BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part II. Special Biotechnologies

Chapter 7. BIOTECHNOLOGY OF THE PRODUCTION AND APPLICATION OF IMMOBILIZED PREPARATIONS

7.5. METHODS OF ENZYME IMMOBILIZATION

Existing Methods of Enzyme Immobilization are divided into two groups: Physical and Chemical methods.

7.5.1. Physical methods of immobilization

7.5.1.1. Enzyme immobilization by adsorption on insoluble carriers

Adsorption immobilization is the oldest of all existing methods. For instance, as early as 1916, J. Nelson and E. Griffin successfully immobilized invertase via adsorption on activated charcoal and aluminum hydroxide gel.

Enzyme immobilization by adsorption on insoluble carriers involves bringing an aqueous enzyme solution into contact with carriers of organic and inorganic origin. The retention of the adsorbed enzyme molecule on the carrier surface is ensured by non-specific Structure/103.html">Van der Waals interactions, Electrostatic Interactions, Hydrogen Bonds, and hydrophobic interactions between the carrier and the surface groups of the enzyme protein. The type of bond depends on The Nature of the carrier and the functional groups On the surface of the enzyme molecule.

Methods of adsorption immobilization. In practice, the following methodological approaches are used to obtain Enzymes immobilized by adsorption.

Static method (Fig. 7.3, a) is the simplest and consists in adding the carrier to an aqueous enzyme solution and leaving the resulting mixture for a certain time without stirring. Immobilization is achieved through random diffusion of the enzyme to the carrier surface followed by adsorption. A disadvantage of this method is that obtaining a preparation with a high content of the adsorbed enzyme requires a significant amount of time.

Method with stirring, or dynamic method. In this approach, the carrier is suspended in the enzyme solution, and the resulting mixture is continuously stirred using a magnetic or mechanical stirrer, or on a laboratory shaker (Fig. 7.3, b). This method is more efficient than the previous one.

Separation of the immobilized enzyme is carried out by filtration or centrifugation. After washing away the unadsorbed enzyme, the preparation is ready for use.

Electrodeposition method. In this case, two electrodes, with a layer of carrier applied to The surface of one of them, are immersed in the enzyme solution. When the electric current is turned on, the enzyme molecules, due to the charged groups on their surface, begin to move in the solution and deposit on the carrier surface (Fig. 7.3, c).

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Fig. 7.3. Methods of enzyme adsorption immobilization

(according to I. V. Berezin et al., 1987)

Column coating method. This is the most convenient for the technological application of the immobilized enzyme. An enzyme solution is pumped through a column filled with the carrier at a flow rate such that the particles remain suspended, forming a "fluidized bed". Washing is also performed directly in the column (Fig. 7.3, d, e).

The adsorption process and the strength of the enzyme-carrier bond largely depend on the conditions under which immobilization is carried out. The main factors influencing enzyme adsorption are the specific surface area and porosity of the carrier, the pH value and Ionic strength of the enzyme solution, its concentration, and the Temperature of the adsorption process.

Specific surface area and porosity of the carrier. The sorption capacity of the carrier is proportional to its specific surface area when the carrier is non-porous or when the pore diameter is approximately twice the size of the protein molecule. If the pores are too small, they cannot accommodate the enzyme molecule, and the sorption capacity of the carrier will be negligible. At the same time, it is considered that the Molecular dimensions of the substrate are much smaller than those of the enzyme, allowing the substrate molecule to penetrate into the pore where the adsorbed enzyme is located. When the substrate is a substance with a very high molecular weight, the choice of the carrier pore size is dictated by the dimensions of the substrate molecule itself. Moreover, the high-molecular-weight substrate can serve as a carrier for enzyme immobilization itself. For example, the substrate of the cellulase complex—Cellulose—was successfully used as a carrier for the adsorption immobilization of its enzymes.

pH value. The reaction of the medium strongly influences the efficiency of enzyme sorption on the carrier surface, especially if sorption occurs mainly through electrostatic interactions. This is because changes in pH alter the ionization state of the ionogenic groups of the carrier and the protein responsible for adsorption. When using non-ion-exchanger carriers, maximum adsorption is achieved at the isoelectric point of the protein.

Ionic strength. This value affects the strength of the enzyme-carrier bond. At high salt concentrations, ions present in the solution displace the enzyme protein molecules bound via

electrostatic interactions from the carrier surface. In other words, an increase in the ionic strength of the solution generally induces desorption of the enzyme.

Enzyme Concentration. As the enzyme concentration in the solution increases, The amount of enzyme adsorbed on the carrier increases, and the specific catalytic activity of the immobilized preparation grows accordingly. However, this pattern holds only up to a certain limit. A further increase in enzyme concentration does not lead to an increase in enzyme sorption on the carrier, meaning that carrier "saturation" occurs.

