Enzymatic. bacterial enzymes

Enzymes are catalysts for chemical reactions of a protein nature, differing in the specificity of their action in relation to the catalysis of certain chemical reactions. They are products of the biosynthesis of all living soil organisms: woody and herbaceous plants, mosses, lichens, algae, microorganisms, protozoa, insects, invertebrates and vertebrates, represented in the natural setting by certain aggregates - biocenoses.

The biosynthesis of enzymes in living organisms is carried out due to genetic factors responsible for the hereditary transmission of the type of metabolism and its adaptive variability. Enzymes are the working apparatus by which the action of genes is realized. They catalyze thousands of chemical reactions in organisms, which ultimately form cellular metabolism. Thanks to them, chemical reactions in the body are carried out at a high speed.

Currently, more than 900 enzymes are known. They are divided into six main classes.

1. Oxireductases catalyzing redox reactions.

2. Transferases catalyzing the reactions of intermolecular transfer of various chemical groups and residues.

3. Hydrolases catalyzing reactions of hydrolytic cleavage of intramolecular bonds.

4. Lyases that catalyze the addition of groups to double bonds and the reverse reactions of abstraction of such groups.

5. Isomerases catalyzing isomerization reactions.

6. Ligases that catalyze chemical reactions with the formation of bonds due to ATP (adenosine triphosphoric acid).

When living organisms die and rot, some of their enzymes are destroyed, and some, getting into the soil, retain their activity and catalyze many soil chemical reactions, participating in the processes of soil formation and in the formation of a qualitative sign of soils - fertility. In different types of soils under certain biocenoses, their own enzymatic complexes were formed, differing in the activity of biocatalytic reactions.

VF Kuprevich and TA Shcherbakova (1966) note that an important feature of soil enzymatic complexes is the orderliness of the action of the existing groups of enzymes, which is manifested in the fact that the simultaneous action of a number of enzymes representing different groups is ensured; the formation and accumulation of compounds present in the soil in excess are excluded; excess accumulated mobile simple compounds (for example, NH 3) are temporarily bound in one way or another and sent to cycles culminating in the formation of more or less complex compounds. Enzymatic complexes are balanced self-regulating systems. Microorganisms and plants play the main role in this, constantly replenishing soil enzymes, since many of them are short-lived. The number of enzymes is indirectly judged by their activity over time, which depends on the chemical nature of the reactants (substrate, enzyme) and on the interaction conditions (concentration of components, pH, temperature, composition of the medium, action of activators, inhibitors, etc.).

This chapter discusses the participation in some chemical soil processes of enzymes from the class of hydrolases - the activity of invertase, urease, phosphatase, protease and from the class of oxidoreductases - the activity of catalase, peroxidase and polyphenol oxidase, which are of great importance in the conversion of nitrogen- and phosphorus-containing organic substances, substances of a carbohydrate nature and in the processes of humus formation. The activity of these enzymes is a significant indicator of soil fertility. In addition, the activity of these enzymes in forest and arable soils of various degrees of cultivation will be characterized by the example of soddy-podzolic, gray forest and soddy-calcareous soils.

CHARACTERISTICS OF SOIL ENZYMES

Invertase - catalyzes the reactions of hydrolytic cleavage of sucrose into equimolar amounts of glucose and fructose, also acts on other carbohydrates with the formation of fructose molecules - an energy product for the vital activity of microorganisms, catalyzes fructose transferase reactions. Studies by many authors have shown that the activity of invertase better than other enzymes reflects the level of soil fertility and biological activity.

Urease - catalyzes the reactions of hydrolytic cleavage of urea into ammonia and carbon dioxide. In connection with the use of urea in agronomic practice, it must be borne in mind that urease activity is higher in more fertile soils. It rises in all soils during periods of their greatest biological activity - in July - August.

Phosphatase (alkaline and acid) - catalyzes the hydrolysis of a number of organophosphorus compounds with the formation of orthophosphate. Phosphatase activity is inversely related to the provision of plants with available phosphorus, so it can be used as an additional indicator when determining the need for phosphate fertilizers to be applied to soils. The highest phosphatase activity is in the rhizosphere of plants.

