Lecture 10 - Immobilization of Enzyme PDF

Enzymology 15316 Immobilization of Enzyme Immobilization is an essential prerequisite for the technical application of enzymes. Enzymes as biocatalysts are present in reaction solutions in small quantities, and recovery is not economical; however, such losses cannot be...

Enzymology 15316 Immobilization of Enzyme Immobilization is an essential prerequisite for the technical application of enzymes. Enzymes as biocatalysts are present in reaction solutions in small quantities, and recovery is not economical; however, such losses cannot be ignored when dealing with expensive enzyme preparations. The aim of enzyme immobilization is to develop a robust biocatalyst able to work under nonnative and harsh operational conditions with an extended lifetime. Fixation of enzymes to solid supports (matrix) may solve this problem. It allows complete recovery and repeated utilization of the same enzyme preparation. Advantages of Enzyme Immobilization 1- The efficiency of the enzymatic reaction is increased. 2- The enzyme can be reused. 3- Reduce the cost of operation by reducing the cost of extraction and purification; and reducing the cost of purchasing new enzyme each time. 4- The reaction time is reduced. 5- The production operations are improved. 6- Fit for industrial and medical application. Disadvantages of Enzyme immobilization 1- Although this process is commercially effective, few enzymes have been immobilized and used on a commercial scale, and the reason for that lies mainly in the difficulty and complexity of the immobilization process. Often immobilization require sophisticated technology. 2- Enzyme activity is lost partly or completely as a result of fixation. 3- Some enzymes lose their stability after fixation. ▪ Modes (methods) of Enzyme Immobilization In 1916, adsorption of invertase onto charcoal was the first example of an immobilized enzyme; this was followed by an extensive investigation of this field. Among thousands of different enzymes are exist, only a few (approximately 15–20) are, in fact, involved in large-scale processes. This fact reflects the difficulties of immobilizing enzymes. The major components for an enzyme immobilization include (1) An enzyme, (2) Support matrix/material, and (3) Mode of attachment. Because each enzyme has its own special features, general rules for immobilization cannot be given. Various modes of immobilization are described, but no really perfect method exists; advantages and disadvantages of each have to be weighed. For example, gentle fixation to take care of the sensitivity of enzymes comes at the cost of stability of the fixation. On the other hand, strong fixation often impairs the enzyme activity. Dr Hassan Aboufarrag 1 Enzymology 15316 The free diffusion of the substrate to and product from the immobilized enzyme must be considered. Therefore, technical considerations such as compressibility of solid support must be taken into account. The stability of material such as resistance against oxidation and microbial attack) of the material must be considered. ▪ Classification of matrix (support materials) Based on the chemical composition, a support matric is generally of two types (Organic and Inorganic carriers). 1. Organic matrix: It subdivides into natural and synthetic polymers. Natural polymers: It shows favorable compatibility with proteins. Polysaccharides (Cellulose, dextran, agar, agarose, chitin, alginate etc.), proteins (Collagen, albumin) and carbon are the natural polymers. Synthetic polymers: It shows high chemical and mechanical stability. Polystyrene, polyacrylate, polyacrylamide, polyamides etc. are examples of synthetic polymers. 2. Inorganic matrix: It subdivides into Natural and Processed minerals. Natural minerals: E.g., celite, silica, charcoal etc. Processed materials: E.g., Porous glass, metals and metal oxides. 1. Adsorption The simplest method of immobilization the adsorption to solid supports and this is also used for purification. Various chromatographic methods, such as ion exchange, adsorption, hydrophobic, and affinity chromatography, are based on the binding of proteins to solid matrices under the appropriate conditions. If the conditions of binding are maintained instead of elution, the enzyme can be regarded as immobilized. Binding of proteins strongly depends on pH and ionic strength, with lower ionic strength favoring binding, and higher ionic strength favoring elution. Besides the advantage of noncovalent fixation, the enzyme can be quickly substituted by a fresh preparation, under elution conditions. However, the enzyme may be unintentionally released from the matrix, for example when the ionic strength or the pH changes owing to the reaction conditions. Reaction components and additives such as metal ions or sulfhydryl reagents can also bind to the ion-exchange material and modify its features. Binding of enzymes to affinity material is highly specific and, thus, more stable. Unspecific interactions do not counteract the binding. However, the affinity component interacts mostly with the active site of the enzyme so that it cannot act as an immobilized catalyst. Advantages 1- The diffusion of the substrate and product into and from immobilized enzyme matrix is large. 2- Easy to apply and does not need many or long steps to perform. Dr Hassan Aboufarrag 2 Enzymology 15316 3- Does not need any special reagents or chemical reactions to fix the enzyme on the carrier. 4- Relatively cheap compared to other methods. 