Enzymes are proteins that accelerate many biochemical and chemical reactions [1]. They are can be referred to as natural catalysts because they are biological macromolecules produced by living organisms (plants, animals and microorganisms) which act as catalysts to bring about specific biochemical reactions [2]. They can be classified as either simple or complex.  Simple enzymes are composed only of proteins while complex enzymes are composed of both proteins and a relatively small group of organic molecule known as the prosthetic group [3]. Enzymes have the ability to catalyse reactions under very mild conditions with a very high degree of substrate specificity, thereby decreasing the formation of by-products which reduce material costs and downstream environmental burdens [4]. They can catalyse reactions in different states: as individual molecules in solution, in aggregates with other entities and as attached to surfaces [5]. The use of enzymes in industrial applications has been limited because of relative instability and cost of recovery of most of them [6] These 2 critical  issues, contribute to    the need to immobilise them.

Enzyme immobilization is a technique where enzymes are physically confined or localized in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously [7].Immobilized enzyme is an enzyme whose movement in space has been restricted either completely or to a small region in a support or matrix [8]. The first immobilization technology was achieved with aminoacylases by Apergillus oryzae for the production of L-amino acids in Japan [4]. Immobilizing an enzyme allows for an increased resistance to variables such as temperature or pH, increased functional efficiency and enhanced reproducibility. Furthermore, it enables enzymes to be stationary throughout the process which in turn makes it much easier for them to be separated and reused. High-fructose corn syrup, amino acid production, semi-synthetic penicillins, acrylamide, hydrolyzed lactose (whey) (from enzyme glucose isomerase, amino acid acylase, penicillin acylase, nitrile hydratase and β-galactosidase respectively) are examples of products obtained using immobilized enzymes.

There are various advantages of using immobilised enzymes which include; the reusability of enzymes and easy separation of products, continuous operation of processes which can be readily controlled with less chance   of   contamination   in   products, minimization of effluent problems and materials handling and, in some cases, favourable alteration of enzyme properties (activity and stability) by immobilization amongst others. In as much as enzymes immobilization has several advantages they also have some limitations, which as are follows; the cost of enzymes carriers and the immobilization process are quite expensive, additional cost is required for isolation, purification and recovery of active enzymes, there could be changes in the properties of some enzymes thereby reducing its specificity/selectivity, which could possibly result in loss of enzymes activity loss [9]. In addition, some enzymes can become unstable and inactivated by heat generated in the bioreactor. 

Components of an Enzyme Immobilization

The major components of an enzyme immobilization include the enzyme, carrier and mode of interaction of enzyme with the carrier. The matrix or support immobilizes the enzyme by holding it permanently or temporarily for a brief period of time. The characteristics of an enzyme carrier include large surface area, permeability, hydrophilic character, insolubility, chemical, mechanical, and thermal stability, high rigidity, suitable shape and particle size, resistance to microbial attack, and regenerability (Singh, 2009). Carrier/ support could be organic or inorganic. Organic could be natural (e.g Polysaccharides-cellulose, dextran, chitin, chitosan, alginate; Proteins- collagen albumin; Carbon) or synthetic (polystyrene, polyamides, vinyl, polyacrylate etc. Inorganic could be natural materials (silica, ceramic, activated charcoal) or processed materials (glass, oxides) [10].

Methods of Immobilization

Immobilized enzymes are modified to a water-insoluble form by suitable techniques. Enzyme immobilization techniques can be categorized into reversible and irreversible methods. In the reversible method, the cell can  easily  be detached  from the  matrix/support while in the irreversible method the cells cannot be detached  from  the matrix/support  without  either destroying  the cell  or  the support  [11]. Examples of some reversible enzyme immobilization methods are physical attachment and ionic bonding while examples of irreversible methods include, entrapment methods, encapsulation, covalent bonding, cross linking etc. There are several factors to be considered when choosing immobilization techniques, they include:

• Enzyme’s tolerance to immobilization chemical and physical environment,

• Surface functional groups on the protein

• Size of the enzyme

• Charge of the protein 

• Polarity of the protein (hydrophobic/hydrophilic regions) 

• Substrate/product transport needs [1]

Physical Adsorption

This is the oldest and simplest method. It is used to immobilize enzyme by the attachment of enzyme on carrier surface via weak forces, such as van der Walls force, electrostatic force, hydrophobic interaction, and hydrogen bond [12] and it does not result in large loss of enzyme activity. The carrier may be organic or inorganic. Activated charcoal, alumina, cellulose, Sephadex, agarose, collagen and starch are examples of carriers used in adsorption [13] while catalase and invertase are enzymes that have been immobilized using physical adsorption.

This method of immobilization is simple, economical with limited loss of activity. It can be recycled, regenerated and reused, though it relatively has low surface area for binding, and could cause exposure of enzyme to microbial attack. Despite many merits of adsorption immobilization technique, it also presents some drawbacks, the immobilized enzyme has poor operation stability; significant enzyme loss cannot be avoided in this technique as the binding forces are weak [14], the amount of adsorbed enzyme is more susceptible to the immobilization parameters such as temperature, ionic strength, and pH; and enzyme can be stripped off easily from the carrier because of the weak forces between them [15].

Ionic Binding

This is a reversible method of immobilisation that involves ionic interaction between the enzyme and the support. The support used is generally charged, such that the protein to be bound has an opposite charge. It is easily reversed by altering the pH or ‘salting out’ of the enzyme [1].Ionic binding is easy and inexpensive, it can be reversed by easy manipulation of the acidity or alkalinity as the matrix is stably charged [14]. 

