The concept of immobilized enzymes first arose in the second half of the 20th century. Meanwhile, back in 1916 it was found that sucrose adsorbed on coal retained catalytic activity. In 1953, D. Schleit and N. Gruhofer carried out the first binding of pepsin, amylase, carboxypeptidase, and RNase to 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 consider this category in more detail.
General information
And mobilized enzymes are compounds that artificially bind 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 limited freedom of movement of protein molecules.
Advantages
Scientists have identified certain benefits of immobilized enzymes . Acting as heterogeneous catalysts, they can easily be separated from the reaction medium. In the framework of studies, it was found that the use of immobilized enzymes can be repeated. In the process of binding, compounds change their properties. They acquire substrate specificity, stability. Moreover, their activity begins to depend on environmental conditions. Immobilized enzymes are characterized by durability and a high degree of stability. It is more than, for example, free enzymes thousands, tens of thousands of times. All this provides high efficiency, competitiveness and cost-effectiveness of technologies in which immobilized enzymes are present.
Carriers
J. Poratu identified the key properties of ideal materials that should be used in immobilization. Carriers must have:
- Insolubility.
- High biological and chemical resistance.
- The ability to quickly activate. Carriers should readily transform into reactive form.
- Significant hydrophilicity.
- Permeability required. Its indicator should be equally acceptable for enzymes, and for coenzymes, reaction products and substrates.
Currently, there is no material that fully complies with these requirements. Nevertheless, in practice, carriers are used that are suitable for immobilization of a certain category of enzymes in specific conditions.
Classification
Depending on their nature, the materials with which the compounds are converted into immobilized enzymes are separated into inorganic and organic. The binding of many compounds is carried out with polymer 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 from glass, ceramics, clay, silica gel, graphite soot. When working with materials, methods of dry chemistry are popular. Immobilized enzymes are obtained by coating the supports with a film of titanium, aluminum, zirconium, hafnium oxides or by treatment 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, structural polymers are worth highlighting. These primarily include collagen, fibrin, keratin, and gelatin. Such proteins are quite widespread 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 you to expand the use of immobilized enzymes in medicine . Meanwhile, proteins have negative properties. The disadvantages of using immobilized enzymes on protein carriers are the high immunogenicity of the latter, as well as the ability to introduce only certain groups of them into the reaction.
Polysaccharides, Aminosaccharides
Of these materials, chitin, dextran, cellulose, agarose and their derivatives are most often used. To make the polysaccharides more resistant to reactions, their linear chains are cross-linked with epichlorohydrin. Different ionogenic groups are introduced into the mesh structures quite freely. Chitin in large quantities accumulates as waste in the industrial processing of shrimp and crab. This substance is chemically resistant and has a well-defined porous structure.
Synthetic polymers
This group of materials is very diverse and affordable. It includes polymers based on acrylic acid, styrene, polyvinyl alcohol, polyurethane and polyamide polymers. Most of them differ in mechanical strength. In the process of conversion, they provide the possibility of varying the pore size over a fairly wide range, introducing a variety of functional groups.
Binding Methods
Currently, there are two fundamentally different from each other immobilization options. 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 it, immobilized enzymes are obtained by holding the drug on the surface of the carrier due to dispersion, hydrophobic, electrostatic interactions and hydrogen bonds. Adsorption was the first way to limit the mobility of elements. However, at present, this option has not lost its relevance. Moreover, adsorption is considered the most common immobilization method in industry.
Features of the method
Scientific publications describe more than 70 enzymes obtained by the adsorption method. The carriers mainly consisted of porous glass, various clays, polysaccharides, aluminum oxides, synthetic polymers, titanium and other metals. Moreover, the latter are used most often. The effectiveness of the adsorption of the drug on the carrier is determined by the porosity of the material and specific surface area.