5- Easy replacement and substitution of old enzyme with fresh one. 2. Entrapment Entrapment into a narrow network is a very gentle immobilization technique. This mean the enzyme is captured within a polymeric network. Network barriers retain the enzyme, its native structure is maintained, and no direct interaction with the matrix is required. The mesh width of the network must be narrow to prevent release of the enzyme, but large enough to allow diffusion of substrates and products. Therefore, this method is not applicable to enzymes with macromolecular substrates such as proteases or RNases. However, even diffusion of small compounds can be stuck within the network, forming a zone of substrate depletion and product accumulation around the enzyme. This effect causes an apparent reduction of the enzyme activity due to decreased substrate and product inhibition. Materials such as agarose, gelatin or polyacrylamide, and polysaccharides from algae (alginate, κ- carrageenan) are applied for entrapment. The formulation of the network requires heating of the material in the presence of the enzyme to form the gel and entrap the enzyme inside the network after solidification of the gel. This can be harmful for thermosensitive enzymes especially if polyacrylamide polymer is used because the temperature increases due to the polymerization reaction. Polymerization is performed directly in the concentrated enzyme solution under defined conditions. The solidified gel is minced into smaller particles or beads and filled in an enzyme reactor or a column to enlarge the accessible surfaces for the substrate and product exchange. The entrapment technique is also used for immobilization of cells as a source of enzyme. This has the advantage of not requiring elaborate enzyme purification, but the problems of diffusion limitation for the substrate and product are even more serious – unfavorable side reactions cannot be excluded. 3. Encapsulation This method is relatively similar to the entrapment method. However, the enzyme is entrapped in a capsule or a bag instead of polymer or network. The enzyme solution is enclosed in a membrane capsule (porous membrane that allow the diffusion of substrate and product). The inert shell (capsule) protects and shields the protein from the external environment. Therefore, this technique is particularly suited for the immobilization of sensitive enzymes and cells, when any interaction with the native enzyme structure should be avoided. A simple model for encapsulation is a dialysis bag containing the enzyme solution immersed in a substrate solution. The enzyme is big so it cannot leave the bag allowing reusing it again. Polyamide or nitrocellulose are commonly used as a material for the capsule; these substances are directly formed in the concentrated enzyme solution. The walls of the capsule must be permeable for substrates and products, but impermeable for the enzyme. Dr Hassan Aboufarrag 3 Enzymology 15316 There is the problem of diffusion limitation through the capsule wall, especially in case of dialysis bag. This is not a very efficient device, because of large diffusion distances for the substrate and product into and out of the bag, respectively. The relatively small surface of the bag involved in maintaining an effective substrate–product exchange is another problem. Microencapsulation (substances is coated by extremely small capsules) at least partly overcomes these problems. 4. Cross Linking This method does not require a support material. In this method, enzymes can be covalently bound with each other through the enzyme function groups. Therefore, enzyme molecules are aggregated to large particles, which can be easily collected by centrifugation or filtration. The aggregation method is performed in concentrated enzyme solutions. However, if concentrated solutions of the enzyme are not available, particles can also be formed by co-immobilization with inert proteins, such as BSA, which often have a stabilizing effect. In addition to BSA, a broad variety of bivalent reagents can be used as cross-linking reagent. Because they are bivalent, these materials can cross-link several enzyme molecules with each other. The most common agents for cross linking, glutaraldehyde and dimethyl suberimidate. Cross-linking reagents differ both with respect to the reactive groups as well as the chain length. Most reagents are directed against NH3 groups and others react with COOH, SH, or OH groups of the protein. The chain length of the reagent determines the distance and flexibility of cross linking. Although, cross-linking is a simple and cheap method, it is not a frequently applied method. The covalent binding can influence the native structure and activity of the enzyme. 5. Covalent Immobilization to Solid Supports Covalent fixation of enzymes to solid supports is the most important immobilization method in which the enzyme is bound to matrix by covalent bonds between some chemical groups in the enzyme molecule and others in the matrix. The advantages: 1- Because the covalent bonds are strong, the immobilized enzyme preparation resist harsh condition, which cannot be completely avoided in technical processes. 2- The enzyme fixation is stable. So, there is no enzyme loss or leakage during the process. 3- It’s a simple and widely used method. 4- The process is not affected by the pH or ionic strength. Disadvantages: 1- The covalent binding can influence the native structure and activity of the enzyme. 2- Sometimes, the enzyme binds to the support materials through the function groups in its active site which may lead to the complete inhibition of the enzyme. To overcome this problem, the substrate is mixed with enzyme before the immobilization. The substrate will Dr Hassan Aboufarrag 4 Enzymology 15316 occupy the active site which guarantee that the enzyme will only bind through other function groups away from its active site. 3- The properties of the enzyme of interest should be well known. Poor knowledge of enzyme structure may lead to the lengthy, labor-intensive process and failure immobilization steps. Solid Support Used in Covalent Immobilization: The materials employed must possess the following features: 1- Should have chemical groups that are compatible to those in the enzyme 2- Contains chemical groups with high binding capacity. 3- Do not interfere in the enzymatic reaction and are not affected by the reaction products or reaction conditions. 4- Be stable under enzymatic reaction conditions. For laboratory use, more gentle materials are preferred, such as polymeric carbohydrates (agarose, dextrans, chitins, cellulose) and proteins (collagen, gelatin, albumin). These materials are not suitable for large-scale application because they are too sensitive, expensive, susceptible to microbial attack and do not resist harsh conditions of processing such as high pressure or fast flow rates. For large scale application, more stable materials are applied including silicon and aluminum oxide, glass, ceramics, and synthetic polymers such as polyamide, polystyrene, polyacrylate, polyacrylamide, polyester. They endure rough processing conditions such as high pressure. However, many enzymes cannot tolerate such supports and suffer considerable activity losses upon immobilization. Dr Hassan Aboufarrag 5