Immobilized enzymes and their application

For the first time the concept of immobilized enzymes arose in the second half of the 20th century. Meanwhile, as early as 1916, it was found that sucrose sorbed on the carbon retained its catalytic activity. In 1953 D. Schleit and N. Grubhofer carried out the first bindings 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. рассматривается в более широком смысле, чем это было в конце 20 века. At the present time, the concept of immobilized enzymes is considered in a broader sense than it was at the end of the 20th century. Consider this category in more detail.

General information

– соединения, которые искусственно связываются с нерастворимым носителем. And immobilized enzymes are compounds that are artificially bound to an insoluble carrier. At the same time they retain their catalytic properties. At present, this process is considered in two aspects - within the framework of partial and complete limitation of freedom of movement of protein molecules.

Advantages

. Scientists have established certain advantages of immobilized enzymes . Acting as heterogeneous catalysts, they can easily separate from the reaction medium. может быть многократным. Within the framework of the research it was established that the use of immobilized enzymes can be repeated. During the binding process, the compounds change their properties. They acquire substrate specificity, stability. In this case, their activity begins to depend on environmental conditions. отличаются долговечностью и высокой степенью стабильности. Immobilized enzymes are distinguished by their durability and high degree of stability. It is more than, for example, free enzymes in thousands, tens of thousands of times. All this ensures high efficiency, competitiveness and economy of technologies in which immobilized enzymes are present.

Media

G. Poratu defined the key properties of ideal materials that should be used for immobilization. Carriers must possess:

1. Insolubility.
2. High biological and chemical resistance.
3. The ability to quickly activate. Carriers should easily transform into a reactive species.
4. Significant hydrophilicity.
5. Necessary permeability. Its index should be equally acceptable for enzymes, and for coenzymes, reaction products and substrates.

At present, there is no material that fully meets these requirements. Nevertheless, in practice, carriers are used that are suitable for immobilization of a certain category of enzymes in specific conditions.

Classification

, разделяются на неорганические и органические. Depending on their nature, the materials, in connection with which the 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 composed of glass, ceramic, clay, silica gel, graphite black. When working with materials, the methods of dry chemistry are popular. Immobilized enzymes are obtained by coating carriers with a film of titanium, aluminum, zirconium, hafnium oxides or 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, it is necessary to distinguish structural polymers. They primarily include collagen, fibrin, keratin, as well as 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 differ in their ability to biodegrade. . This makes it possible to expand the use of immobilized enzymes in medicine . Meanwhile, there are also negative properties of proteins. на протеиновых носителях заключаются в высокой иммуногенности последних, а также возможность внедрять в реакции только определенные их группы. The disadvantages of using immobilized enzymes on protein carriers are the high immunogenicity of the latter, and the ability to introduce only certain groups in the reaction.

Polysaccharides, aminosaccharides

Of these materials, chitin, dextran, cellulose, agarose and their derivatives are most often used. For polysaccharides to be more resistant to reactions, their linear chains are cross-linked with epichlorohydrin. Different ionogenic groups are freely introduced into the mesh structures. Chitin is accumulated in large quantities in the form of waste during industrial processing of shrimps and crabs. This substance is chemically 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 differ in mechanical strength. In the process of transformation, they provide the possibility of varying the size of the pores in a sufficiently wide range, the introduction of a variety of functional groups.

Ways of binding

Currently, there are two fundamentally different versions of immobilization. The first is the preparation of compounds without covalent bonds with the carrier. This method is physical. Another option involves the formation of a covalent bond with the material. This is a chemical method.

Adsorption

получают путем удерживания препарата на поверхности носителя благодаря дисперсионным, гидрофобным, электростатическим взаимодействиям и водородным связям. With it, immobilized enzymes are obtained by keeping the drug on the surface of the carrier due to dispersion, hydrophobic, electrostatic interactions and hydrogen bonds. Adsorption became 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 way of immobilization in industry.

Features of the method

In scientific publications more than 70 enzymes, obtained by adsorption method, are described. The carriers were mainly porous glass, various clays, polysaccharides, aluminum oxides, synthetic polymers, titanium and other metals. The latter are most often used. The effectiveness of the adsorption of the drug on the carrier is determined by the porosity of the material and the specific surface area.