Entrapment

This involves the physical entrapment of enzyme in a porous matrix using covalent or non-covalent bond [14]. It involves the inclusion in gels, fibres or microcapsules. Moreover, accurate pore size selection of the support is crucial (the smaller the pores, lesser the enzyme entrapped, while larger the pores, more the leaking of the enzyme) [13]. Examples of water-soluble matrix used in entrapment include polyacrylamide gels, agar, gelatine, alginate, carrageenan. However, efficient encapsulation has been achieved with alginate–gelatin–calcium hybrid carriers that prevented enzyme leakage and provided increased mechanical stability [16]. The enzymes or cells are not directly attached to the support surface, but simply trapped inside the polymer matrix. Protein is retained while allowing penetration of substrate. It can be classified into Lattice and microcapsule types. Enzymes immobilized by entrapment are stable (less chances of confrontational changes) [17] but the enzyme may leak from the pore. And there could be chance of microbial contamination.

Encapsulation

Here, the enzyme is enclosed in the internal structure of polymer material (membrane capsule) made of semi permeable structure e.g., nitrocellulose [18]. This encapsulation immobilization preserves the mobility of the enzyme and allows to increase its activity [15]. This method is inexpensive and allows for large amounts of enzymes to be immobilized, it maintains the enzyme structure in its native form and protects enzymes from the harsh conditions of the medium [21]. The limitations of these methods include pores size limitation (only small substrate molecule is able to cross the membrane); enability small substrate molecules to diffuse in and the product molecules to diffuse out.

Table 1: Some Enzymes, Support, Immobilization Technique and Application

Covalent Binding

This involves the chemical binding between functional groups of the enzyme and support (formation of covalent bond between enzyme and support) e.g., bonding between amino or hydroxyl group of carriers and that of the enzyme [14]. Functional groups that may take part in this binding are amino group, carboxyl group, hydroxyl group, imidazole group, phenolic group, Thiol group etc. The binding force between enzyme and carrier is so strong that no leakage of the enzyme occurs, even in the presence of substrate or solution of high ionic strength [1]. The presence of different functional groups allows wide applicability of the immobilized enzymes. The covalent binding may alter the conformational structure and active centre of the enzyme resulting in major loss of activity and/or changes of the substrate [15].

Cross Linking

Cross linking involves intermolecular cross linking of enzyme molecules in the presence or absence of solid support. The method produces a 3-dimensional cross linked enzyme aggregate (insoluble in water) by means of a multifunctional reagent that links covalently to the enzyme molecules. There are two methods of cross linking in use

• Cross Linking Enzyme Aggregate (CLEA)

• Cross Linking Enzyme Crystals (CLEC) [14]. 

Cross linking agents include: glutaraldehyde, dextran polysaccharide, bis-isocyanate, bis-diazobenzidine, diazonium salts [22]. The CLEC or CLEA are added to the reaction mixture and can be later removed from the mixture during product purification. The cross-linking method has very little desorption (enzymes strongly bound), higher stability (i.e. pH, ionic and substrate concentration) hence it can be widely used in commercial preparations and industrial applications. Although cross linking may cause significant changes in the active site thereby resulting in the loss of activity moreover, it is time consuming and expensive [9,23].

Application of Immobilized Enzymes

Immobilised enzymes can be applied in the following ways:

• Brewing/beverage industry: the brewing industry has used immobilized enzymes to create high-quality beer. The use of immobilized yeast cells made it possible for the industry to transition from making traditional batches to brewing continual batches. 

• Biomedical applications: immobilized enzymes are widely used in the diagnosis and treatment of many diseases. They can be used to overcome inborn metabolic disorders. inborn disorder [18].

• Pharmaceuticals: Enzymes such as streptokinase, urokinase, asparaginase, deoxyribonuclease I, and hyaluronidase are very important to the pharmaceutical industry. Immobilized enzymes have been used to create life-saving medications such as penicillin. 

• Textile industry: immobilized enzymes can be used in textile industry-scouring, biopolishing and desizing of fabrics [14]. Cellulase is used for cotton softening, denim finishing, laccase (for bleaching), etc. Immobilization of lipase enzyme for effective dirt removal from cloths [18].

• Wastewater management: immobilized enzymes are useful in the treatment of sewage and industrial effluents. Bacterial strains were immobilized to enhance the biological oxidation of toxic pollutants in wastewater. Horseradish peroxide (HRP) has been immobilized to remove phenol from wastewater at high levels [24]

• Detergent industry: immobilised lipase has been used in the detergent industry for effective dirt removal from cloths using [18]. In addition, immobilised alkaline protease is commonly used in detergent, this allows the detergents to be more stable in higher temperatures, which makes for more efficient washing conditions

• Manufacturing industry: immobilised enzymes are used in the industrial production of antibiotics, beverages, amino acids, production  of High  Fructose  Corn Syrup (HFCS), they can also be used for the production of biotechnology products such as Glucose isomerase High-fructose corn syrup, Amino acid acylase Amino acid production, Penicillin acylase Semi-synthetic penicillin etc. [8].

• Food industry: pectinase and cellulase immobilized on suitable carriers are used for the production of jams, jelly syrup form fruits and vegetables [4,24]

It is reported recently that many have nano particles as support. According to  Ahmad et al. [23] these include Lysozyme(chitosan nanofibers), superoxide dismutase(NanoFeOcoated), Mucor lipase(nano sized magnetite), Thermoanaerobacter brockii alcohol dehydrogenase (gold and silver)

Enzymes are not easily isolated from the product because they are usually lost after first use. Immobilization of enzyme empowers the reusability of enzymes thereby prevent loss after first use. The reusability of these immobilised enzymes has helped in reducing the cost of enzyme and in turn product since the immobilised enzymes are reuseable (enzymes can be reused as many times as possible) and as a result the buying of more enzymes at expensive prices for production purpose will be eliminated. Moreover, immobilisation of enzymes will also ensure increased functional efficiency and enhanced reproducibility.

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