Proteases are a group of enzymes, with the participation of which proteins are broken down into polypeptides and amino acids, then they are hydrolyzed to ammonia, carbon dioxide and water. In this regard, proteases are of great importance in the life of the soil, since they are associated with changes in the composition of organic components and the dynamics of nitrogen forms assimilated by plants.

Catalase - as a result of its activating action, hydrogen peroxide, which is toxic to living organisms, is split into water and free oxygen. Vegetation has a great influence on the catalase activity of mineral soils. As a rule, soils under plants with a powerful deep-penetrating root system are characterized by high catalase activity. A feature of catalase activity is that it changes little down the profile, has an inverse relationship with soil moisture and a direct relationship with temperature.

Polyphenol oxidase and peroxidase - they play an important role in the processes of humus formation in soils. Polyphenol oxidase catalyses the oxidation of polyphenols to quinones in the presence of free atmospheric oxygen. Peroxidase catalyzes the oxidation of polyphenols in the presence of hydrogen peroxide or organic peroxides. At the same time, its role is to activate peroxides, since they have a weak oxidizing effect on phenols. Further condensation of quinones with amino acids and peptides can occur with the formation of a primary molecule of humic acid, which can further become more complex due to repeated condensations (Kononova, 1963).

It was noted (Chunderova, 1970) that the ratio of the activity of polyphenol oxidase (S) to the activity of peroxidase (D), expressed as a percentage (), is related to the accumulation of humus in soils, therefore this value is called the conditional coefficient of humus accumulation (K). In arable poorly cultivated soils of Udmurtia for the period from May to September, it was: in soddy-podzolic soil - 24%, in gray forest podzolized soil - 26% and in soddy-calcareous soil - 29%.

ENZYMATIVE PROCESSES IN SOILS

The biocatalytic activity of soils is in significant agreement with the degree of their enrichment with microorganisms (Table 11), depends on the type of soils, and varies across genetic horizons, which is associated with changes in the humus content, reaction, Red-Ox potential, and other indicators along the profile.

In virgin forest soils, the intensity of enzymatic reactions is mainly determined by the horizons of the forest litter, and in arable soils, by arable layers. Both in some and in other soils, all biologically less active genetic horizons located under the A or A p horizons have low enzyme activity, which slightly changes in a positive direction when soils are cultivated. After the development of forest soils for arable land, the enzymatic activity of the formed arable horizon in comparison with the forest litter turns out to be sharply reduced, but as it becomes cultivated, it increases and, in highly cultivated species, approaches or exceeds that of the forest litter.

11. Comparison of biogenicity and enzymatic activity of soils in the Middle Cis-Urals (Pukhidskaya and Kovrigo, 1974)

section number, soil name	Horizon, sampling depth, cm	The total number of microorganisms, thousand per 1 g abs. dry soil (average for 1962, 1964-1965)	Enzyme activity indicators (average for 1969-1971)
			Invertase, mg glucose per 1 g of soil for the first day	Phosphatase, mg phenolphthalein per 100 g of soil for 1 hour	Urease, mg NH, per 1 g of soil for 1 day	Catalase, ml 0 2 per 1 g of soil for 1 min	Polyphenol oxidase	Peroxidase
			Invertase, mg glucose per 1 g of soil for the first day		Urease, mg NH, per 1 g of soil for 1 day	Catalase, ml 0 2 per 1 g of soil for 1 min	mg purpurogallin per 100 g soil
3. Sod-medium podzolic medium loamy (under forest)


				Not defined
1. Soddy medium podzolic medium loamy poorly cultivated


10. Gray forest podzolized heavy loamy poorly cultivated


2. Sod-carbonate, slightly leached, light loamy, poorly cultivated

The activity of biocatalytic reactions in soils changes. It is lowest in spring and autumn, and the highest is usually in July-August, which corresponds to the dynamics of the general course of biological processes in soils. However, depending on the type of soils and their geographical location, the dynamics of enzymatic processes is very different.

Control questions and tasks

1. What compounds are called enzymes? What are their production and significance for living organisms? 2. Name the sources of soil enzymes. What role do individual enzymes play in soil chemistry? 3. Give the concept of the enzymatic complex of soils and its functioning. 4. Give a general description of the course of enzymatic processes in virgin and arable soils.

