INTRODUCTION:

In order to make enzyme utilization in biotechnological processes more favorable, different methods for cost reduction have been put into practice and, immobilization is one of them. The word “immobilize” means to make anything unable to move at its own. The term ‘immobilized enzymes’ refers to ‘enzymes 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[1].

Immobilization provides a facile separation of the enzyme from the product[2] hence protein contamination of the product is minimized or avoided altogether. The principal components of an immobilized enzyme system are the enzyme, the matrix and the mode of attachment. The driving forces for enzyme immobilization are the improvement of enzyme stability, increment of volume specific enzyme loading and simplification of biocatalyst recycling and downstream processing[3].The sodium alginate is a versatile functional biomaterial for viscosity enhancement, stabilizer, matrixing agent, encapsulation polymer, bio adhesive and film former in transdermal and transmucosal drug delivery. In this review article, the various aspects of pharmaceutical microemulsion where compile together and the target audience are specifically the M. pharm and B .pharm student so that their knowledge towards the subject concern can be enhanced and also at the same time can be motivated towards the publication.

Criteria for enzyme immobilization

· When the reaction is a single step

· When co-enzyme is not involved

· When there is use of single enzyme

Advantages of immobilized enzymes:

· Stable and more efficient in function.

· Can be reused again and again.

· Products are enzyme-free.

· Ideal for multi-enzyme reaction systems.

· Control of enzyme function is easy.

· Suitable for industrial and medical use.

· Minimize effluent disposal problems.

· Immobilized enzymes are generally preferred over immobilized cells due to specificity to yield the products in pure form.

Table 1: Major products obtained using Immobilized Enzymes

S.NO	ENZYMES	PRODUCT
1	Glucose isomerase	High-fructose corn syrup
2	Amino acid acylase	Amino acid production
3	Penicillin acylase	Semi-synthetic penicillins
4	Nitrile hydratase	Acrylamide
5	B-Galactosidase	Hydrolysed lactose (whey)

Properties of Immobilized Enzyme

Some properties of the enzyme molecule, such as its catalytic activity or thermal stability, become altered with respect to those of its soluble counterpart [4,5]. This modification of the properties may be caused either by changes in the intrinsic activity of the immobilized enzyme or by the fact that the interaction between the immobilized enzyme and the substrate takes place in a microenvironment that is different from the bulk solution. One of the main problems associated with the use of immobilized enzymes is the loss of catalytic activity, especially when the enzymes are acting on macromolecular substrates. Because of the limited access of the substrate to the active site of the enzyme, the activity may be reduced to accessible surface groups of the substrate only.

Support or Matrix used in immobilization technology

The matrix or support immobilizes the enzyme by holding it permanently or temporaily for a brief period of time. There are wide variety of support or carrier available for immobilization..Both organic (mainly polysaccharides, polyacrylic and polyvinylic materials) and inorganic supports (mainly silica or other metal-oxide-based) have been described as efficient carriers for enzyme immobilization[6,7]. Several chemical functions can be inserted on the surface of the material (i.e., –NH2, alcoholic –OH, –COOH, –SH) capable of covalently reacting with enzymes under proper conditions. Typically, following functionalization, activation of supports with specific activating agents (such as organic and inorganic halides, glutaraldehyde, carbodiimides, various bifunctional Molecules 2014, 19 14141 agents) is necessary, to achieve enzyme immobilization.( functionalization with –NH2 groups and activation with cyanogen bromide is reported as an example in fig-2)

Figure 1 Classification of enzyme carriers

TECHNIQUES OF IMMOBILIZATION

Basically immobilization techniques can be divided into two general classes namely, the chemical and physical methods. Physical methods are characterized by weaker, monocovalent interactions such as hydrogen bonds, hydrophobic interactions, Vander Waals forces, affinity binding, ionic binding of the enzyme with the support material, or mechanical containment of enzyme within the support. In the chemical method, formation of covalent bonds achieved through ether, thio-ether, amide or carbamate bonds between the enzyme and support material are involved[8]. Based on support or matrix and the type of bonds involved, there are five different methods of immobilization of enzyme:

1. Adsorption

2. Encapsulation

3. Entrapment

4. Covalent bonding

5. Cross linking

Figure 2: Scheme of functionalization and activation of inorganic supports during covalent immobilization Support or Matrix used in immobilization technology

1. Adsorption (non- covalent interaction)

The physical adsorption method can be defined as one of the straightforward methods of reversible immobilization that involve the enzymes being physically adsorbed or attached onto the support material as shown in Fig 4. Enzyme immobilization through the technique of physical adsorption is quite simple and may have a higher commercial potential due to its simplicity, low cost and retaining high enzyme activity as well as a relatively chemical-free enzyme binding.

1.1 Non-specific adsorption

The simplest immobilization method is nonspecific adsorption, which is mainly based on physical adsorption or ionic binding [10]. In physical adsorption the enzymes are attached to the matrix through hydrogen bonding, van der Waals forces, or hydrophobic interactions.

