The concept of immobilized enzymes first appeared in the second half of the 20th century. Meanwhile, back in 1916, it was found that sucrose sorbed on carbon retained its catalytic activity. In 1953, D. Schleit and N. Grubhofer carried out the first binding of pepsin, amylase, carboxypeptidase and RNase with an insoluble carrier. The concept of immobilized enzymes was legalized in 1971. This happened at the first conference on engineering enzymology. At present, the concept of immobilized enzymes is considered in a broader sense than it was at the end of the 20th century. Let's take a closer look at this category.
General information
Immobilized enzymes are compounds that are artificially bound to an insoluble carrier. However, they retain their catalytic properties. Currently, this process is considered in two aspects - within the framework of partial and complete limitation of the freedom of movement of protein molecules.
Dignity
Scientists have established certain benefits of immobilized enzymes. Acting as heterogeneous catalysts, they can easily be separated from the reaction medium. As part of the research, it was found that the use of immobilized enzymes can be repeated. During the binding process, connections change their properties. They acquire substrate specificity and stability. At the same time, their activity begins to depend on environmental conditions. Immobilized enzymes are durable and have a high degree of stability. It is greater than, for example, that of free enzymes by thousands, tens of thousands of times. All this ensures high efficiency, competitiveness and economy of technologies in which immobilized enzymes are present.
Media
J. Poratu identified the key properties of ideal materials to be used in immobilization. Bearers must have:
- Insolubility.
- High biological and chemical resistance.
- The ability to quickly activate. The carriers should easily become reactive.
- Significant hydrophilicity.
- Necessary permeability. Its indicator should be equally acceptable for both enzymes and coenzymes, reaction products and substrates.
Currently there is no material that fully meets these requirements. Nevertheless, in practice, carriers are used that are suitable for immobilization.certain category of enzymes under specific conditions.
Classification
Depending on their nature, the materials, in connection with which compounds are converted into immobilized enzymes, are divided into inorganic and organic. The binding of many compounds is carried out with polymeric carriers. These organic materials are divided into 2 classes: synthetic and natural. In each of them, in turn, groups are distinguished depending on the structure. Inorganic carriers are mainly represented by materials made of glass, ceramics, clay, silica gel, and graphite black. When working with materials, dry chemistry methods are popular. Immobilized enzymes are obtained by coating carriers with a film of titanium, aluminum, zirconium, hafnium oxides or by processing with organic polymers. An important advantage of materials is the ease of regeneration.
Protein carriers
The most popular are lipid, polysaccharide and protein materials. Among the latter, it is worth highlighting structural polymers. These primarily include collagen, fibrin, keratin, and gelatin. Such proteins are widely distributed in the natural environment. They are affordable and economical. In addition, they have a large number of functional groups for binding. Proteins are biodegradable. This allows expanding the use of immobilized enzymes in medicine. Meanwhile, proteins also have negative properties. The disadvantages of using immobilized enzymes on protein carriers are the high immunogenicity of the latter, as well asthe ability to introduce only certain groups of them into reactions.
Polysaccharides, aminosaccharides
Of these materials, chitin, dextran, cellulose, agarose and their derivatives are most often used. To make polysaccharides more resistant to reactions, their linear chains are cross-linked with epichlorohydrin. Various ionogenic groups are freely introduced into the network structures. Chitin accumulates in large quantities as waste during the industrial processing of shrimp and crabs. This substance is chemical resistant and has a well-defined porous structure.
Synthetic polymers
This group of materials is very diverse and accessible. It includes polymers based on acrylic acid, styrene, polyvinyl alcohol, polyurethane and polyamide polymers. Most of them are mechanically strong. In the process of transformation, they provide the possibility of varying the pore size within a fairly wide range, introducing various functional groups.
Binding Methods
Currently, there are two fundamentally different options for immobilization. The first is to obtain compounds without covalent bonds with the carrier. This method is physical. Another option involves the emergence of a covalent bond with the material. This is a chemical method.
Adsorption
With the help of it, immobilized enzymes are obtained by holding the drug on the surface of the carrier due todispersion, hydrophobic, electrostatic interactions and hydrogen bonds. Adsorption was the first way to limit the mobility of elements. However, even now this option has not lost its relevance. Moreover, adsorption is considered the most common immobilization method in the industry.
Features of the method
Scientific publications describe more than 70 enzymes obtained by the adsorption method. The carriers were mainly porous glass, various clays, polysaccharides, aluminum oxides, synthetic polymers, titanium and other metals. The latter are the most commonly used. The effectiveness of adsorption of the drug on the carrier is determined by the porosity of the material and the specific surface area.
Mechanism of action
Enzyme adsorption on insoluble materials is simple. It is achieved by contact of an aqueous solution of the drug with the carrier. It can pass in a static or dynamic way. The enzyme solution is mixed with fresh sediment, for example, titanium hydroxide. The compound is then dried under mild conditions. Enzyme activity during such immobilization is retained by almost 100%. At the same time, the specific concentration reaches 64 mg per gram of carrier.