The enzymatic activity of microorganisms is rich and varied. Using it, one can establish not only the species and type of microbe, but also determine its variants (the so-called biovars). Consider the main enzymatic properties and their qualitative definition.

The breakdown of carbohydrates (saccharolytic activity), i.e., the ability to break down sugars and polyhydric alcohols with the formation of acid or acid and gas, is studied on Hiss media, which contain one or another carbohydrate and indicator. Under the action of the acid formed during the breakdown of the carbohydrate, the indicator changes the color of the medium. Therefore, these media are called "variegated series". Microbes that do not ferment this carbohydrate grow on the medium without changing it. The presence of gas is established by the formation of bubbles in media with agar or by its accumulation in a "float" on liquid media. "Float" - a narrow glass tube with a sealed end facing upwards, which is placed in a test tube with a medium before sterilization.

In addition, saccharolytic activity is studied on Endo, EMS, Ploskirev media. Microorganisms, fermenting milk sugar (lactose) in these media to acid, form colored colonies - the acid changes the color of the indicator present in the medium. Colonies of microbes that do not ferment lactose are colorless.

Milk with the growth of lactose fermenting microbes coagulates.

With the growth of microorganisms that form amylase, on media with soluble starch, it is cleaved. This is known by adding a few drops of Lugol's solution to the culture - the color of the medium does not change. Undigested starch gives a blue color with this solution.

Proteolytic properties (i.e., the ability to break down proteins, polypeptides, etc.) are studied on media with gelatin, milk, whey, peptone. With the growth of gelatin-fermenting microbes on the gelatin medium, the medium liquefies. The nature of the liquefaction caused by different microbes is different. Microbes that break down casein (milk protein) cause milk peptonization - it takes on the appearance of whey. During the splitting of peptones, indole, hydrogen sulfide, ammonia can be released. Their formation is established using indicator pieces of paper. The filter paper is pre-impregnated with certain solutions, dried, cut into narrow strips 5–6 cm long, and after sowing the culture on the BCH, placed under the cork between it and the test tube wall. After incubation in a thermostat, the result is taken into account. Ammonia causes litmus paper to turn blue; when hydrogen sulfide is released on a paper soaked in a 20% solution of lead acetate and sodium bicarbonate, lead sulfate is formed - the paper turns black; indole causes reddening of a piece of paper soaked in a solution of oxalic acid.

In addition to these media, the ability of microorganisms to break down various nutrient substrates is determined using paper discs impregnated with certain reagents (paper indicator systems "SIB"). These disks are dipped into test tubes with the culture under study, and after 3 hours of incubation in a thermostat at 37 ° C, the decomposition of carbohydrates, amino acids, proteins, etc. is judged by the change in the color of the disks.

Hemolytic properties (the ability to destroy red blood cells) are studied on media with blood. In this case, liquid media become transparent, and a transparent zone appears around the colony on dense media. When methemoglobin is formed, the medium turns green.

PRESERVATION OF CROPS

Isolated and studied cultures (strains) of value for science or production are stored in museums of living cultures. The All-Union Museum is located in the State Research Institute for Standardization and Control of Medical Biological Preparations named after V.I. L. A. Tarasevich (GISK).

The task of storage is to maintain the viability of microorganisms and prevent their variability. To do this, it is necessary to weaken or stop the exchange in the microbial cell.

One of the most advanced methods of long-term preservation of cultures - lyophilization - drying in a vacuum from a frozen state allows you to create a state of suspended animation. Drying is carried out in special devices. Cultures are stored in sealed ampoules at a temperature of 4 °C, preferably at -30-70 °C.

Recovery of dried crops.

The tip of the ampoule is strongly heated in the flame of the burner and touched with a cotton swab slightly moistened with cold water so that microcracks form on the glass, through which air slowly seeps into the ampoule. At the same time, passing through the heated edges of the cracks, the air is sterilized.

Do not forget that there is a vacuum in the sealed ampoule. If air enters it immediately through a large hole, the culture in the ampoule can be sprayed and ejected.

After allowing air to enter, quickly break with tweezers and remove the top of the ampoule. The hole is lightly burned and a solvent (broth or isotonic solution) is introduced into the ampoule with a sterile Pasteur pipette or syringe. Mix the contents of the ampoule and inoculate on the media. The growth of regenerated crops in the first crops may be slowed down.