Mechanism of action

Adsorption of enzymes on insoluble materials is simple. It is achieved by contacting the 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 enzyme activity with this immobilization is 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 the carrier. In the process of changes in the reaction conditions, there may be a loss of elements, contamination of the products, desorption of the protein. To increase the bond strength, the carriers are pre-modified. In particular, the materials are treated with metal ions, polymers, hydrophobic compounds and other polyfunctional agents. In some cases, the modification is subjected to the drug itself. But often this leads to a decrease in its activity.

Incorporation into gel

This option is quite common due to its uniqueness and simplicity. This method is suitable not only for individual elements, but also for multienzyme complexes. Inclusion in the gel can be performed in two ways. In the first case, the preparation is combined with an aqueous solution of the monomer, followed by polymerization. As a result, a spatial structure of the gel appears, containing the molecules of the enzyme in the cells. In the second case, the preparation is added to the solution of the finished polymer. Then it is transferred to the gel state.

Embedding in semi-transparent structures

The essence of this method of immobilization is the separation of the aqueous enzyme solution from the substrate. For this, a semipermeable membrane is used. It passes low-molecular elements of cofactors and substrates and delays large molecules of enzymes.

Microencapsulation

There are several options for introducing into semi-transparent structures. Of greatest interest are microencapsulation and the incorporation of proteins into liposomes. The first version 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 a semipermeable polymer. The appearance of the membrane on the surface is due to the reaction of interfacial polycondensation of the compounds. One of them is dissolved in the organic, and the other - in the water phase. An example is the formation of a microcapsule obtained by polycondensation of the halogenohydride of the sebacic acid (organic phase) and hexamethylenediamine-1,6 (respectively, the aqueous phase). The thickness of the membrane is calculated in hundredths of a micrometer. The size of capsules is hundreds or tens of micrometers.

Incorporation into liposomes

This method of immobilization is close to microencapsulation. Liposomes are represented in lamellar or spherical systems of lipid bilayers. This method was first applied in 1970. Evaporation of an 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 takes place. . 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. являются важнейшим исследовательским материалом, позволяющим изучать и описывать закономерности процессов жизнедеятельности. Included in the liposome immobilized enzymes in medicine are the most important research material, allowing to study and describe the patterns of life processes.

Formation of new links

Immobilization by formation of new covalent chains between enzymes and carriers is considered to be the most mass method of obtaining 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. At the same time, the location of the enzyme at a distance of the first covalent bond relative to the carrier creates certain difficulties in performing the catalytic process. The molecule is separated from the material by means of an insert. Poly-and bifunctional agents often act as it. They are, in particular, hydrazine, bromocyan, glutaric dialdride, sulfuryl chloride, etc. For example, to remove the galactosyltransferase, the following sequence -CH ₂ -NH- (CH ₂ ) ₅ -CO- is inserted between the carrier and the enzyme. In such a situation, there is an insert, a molecule and a carrier in the structure. All of them are connected by covalent bonds. Of fundamental importance is the need for introducing 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. As a result, more stable and active immobilized enzymes are obtained .

Cells

The methods described above are considered to be universal for all types of biocatalysts. Among them, among others, include cells, subcellular structures, whose immobilization has recently become widespread. This is due to the following. When immobilizing cells, there is no need to isolate and purify enzyme preparations, to introduce cofactors into the reaction. As a result, it becomes possible to obtain systems that carry out multistage continuous processes.

The use of immobilized enzymes

, промышленности, других хозяйственных отраслях достаточно популярны препараты, полученные указанными выше способами. In veterinary medicine , industry, other economic branches, the preparations obtained by the above methods are quite popular. The approaches developed in practice provide the solution to the problems of implementing targeted drug delivery in the body. Immobilized enzymes made it possible to obtain long-acting medicines with minimal allergenicity and toxicity. At present, scientists are solving problems related to 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 control over the state of the environment should belong to new types of analysis. In particular, we are talking about bioluminescent and immunoenzymatic methods. Of particular importance are advanced approaches in the processing of lignocellulosic raw materials. It is possible to use immobilized enzymes as amplifiers of weak signals. The active center may be under the influence of a carrier under ultrasound, mechanical stress, or susceptible to phytochemical transformations.