Mechanism of action
The adsorption of enzymes on insoluble materials is simple. It is achieved by contact of an aqueous solution of the drug with the carrier. It can take place in a static or dynamic way. The enzyme solution is mixed with a fresh precipitate, for example, titanium hydroxide. Then, under mild conditions, the compound is dried. The activity of the enzyme with such immobilization is maintained at almost 100%. In this case, the specific concentration reaches 64 mg per gram of carrier.
Negative moments
The disadvantages of adsorption include low strength upon binding of the enzyme and the carrier. In the process of changing reaction conditions, loss of elements, contamination of products, desorption of protein can be noted. To increase the bonding strength, the carriers are pre-modified. In particular, the materials are treated with metal ions, polymers, hydrophobic compounds and other multifunctional agents. In some cases, the drug itself is modified. But quite often this leads to a decrease in its activity.
Gel inclusion
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. Inclusion in the gel can be performed by two methods. In the first case, the drug is combined with an aqueous solution of monomer, after which polymerization is performed. As a result, a spatial structure of the gel appears, which contains enzyme molecules in the cells. In the second case, the drug is introduced into the solution of the finished polymer. Then it is transferred to a gel state.
Intrusion into translucent structures
The essence of this method of immobilization is to separate the aqueous enzyme solution from the substrate. A semipermeable membrane is used for this. It passes low molecular weight elements of cofactors and substrates and retains large molecules of enzymes.
Microencapsulation
There are several options for implementation 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 lies in the fact that the enzyme solution is introduced into a closed capsule, the walls of which are made of a semipermeable polymer. The appearance of a membrane on the surface is determined by the reaction of interfacial polycondensation of compounds. One of them is dissolved in the organic, and the other in the aqueous phase. As an example, we can mention the formation of a microcapsule obtained by polycondensation of sebacic acid halide to-you (organic phase) and hexamethylene diamine-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.
Inclusion in liposomes
This immobilization method is close to microencapsulation. Liposomes are presented in lamellar or spherical systems of lipid bilayers. This method was first applied in 1970. Evaporation of the organic solvent is carried out to isolate liposomes from the lipid solution. 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 in medicine are quite popular. This is due to the fact that most of the molecules are localized in the lipid matrix of biological membranes. Immobilized enzymes included in liposomes in medicine are the most important research material that allows us to study and describe the laws of vital processes.
The formation of new ties
Immobilization through the formation of new covalent chains between enzymes and carriers is considered the most widespread method for producing industrial biocatalysts. Unlike physical methods, this option provides an irreversible and strong bond between the molecule and the material. Her education is often accompanied by stabilization of the drug. However, the location of the enzyme at the distance of the 1st covalent bond relative to the carrier creates certain difficulties in the performance of the catalytic process. The molecule is separated from the material using the insert. As it is often poly and bifunctional agents. They are, in particular, hydrazine, bromine cyan, glutaral dialhydride, sulfuryl chloride, etc. For example, to remove galactosyl transferase between the carrier and the enzyme, the following sequence —CH 2 —NH— (CH 2 ) 5 —CO— is inserted. 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 functional groups in the reaction that are not essential for the catalytic function of the element. So, as a rule, glycoproteins attach 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 others, 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, introduce cofactors in the reaction. As a result, it becomes possible to obtain systems that carry out multi-stage continuous processes.
The use of immobilized enzymes
In veterinary medicine , industry, and other economic sectors, preparations obtained by the above methods are quite popular. The approaches developed in practice provide a solution to the problems of implementing targeted drug delivery in the body. Immobilized enzymes made it possible to obtain drugs with 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 makes a significant contribution to the work. Prospects for development are represented by scientists as quite broad. So, in the future, one of the key roles in the process of environmental control should belong to new types of analysis. In particular, we are talking about bioluminescent and enzyme immunoassay methods. Of particular importance are advanced approaches in the processing of lignocellulosic raw materials. Immobilized enzymes can be used as amplifiers for weak signals. The active center may be under the influence of a carrier under ultrasound, mechanical stress, or prone to phytochemical transformations.