It is also possible to store cultures for a long time in liquid nitrogen (-196 ° C) in special devices.

Methods for short-term preservation of cultures are as follows: 1) subcultivation (periodic transfers to fresh media) at intervals depending on the properties of the microorganism, the medium and cultivation conditions. Cultures are stored at 4°C between reseedings; 2) preservation under a layer of oil. The culture is grown in agar in a column 5-6 cm high, poured with sterile vaseline oil (oil layer about 2 cm) and stored vertically in the refrigerator. The shelf life of different microorganisms is different, therefore, a culture is periodically sown from test tubes to check its viability; 3) storage at -20-70 °C; 4) storage in sealed tubes. As needed, the stored material is sown on a fresh medium.

Chapter 8. FAGI

Phages are viruses of bacteria and a number of other microorganisms. Under certain conditions, they cause lysis (dissolution) of their hosts. The action of phages manifests itself in nature and is used in practice.

Fig.42 Bacteriophages

History of the discovery and study of the phage. In 1898, N. F. Gamaleya showed that the filtrate of anthrax bacilli causes lysis of fresh cultures of these microorganisms. bacterial filters, while maintaining the ability to dissolve fresh cultures of microorganisms. The phenomenon of lysis of microorganisms has been described, but its nature has not been studied. That is why the honor of discovering the bacteriophage belongs to the Canadian scientist d'Herelle.

D "Erell (1917) studied stool filtrates, which he took daily from a patient with dysentery and introduced into test tubes with a freshly planted culture of the causative agent of this disease. After incubation in a thermostat, the culture grew. But one day it did not grow, but dissolved. This coincided with the beginning of the patient's recovery.

D "Erell showed that the lysing ability of stool filtrates increased with successive passages on fresh cultures of bacteria. From this, the scientist concluded that it dissolves their living agent passing through bacterial filters, i.e. the virus. Currently, his point of view is accepted by the majority scientists.

The open virus d "Erell called a bacteriophage-devourer of bacteria (from the Greek phagos - devouring), and the phenomenon - bacteriophagy.

With the discovery of the electron microscope, the corpuscular nature of the phage was confirmed and its morphology was studied.

d'Herelle's discovery attracted the attention of physicians who used phages to treat and prevent a number of infectious diseases. Currently, phages are widely used in medical practice and in various biological studies. Bacteriologists, virologists, biochemists, geneticists, biophysicists, molecular biologists, experimental oncologists are engaged in phages , specialists in genetic engineering and biotechnology, etc. The study of the phage continues as one of the most interesting chapters of biology.

PROPERTIES OF PHAGES

Phage morphology. Most phages consist of a head and a tail, so they are compared to tadpoles or spermatozoa. The T-phages of Escherichia coli are the most studied). Their process is a hollow cylinder (rod) covered with a sheath and ending in a basal plate with spines and fibrils. The size of phages, the shape and size of the head, the length and structure of the process are different in different phages. For example, there are phages with a long process, the sheath of which does not contract, phages with a short process, without a process, and filamentous

Chemical composition of phages. Like all viruses, phages are composed of a single type of nucleic acid (DNA phages are more common) and a protein. The nucleic acid molecule, twisted into a spiral, is located in the head of the phage. The shell of the phage (capsid) and the process are of a protein nature. The free end of the process contains a lytic enzyme, usually lysozyme or hyaluronidase.

Interaction of a phage with a sensitive cell goes through successive stages. The whole cycle takes in different systems phage-bacteria from several minutes to 1-2 hours. Let us analyze the sequence of this process using the example of the T-even phage of Escherichia coli.

Stage I - adsorption of phage particles on the surface receptors of the cell is carried out with the help of filaments of the tail process. Hundreds of phages can be adsorbed on one cell (one is enough for cell lysis). Phage adsorption is specific.

Stage II - the penetration (injection) of the nucleic acid of the phage into cells in different phages occurs in different ways. In E. coli T-phages, the spikes of the basal lamina are in contact with the cell wall. The rod "pierces" the cell wall. An enzyme found in the process, most often lysozyme, destroys the cytoplasmic membrane. At the same time, the sheath of the process contracts, and the nucleic acid of the phage is "injected" into the cell through the channel of the rod. The empty protein shell of the phage (“shadow”) remains outside.

