceutical application, immobilized enzymes are also used as a biosensor, in cosmetic and dyeing industries, in wastewater treatment^35,36,37.

CONCLUSION

Immobilized enzymes can be producedrelatively easily,and they are highly specific in their activity and also are environmentally friendly. They are widely used in different areasof science. Immobilized enzymes have their very own specific properties- biochemical, chemical, Kinetic and mechanical.Immobilization of enzymes can be done by the methods such as entrapmentadsorption, covalent bonding, crosslinking of enzyme crystals, ionic binding, affinity binding,etc and different polymeric materials are used for this purpose such as synthetic organic polymers, biopolymers, smart polymers, hydrogels and inorganic supports.Immobilized enzymes are used in biomedicine, antibiotic production, food industry, biodiesel production, bioremediation, the cosmetics industry and many others fields as is cheaper and reusable technology. In all, it was good to see that the students were propelled towards the said target and they have now know the basics of the publication process. Henceforth, this article will prove to be a milestone in their future research carrier

ACKNOWLEDGMENT:

The author wants to show a sincere gratitude to the Rungta College of Pharmaceutical Sciences and Research for providing necessary facilities for the completion of work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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