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Multiple-enzyme immobilization including co-factor regeneration and cell immobilization. Example: production of L-aminoacids from keto-acids in membrane reactors.

The major components of an immobilized enzyme system are the enzyme, the matrix, and the mode of attachment of the enzyme to the matrix. The terms solidphase support, carrier, and matrix are used synonymously.

3. Choice of Supports

The characteristics of the matrix are of paramount importance in determining the performance of the immobilized enzyme system. Ideal support properties include physical resistance to compression, hydrophilicity, inertness toward enzymes ease of derivatization, biocompatibility, resistance to microbial attack, and availability at low cost (12-14).

Supports can be classified as inorganic and organic according to their chemical composition (Table 4). The organic supports can be subdivided into natural and synthetic polymers (15).

The physical characteristics of the matrices (such as mean particle diameter, swelling behavior, mechanical strength, and compression behavior) will be of major importance for the performance of the immobilized systems and will determine the type of reactor used under technical conditions (i.e., stirred tank, fluid-ized, fixed beds). In particular, pore parameters and particle size determine the total surface area and thus critically affect the capacity for binding of enzymes. Nonporous supports show few diffusional limitations but have a low loading capacity. Therefore, porous supports are generally preferred because the high surface area allows for a higher enzyme loading and the immobilized enzyme receieves greater protection from the environment. Porous supports should have a controlled pore distribution in order to optimize capacity and flow properties. In spite of the many advantages of inorganic carriers (e.g., high stability against physical, chemical, and microbial degradation), most of the industrial applications are performed with organic matrices. The hydrophilic character is one of the most important factors determining the level of activity of an immobilized enzyme (16).

An excellent matrix that has been extensively used is agarose. In addition to its high porosity, which leads to a high capacity for proteins, some other advantages of using agarose as a matrix are hydrophilic character, ease of derivatization, ab-

Table 4

Classification of Supports

Organic

Natural polymers

• Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate

• Proteins: collagen, albumin

• Carbon Synthetic polymers

• Polystyrene

• Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides, vinyl, and allyl-polymers

Inorganic

Natural minerals: bentonite, silica

Processed materials: glass (nonporous and controlled pore), metals, controlled pore metal oxides sence of charged groups (which prevents nonspecific adsorption of substrate and products), and commercial availability. However, an important limitation in the use of agarose and other porous supports is the high cost. This problem can be circumvented by employing reversible methods that allow matrix regeneration and re-use.

The enzymes can be attached to the support via interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. One way of classifying the various approaches to immobilizing enzymes is in two broad categories: irreversible and reversible methods (17). The strength of the binding is usually inversely related to the ease with which it can be reversed. These two conflicting objectives—stability and reversibility—are difficult to fulfill simultaneously. The traditional approach has been to make the bond as strong as possible and sacrifice reversibility.

4. Methods of Irreversible Enzyme Immobilization

The concept of irreversible immobilization means that once the biocatalyst is attached to the support it cannot be detached without destroying either the biological activity of the enzyme or the support. The most common procedures of irreversible enzyme immobilization are covalent coupling, entrapment or micro-encapsulation, and cross-linking (see Fig. 1).

4.1. Formation of Covalent Bonds

Immobilization of proteins by methods based on the formation of covalent bonds are among the most widely used. An advantage of these methods is that, because of the stable nature of the bonds formed between enzyme and matrix, the enzyme is not released into the solution upon use. However, in order to achieve

Enzyme Immobilisatiobn
Fig. 1. Approaches to enzyme immobilization, irreversible methods.

high levels of bound activity, the amino acid residues essential for catalytic activity must not be involved in the covalent linkage to the support; this may prove a difficult requirement to fulfill in some cases. A simple procedure that sometimes improves the activity yield is to carry out the coupling reaction in the presence of substrate analogs (18). Covalent methods for immobilization are employed when there is a strict requirement for the absence of the enzyme in the product.

A wide variety of reactions have been developed depending on the functional groups available on the matrix (19). Coupling methods in general can be divided in two main classes: (1) activation of the matrix by addition of a reactive function to a polymer and (2) modification of the polymer backbone to produce an activated group (Table 5). The activation processes are generally designed to generate electrophilic groups on the support which, in the coupling step, react with the strong nucleophiles on the proteins. The basic principles controlling the course of covalent coupling to the matrices are analogous to those used for the chemical modification of proteins. The most frequently used reactions involve the following side chains of the amino acids: lysine (e-amino group), cysteine (thiol group), and aspartic and glutamic acids (carboxylic group).

Table 5A

Covalent Coupling Methods of Enzymes: Activation of Matrix Hydroxy! Functions

Table 5A

Covalent Coupling Methods of Enzymes: Activation of Matrix Hydroxy! Functions

Activation method

Group that reacts (with activated matrix)

References

Tresyl chloride,

Thiol, amines

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