1.2 Ionic binding

In ionic bonding the enzymes are bound through salt linkages. The nature of the forces involved in non covalent immobilization results in a process can be reversed by changing the conditions that influence the strength of the interaction (e.g., pH, ionic strength, temperature, or polarity of the solvent). The method is simple and reversible but, in general, it is difficult to find conditions under which the enzyme remains both strongly bound and fully active.

1.3 Hydrophobic Adsorption

In this method, it is not the formation of chemical bonds but rather an entropically driven interaction that takes place. Hydrophobic adsorption has been used as a chromatographic principle for more than three decades. The strength of interaction relies on both the hydrophobicity of the adsorbent and the protein. The hydrophobicity of the adsorbent can be regulated by the degree of substitution of the support and by the size of the hydrophobic ligand molecule[11].

Figure 3: Diagram representing Immobilization by Adsorption

1.4 Affinity binding

The principle of affinity between complementary biomolecules has been applied to enzyme immobilization. The remarkable selectivity of the interaction is a major benefit of the method.

Example: Adsorption of fungus on the wood chip The adsorbent on which the cells to be immobilized were kept in a cultivated medium to promote the growth/attachment of cells on the surface of adsorbent. The medium is incubated for 3 days at 30ºC. Finally the adsorbent is separated from the medium and dried by vacuum freeze desiccator[12].

Advantages of Adsorption

· It is Easy.

· It is Simple.

· It gives high yield.

Limitations of Adsorption

· Enzyme will leach with change in pH, or ionic strength.

· Substrate with same charge as polymer may not gain access to enzyme except at high concentration, which in turn causes loss of enzyme.

2. Encapsulation

Encapsulation is a type of entrapment. It refers to the process of spherical particle formation wherein a liquid or suspension is enclosed in a semi permeable membrane. The membrane may be polymeric, lipoidal, lipoprotein-based or non-ionic in nature. There are three distinct ways of encapsulation.

1. Building of special membrane reactors.

2. Formation of emulsions.

3. Stabilization of emulsions to form microcapsules.

Microencapsulation is recently being used for immobilization of enzymes and mammalian cells. For instance, pancreatic cells grown in cultures can be immobilized by microencapsulation. Hybridoma cells have also been immobilized successfully by this technique.

Encapsulation is the process of forming a continuous membrane around cells to be immobilized that denote the core of the system [13] in which the inner matrix is protected by means of the outer membrane as depicted in Fig 5. Liquid form of active substance is the core material and polymeric wall is the outer membrane [14]. Microencapsulation of cell can be done by three methods such as emulsification, interface technique and coacervation.

Figure 5: Immobilization by Encapsulation

2.1 Emulsification

Emulsification is the process of dispersing the cell-loaded polymeric solution into an immiscible liquid phase using a low pressure nozzle device to form an emulsion phase (droplet of polymeric solution in immiscible liquid).Finally the microencapsulated beads can be produced by cooling the emulsion to form a membrane[15].

2.2 Interfacial Phenomenon

The organic phase consists of an active substance (microbes) and an oil soluble monomer is dispersed into the surfactant to form the droplets. Water soluble shell monomer is added to this dispersion to initiate the reaction with the oil soluble shell monomer. As a result of reaction between the two monomers, the polymeric shell is formed at the interface of each droplet.

2.3 Coacervation:

Coacervation is the process of forming continuous polymeric membrane around the liquid core. The liquid core present inside the polymeric membrane is called as coacervate. There are two types of phase separation process such as simple and complex coacervation .

Advantages of encapsulation

· Surface area to volume ratio is high.

· Replacement of enzyme is easy.

· Highly viscosity substrate may be use.

Limitations of encapsulation

· Membrane bound enzyme can denature.

· This technique used for low molecular weight compound.

3. Entrapment:

Entrapment is defined as an irreversible method of enzyme immobilization where enzymes are entrapped in a support or inside of fibres, either the lattice structure of a material or in polymer membranes that allows the substrate and products to pass through but retains the enzyme. Entrapment can be achieved by means of addition of one or a combination of gelling or cross linking agent. The polymeric solution loaded with cells is extruded through needle into a hardening solution to form beads. It is considered as one of the easiest, simplest and safest methods of immobilization. The gelation of polyelectrolyte solution occurs in the presence of a multivalent ion of opposite charge[9]. Example: Cell suspension of genes sp. d2 is mixed with 4% sodium alginate solution in the ratio of 1:2. Then the mixture is extruded into 0.2 M CaCl2 solution using syringe or suitable bead forming device to form alginate beads[16].

Figure 6: Immobilization by Entrapment

3.1 Enzyme inclusion in gels:

This is an entrapment of enzymes inside the gels

3.2 Enzyme inclusion in fibres:

The enzymes are trapped in a fibre format of the matrix

3.3 Enzyme inclusion in microcapsules:

In this case, the enzymes are trapped inside a microcapsule matrix. The hydrophobic and hydrophilic forms of the matrix polymerise to form a microcapsule containing enzyme molecules inside. The major limitation for entrapment of enzymes is their leakage from the matrix. Most workers prefer to use the technique of entrapment for immobilization of whole cells. Entrapped cells are in use for industrial production of amino acids (L-isoleucine, L-aspartic acid), L-malic acid and hydroquinone.