Negative moments
The disadvantages of adsorption include low strength when binding the enzyme and carrier. In the process of changing the reaction conditions, loss of elements, contamination of products, and protein desorption can be noted. To improve strengthbinding carriers are pre-modified. In particular, materials are treated with metal ions, polymers, hydrophobic compounds, and other polyfunctional agents. In some cases, the drug itself is modified. But quite often this leads to a decrease in its activity.
Inclusion in the gel
This option is quite common due to its uniqueness and simplicity. This method is suitable not only for individual elements, but also for multi-enzyme complexes. Incorporation into the gel can be done in two ways. In the first case, the drug is combined with an aqueous solution of the monomer, after which polymerization is performed. As a result, a spatial gel structure appears, containing enzyme molecules in the cells. In the second case, the drug is introduced into the solution of the finished polymer. It is then put into a gel state.
Intrusion into translucent structures
The essence of this method of immobilization is the separation of an aqueous enzyme solution from the substrate. For this, a semi-permeable membrane is used. It allows low molecular weight elements of cofactors and substrates to pass through and retains large molecules of enzymes.
Microencapsulation
There are several options for embedding in translucent structures. Of these, microencapsulation and incorporation of proteins into liposomes are of the greatest interest. The first option was proposed in 1964 by T. Chang. It consists in the fact that the enzyme solution is introduced into a closed capsule, the walls of which are made of semi-permeablepolymer. The appearance of a membrane on the surface is caused by the reaction of interfacial polycondensation of compounds. One of them is dissolved in the organic, and the other - in the aqueous phase. An example is the formation of a microcapsule obtained by polycondensation of sebacic acid halide (organic phase) and hexamethylenediamine-1, 6 (respectively, aqueous phase). The thickness of the membrane is calculated in hundredths of a micrometer. The size of the capsules is hundreds or tens of micrometers.
Incorporation into liposomes
This immobilization method is close to microencapsulation. Liposomes are presented in lamellar or spherical systems of lipid bilayers. This method was first used in 1970. To isolate liposomes from a lipid solution, the organic solvent is evaporated. The remaining thin film is dispersed in an aqueous solution in which the enzyme is present. During this process, self-assembly of lipid bilayer structures occurs. Such immobilized enzymes are quite popular in medicine. This is due to the fact that most of the molecules are localized in the lipid matrix of biological membranes. The immobilized enzymes included in liposomes are the most important research material in medicine, which makes it possible to study and describe the patterns of vital processes.
Formation of new bonds
Immobilization by forming new covalent chains between enzymes and carriers is considered the most widespread method for obtaining industrial biocatalysts.destination. Unlike physical methods, this option provides an irreversible and strong bond between the molecule and the material. Its formation is often accompanied by drug stabilization. At the same time, the location of the enzyme at a distance of the 1st covalent bond relative to the carrier creates certain difficulties in the implementation of the catalytic process. The molecule is separated from the material by means of an insert. It is often used as poly- and bifunctional agents. In particular, they are hydrazine, cyanogen bromide, glutaric dialhedride, sulfuryl chloride, etc. For example, to remove galactosyltransferase, the following sequence is inserted between the carrier and the enzyme -CH2-NH-(CH 2)5-CO-. In such a situation, an insert, a molecule, and a carrier are present in the structure. All of them are connected by covalent bonds. Of fundamental importance is the need to introduce into the reaction functional groups that are not essential for the catalytic function of the element. So, as a rule, glycoproteins are attached to the carrier not through the protein, but through the carbohydrate part. The result is more stable and active immobilized enzymes.
Cells
The methods described above are considered universal for all types of biocatalysts. These include, among other things, cells, subcellular structures, the immobilization of which has recently become widespread. This is due to the following. When cells are immobilized, there is no need to isolate and purify enzyme preparations or introduce cofactors into reactions. As a result, it becomes possible tosystems that carry out multi-stage continuous processes.
Use of immobilized enzymes
In veterinary medicine, industry, and other economic sectors, drugs obtained by the above methods are quite popular. Approaches developed in practice provide a solution to the problems of targeted drug delivery in the body. Immobilized enzymes made it possible to obtain drugs of prolonged action with minimal allergenicity and toxicity. Currently, scientists are solving the problems associated with the bioconversion of mass and energy using microbiological approaches. Meanwhile, the technology of immobilized enzymes also makes a significant contribution to the work. Prospects for development seem to be quite broad. So, in the future, one of the key roles in the process of monitoring the state of the environment should belong to new types of analysis. In particular, we are talking about bioluminescent and enzyme immunoassay methods. Advanced approaches are of particular importance in the processing of lignocellulosic raw materials. Immobilized enzymes can be used as weak signal amplifiers. The active center may be under the influence of a carrier that is under ultrasound, mechanical stress, or subject to phytochemical transformations.