Stage III - reproduction of the protein and nucleic acid of the phage inside the cell.

Stage IV - assembly and formation of mature phage particles.

Fig.43 The structure of the phage.

1 - head; 2 - DNA; 3 - rod; 4 - case; 5 - basal plate; 6 spikes; 7 - tail fibrils.

Fig.44 Phage morphology.

1 - phages with a head, a process and a contracting sheath; 2 - head and process, without contractility; 3 - head and short process; 4 - tailless phages; 5-filamentous phages.

Stage V - cell lysis and release of mature phage particles from it. Usually, the cell wall ruptures and several hundred new phages are released into the environment, capable of infecting fresh cells. Such lysis is called lysis from within.

In contrast to lysis from the inside, lysis from the outside occurs when a very large number of phages are adsorbed on the cell at once. They make numerous holes in the cell wall through which the contents of the cell flow out. Thus, during lysis from outside, the phage does not multiply, and the number of its particles does not increase.

According to the nature of the action on microorganisms, virulent and temperate phages are distinguished.

Virulent phages cause the lysis of the infected cell with the release into the environment of a large number of phage particles capable of infecting new cells. In this case, the culture of microorganisms is lysed. The liquid medium becomes transparent - the formation of a phagolysate occurs - a medium in which a large number of phages are located. With the development of a virulent phage in bacteria growing on a dense medium, either transparent areas of continuous lysis are formed, or separate transparent formations grow - phage colonies. They are called negative colonies (plaques). Colonies - different phages differ in size and structure.

Temperate phages do not lyse all cells in the population. With some of them, phages enter into symbiosis: the nucleic acid of the phage (its genome) is integrated into the cell chromosome and is called pro Phage. A single chromosome is formed. The bacterial cell does not die. A prophage that has become part of the cell's genome can be transmitted to an unlimited number of descendants during its reproduction, i.e., to new cells. The phenomenon of symbiosis of a microbial cell with a temperate phage (prophage) is called lysogeny, and a culture in which there is a prophage is called lysogenic. This name reflects the ability of the prophage to spontaneously leave the cell chromosome and, passing into the cytoplasm, turn into a virulent phage. Those culture cells in which the virulent phage was formed die (lyse), the rest remain lysogenic.

Scheme of the main stages of interaction between a phage and a bacterial cell.

1 – insertion of the phage nucleic acid into the clerk; 2-young, breeding

phages; 3 - mature phages; 4 - isolation of phages.

Lysogenic cultures do not differ in their basic properties from the original ones, but they are resistant to re-infection with the phage of the same name. When a lysogenic culture is exposed to penetrating radiation (certain doses and exposure to X-rays, cosmic rays), certain chemicals and a number of other factors, the production of virulent phage and the lysis of culture cells by it increase significantly.

Temperate phages can be detrimental to microbiological production. For example, if the strains producing vaccines, antibiotics, and other biological substances are lysogenic, there is a danger that the temperate phage will become virulent, which will lead to the lysis of the production strain.

Temperate phages are a powerful factor in the variability of microorganisms. A prophage can change some properties of a microbial culture, for example, make it capable of producing toxin, which is observed among diphtheria bacilli, the causative agent of scarlet fever, etc. In addition, by turning into a virulent form and lysing a cell, a phage can capture a part of the host cell's chromosome and transfer this part of the chromosome to another cell, where the phage will again turn into a prophage, and the cell will acquire new properties.

The distribution of phages in nature is ubiquitous. Phages are found where microorganisms sensitive to them are found: in water, soil, sewage, excretions of humans and animals, etc. Almost all known bacteria are hosts of phages specific to them.

The resistance of phages to physical and chemical factors is higher than that of the vegetative forms of their hosts. Phages withstand heating up to .75°C, prolonged drying, pH from 2.0 to 8.5. They are not sensitive to antibiotics, thymol, chloroform and a number of other substances that destroy the accompanying microflora. Therefore, these substances are used in the isolation and preservation of phages. Acids and disinfectants are detrimental to phages.