Advantages of Entrapment

· It is simple.

· It provides variability of pore size for the immobilization of cells or enzyme.

· Mild conditions are used in the preparation of immobilized cells or enzymes.

Figure 7: Different methods of entrapment involves (A) enzyme inclusion in gels, (B) enzyme inclusion in fibres. (C) enzyme inclusion in microencapsules.

Limitations of Entrapment

· Viable cells bursts from gel material.

4. Covalent bonding

Covalent bonding is one of the most widely used methods for irreversible enzyme immobilization. Immobilization of the enzymes can be achieved by creation of covalent bonds between the chemical groups of enzymes and the chemical groups of the support. Covalent binding is often associated with loss of some enzyme activity. The inert support usually requires pretreatment (to form pre-activated support) before it binds to enzyme. The following are the common methods of covalent binding.

Figure 8: Immobilization by Covalent bonding

4.1 .Cyanogen bromide activation:

The inert support materials (cellulose, sepharose, sephadex) containing glycol groups are activated by CNBr, which then bind to enzymes and immobilize them.(fig 9, C)

4.2. Diazotation:

Some of the support materials (amino benzyl cellulose, amino derivatives of polystyrene, aminosilanized porous glass) are subjected to diazotation on treatment with NaNO₂and HCI [17]. They, in turn, bind covalently to tyrosyl or histidyl groups of enzymes .(fig 9, B)

Figure 9: Immobilization by covalent bonding (A) Cyanoden bromide activation (B) Diazotisation (C) Peptide bond formation (D) Activation by functional agents.

4.3. Peptide bond formation:

Enzyme immobilization can also be achieved by the formation of peptide bonds between the amino (or carboxyl) groups of the support and the carboxyl (or amino) groups of enzymes .The support material is first chemically treated to form active functional group.(fig 9, C).

4.4. Activation by bi- or poly-functional reagents:

Some of the reagents such as glutaraldehyde can be used to create bonds between amino groups of enzymes and amino groups of support (e.g. aminoethylcellulose, albumin, amino alkylated porous glass) (fig 9, D).

5 Cross-linking

Cross linking is another irreversible method of enzyme immobilization that does not require a support to prevent enzyme loss into the substrate solution[18]. The method is also called carrier-free immobilization [5] where the enzyme acts as its own carrier and virtually pure enzyme is obtained eliminating the advantages and disadvantages associated with carriers. Technically, cross-linking is performed by formation of intermolecular cross-linkages between the enzyme molecules by means of bi- or multifunctional reagents[19] as shown in Fig 10. The most commonly used cross-linking reagent is glutaraldehyde as it is economical and easily obtainable in large quantities[5].

Figure 10: Immobilization by Cross linking

APPLICATIONS

1) Immobilized Enzyme-aminoacylase used for the first time by the immobilization method for the production of L-amino acids.

2) In food industry, fructose syrup is produce from glucose by use of immobilized enzyme glucose-isomerase.

3) Immobilized enzyme used in biosensor.

4) Immobilized enzymes used in various analytical techniques where one can diagnose clinical problems.

5) Accurate analysis of sample done with the help of specific immobilized enzyme and sensitive chemical analytical techniques uses immobilized enzyme.

6) Immobilized Enzyme or Cells used in industry for the production of various industrial products.[20]

CONCLUSION:

In conclusion, the sodium alginate as biopolymer has wide range of applications in the food and pharmaceutical industry including under immobilization method. It has versatile pharmaceutical utility starting from thickening agent to polymeric backbone in sustained release dosage forms. Being biopolymer of high biological tolerability, it has a special role to play in the formulations of proteins or peptides and other biological products. Its capability of fabrication in all aqueous systems, cross-linking with variety of agents, miscibility with other polymers of biological or synthetic origin offers the most widely applicable polymeric systems avoiding harsh conditions. With this compilation we assure that the content of the article would be a useful tool to understand the in-depth knowledge of this subject concern.

Figure 11: An example showing Immobilized lactase converts lactose into glucose and galactose

ACKNOWLEDGEMENT:

Authors want to acknowledge the facilities provided by the Rungta College of Pharmaceutical Sciences and Research, Kohka, Kurud Road, Bhilai, Chhattisgarh, India. The authors are also grateful to the e-library of Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India, 490001 for providing UGC-INFLIBNET facility. The authors acknowledge Chhattisgarh Council of Science and Technology (CGCOST) for providing financial assistance under mini research project (MRP) vide letter no. 1124/CCOST/MRP/2015; Dated: September 4, 2015 and 1115/CCOST/MRP/2015; Dated: September 4, 2015.

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Received on 22.03.2017 Modified on 14.04.2017

Research J. Pharm. and Tech. 2017; 10(4): 1197-1203.

DOI: 10.5958/0974-360X.2017.00215.3