Temperature. Temperature elevation affects the adsorption process in different ways. On the one hand, strong heating leads to Protein Denaturation and loss of catalytic activity. On the other hand, rising temperatures typically accelerate the process. Consequently, there is an optimal temperature for adsorption immobilization, the exact value of which depends on the nature of both the enzyme and the carrier.

Thus, the efficiency of adsorption enzyme immobilization is determined by a delicate balance of numerous factors. Disrupting this balance due to changes in any of the external parameters can lead to a sharp weakening of the enzyme-support interaction and, consequently, to its desorption.

Sorption efficiency can be enhanced by using pre-modified Supports and enzymes.

Immobilization on pre-modified supports. Preliminary Modification of the support in many cases significantly increases the binding strength of the enzyme to the matrix.

Modifying agents for supports include hydrophobic substances, solutions of metal-complexing ions, as well as substances with a high density of functional groups capable of electrostatic interaction with the protein globule.

1. Treatment of supports with Metal Ions (Ti, Sn, Zn, V, Fe) increases the strength of enzyme binding to the support through The formation of a protein-metal ion complex. The metal ion acts as a bridge connecting the enzyme molecule to the support (Fig. 7.4, a). This method is effective for immobilizing various enzymes on supports such as cellulose, nylon, Glass, filter paper, etc.

Fig. 7.4. Adsorption immobilization of enzymes on pre-modified supports

(according to Berezin I.V. et al., 1987)

2. Modification with hydrophobic compounds also helps to increase sorption efficiency, driven by hydrophobic interactions between the modifier and non-polar patches on the protein globule surface (Fig. 7.4, c). Among the supports, various argaroses covalently modified with hydrophobic groups (alkyl, phenyl, etc.) are most commonly used, along with polysaccharide supports modified with tannin, etc. A charged group may also be present at the end of such a hydrophobic 'spacer arm', ensuring that interaction with the enzyme occurs simultaneously through both electrostatic and hydrophobic forces.

3. Treatment of the support with substances whose molecules contain numerous functional groups capable of interacting with groups on the surface of the protein globule via electrostatic forces and hydrogen bonds (Fig. 7.4, b). For instance, surface polymerization of silochrome with acrylic acid, vinyl acetate, etc., followed by chemical modification of the polymer, results in a support with a high surface concentration of functional groups (hydroxyl, aminoalkyl, aminoaryl, and hydrazide) capable of electrostatic interaction with the protein globule.

Albumin is also frequently used as a modifier; it is adsorbed onto the support and subsequently heat-denatured. The denatured albumin layer forms a 'soft' cushion on the support surface rich in functional groups, which can firmly bind enzyme molecules while providing them with a favorable microenvironment. As a result, albumin treatment often successfully increases sorption efficiency and improves the catalytic performance of the immobilized enzyme.

In addition to increasing sorption efficiency, support modification often improves the catalytic Properties of the immobilized enzyme by creating a favorable microenvironment for its molecules. Furthermore, sometimes without prior support modification, it is entirely impossible to preserve the catalytic activity of the enzyme during adsorption immobilization. For example, if an enzyme has low stability in an acidic pH environment, its adsorption on silica gel may result in a loss of catalytic activity because the surface of this support is acidic (~pH 4). To prevent Enzyme inactivation, the support must be pre-incubated in a buffer solution with a pH value matching the optimum pH for the enzyme.

A similar problem frequently arises during the adsorption immobilization of enzymes that require a metal ion in their Active Site for normal function.

During the immobilization of metalloenzymes, the metal ion may leach out of the active site and bind to the support surface, accompanied by a partial or complete loss of catalytic activity. This undesirable phenomenon can be prevented by treating the support with a solution containing the corresponding metal ions, thereby saturating the metal ion sorption sites on the support.

Enzyme modification similarly involves introducing ionogenic groups (polyacids, carboxymethylcellulose, succinic acid residues, etc.) or treating with hydrophobic substances. In some cases, to enhance the binding of the adsorbed enzyme to the support and prevent its washing off (desorption), treatment with a bifunctional cross-linking reagent is employed. In this case, the support surface becomes covered with a film of cross-linked enzyme molecules. Glutaraldehyde is the most commonly used cross-linking agent.

Advantages and disadvantages of the method. Enzyme immobilization via adsorption is widespread. The method is simple, accessible, inexpensive, and the supports used for adsorption are relatively low-cost. In most cases, enzymes immobilized by adsorption exhibit high catalytic activity and technological advantages.

One of the significant disadvantages of the method is enzyme desorption—its detachment from the support—which leads to the loss of expensive biocatalyst and contamination of the final product. This most frequently occurs at the moment of adding the substrate to the immobilized enzyme. Other drawbacks include a low yield of bound enzyme per unit mass of support, as well as its partial or complete inactivation.

Additionally, a drawback of the adsorption immobilization method is the inability to provide general recommendations that would allow the a priori correct Selection of a support and optimal process conditions for a specific enzyme. This task has to be solved anew each time using trial and error.