PRACTICAL APPLICATIONS OF PHAGES

The use of phages is based on their strict specificity and ability to destroy microbial cells or enter into symbiosis with them.

Phage prophylaxis and phage therapy, the prevention and treatment of infections with the help of phages, are based on the fact that when a phage encounters a pathogen in the patient's body, it destroys it. Currently, phages are widely used in the treatment and prevention of staphylococcal and streptococcal infections, even those that are not amenable to antibiotics, as well as cholera, plague, and a number of other infections, such as infections caused by Escherichia coli and Proteus.

Phage diagnostics includes: a) identification of isolated cultures using known (diagnostic) phages. The culture corresponds to the phage that lysed it. For example, if a cholera phage caused lysis, then this is a culture of vibrio cholerae. The strict specificity of typical phages makes it possible to type variants within a species (fagovars). Phage typing is of great importance in epidemiology, as it makes it possible to establish the source of infection and solve a number of other issues; b) identification of an unknown phage by a test culture of microbes. If phage lyzes the culture of the causative agent of dysentery, then this is a dysentery phage; c) an accelerated diagnostic method using the RNTF phage titer increase reaction does not require the isolation of a pure culture of the pathogen. The test material (from the patient or from environmental objects) and the indicator phage, the titer of which is strictly established, are added to the broth.

Temperate phages are widely used in solving cardinal problems of biology. With their help, the genetic code has been studied, great success has been achieved in genetic engineering, they are used to study tumor growth, as a factor in the variability of microorganisms, and in other studies. Since lysogenic cultures, in contrast to "healthy" cultures, are sensitive to radiation, they serve to determine the reliability of protection of spacecraft from cosmic rays: with unreliable protection, the prophage passes into a virulent form and lyses the culture.

PHAGE PREPARATIONS

In the production of phage preparations, well-studied strains of microorganisms and phages are used, which are usually grown in reactors, which makes it possible to obtain large amounts of phagolysate.

Phages are produced in liquid form (ampoules and vials), in tablets and suppositories. Phage tablets intended for oral administration are coated with an acid-resistant coating that protects the phages from the action of gastric hydrochloric acid.

All phage preparations are subject to mandatory control for the absence of foreign flora, harmlessness and activity (titer), which is carried out at the production facility that produces them. Selective control is carried out at the State Research Institute for Standardization and Control of Medical Biological Preparations named after V.I. L.A. Tarasevich. The released phage is provided with a label, which indicates: the institution that produces it, the name of the phage, series, control number and expiration date. Each package is supplied with instructions for the use and storage of the phage.

Enzyme activity. Under enzyme activity understand its amount, which catalyzes the transformation of a certain amount of substrate per unit of time. To express the activity of enzyme preparations, two alternative units are used: international (IU) and catal (cat). Per international unit of activity the amount of enzyme taken is such that it catalyzes the conversion of 1 µmol of the substrate into the product in 1 min under standard conditions (usually optimal). One rolled denotes the amount of enzyme catalyzing the conversion of 1 mol of substrate in 1 s (1 cat = 6 ∙ 10 7 IU). In the bimolecular reaction A + B = C + D, the amount of enzyme activity that catalyzes the conversion of 1 µmol A or B, or 2 µmol A (if B = A) in 1 min is taken as a unit of enzyme activity.

Often enzyme preparations are characterized by specific activity, which reflects the degree of purification of the enzyme. Specific activity is the number of units of enzyme activity per 1 mg of protein.

Molecular activity (number of enzyme turnovers) is the number of substrate molecules converted by one enzyme molecule in 1 min when the enzyme is completely saturated with the substrate. It is equal to the number of units of enzyme activity divided by the amount of enzyme, expressed in micromoles. The concept of molecular activity is applicable only to pure enzymes.

When the number of active centers in an enzyme molecule is known, the concept is introduced catalytic site activity . It is characterized by the number of substrate molecules that undergoes transformation in 1 min per one active center.

The activity of enzymes strongly depends on external conditions, among which the temperature and pH of the medium are of paramount importance. An increase in temperature in the range of 0–50°C usually leads to a gradual increase in enzymatic activity, which is associated with an acceleration of the processes of formation of the enzyme–substrate complex and all subsequent events of catalysis. For every 10°C increase in temperature, the rate of the reaction approximately doubles (van't Hoff's rule). However, a further increase in temperature (>50°C) is accompanied by an increase in the amount of inactivated enzyme due to the denaturation of its protein part, which is expressed in a decrease in activity. Each enzyme is characterized temperature optimum– the temperature value at which its greatest activity is recorded.

The dependence of enzyme activity on the pH value of the medium is complex. Each enzyme has its own optimum pH environments at which it is most active. As you move away from this value in one direction or another, the enzymatic activity decreases. This is due to a change in the state of the active center of the enzyme (decrease or increase in the ionization of functional groups), as well as the tertiary structure of the entire protein molecule, which depends on the ratio of cationic and anionic centers in it. Most enzymes have an optimum pH in the neutral range. However, there are enzymes that show maximum activity at pH 1.5 (pepsin) or 9.5 (arginase). When working with enzymes, the pH must be maintained with an appropriate buffer solution.

Enzyme activity is subject to significant fluctuations depending on exposure inhibitors(substances that partially or completely reduce activity) and activators(substances that increase activity). Their role is played by metal cations, some anions, carriers of phosphate groups, reducing equivalents, specific proteins, intermediate and final products of metabolism, etc.

Principles of enzymatic kinetics. The essence of kinetic studies is to determine the maximum rate of the enzymatic reaction ( V max) and Michaelis constant K M. Enzymatic kinetics studies the rates of quantitative transformations of some substances into others under the action of enzymes. The rate of an enzymatic reaction is measured by the loss of the substrate or the increase in the resulting product per unit of time, or by the change in the concentration of one of the adjacent forms of the coenzyme.

Influence enzyme concentration on the reaction rate is expressed as follows: if the concentration of the substrate is constant (assuming an excess of the substrate), then the reaction rate is proportional to the concentration of the enzyme. For kinetic studies, an enzyme concentration of 10–8 M of active centers is used. The optimal value of the enzyme concentration is determined from the graph of the dependence of enzyme activity on its concentration. The optimal value is considered to lie on the plateau of the obtained graph in the range of enzyme activity values that depend little on its concentration (Fig. 4.3).

Rice. 4.3. The dependence of the rate of the enzymatic reaction

on enzyme concentration

To study the influence substrate concentration on the rate of the enzymatic reaction, first build a kinetic curve that reflects the change in the concentration of the substrate (S 1) or product (P 1) over time (Fig. 4.4) and measure the initial rate ( V 1) reactions as the tangent of the slope of the tangent to the curve at the zero point.

Rice. 4.4. Kinetic curves of the enzymatic reaction

By constructing kinetic curves for other concentrations of a given substrate (S 2 , S 3 , S 4 , etc.) or product (P 2 , P 3 , P 4 , etc.) and determining the initial rates ( V 2, V 3 , V 4, etc.) of the reaction, build a graph of the dependence of the initial rate of the enzymatic reaction on the concentration of the substrate (at a constant concentration of the enzyme), which has the form of a hyperbola (Fig. 4.5).

Rice. 4.5. Dependence of the initial rate of the enzymatic reaction

from substrate concentration

The kinetics of many enzymatic reactions is described by the Michaelis-Menten equation. At a constant enzyme concentration and low substrate concentrations[S] The initial reaction rate is directly proportional to [S] (Fig. 4.5). In this case, we speak of half-saturation of the enzyme with the substrate, when half of the enzyme molecules are in the form of an enzyme-substrate complex and the reaction rate V = 1/2V max. In relation to the substrate, the reaction has the 1st order (the reaction rate is directly proportional to the concentration of one reactant) or the 2nd order (the reaction rate is proportional to the product of the concentrations of the two reactants).

At high values substrate concentration[S] the reaction rate is almost independent of [S]: with a further increase in [S], the reaction rate grows more slowly and eventually becomes constant (maximum) (Fig. 4.5). In this case, complete saturation of the enzyme with the substrate is achieved, when all enzyme molecules are in the form of an enzyme-substrate complex and V = V max. With regard to the substrate, the reaction has the 0th order (the reaction rate does not depend on the concentration of the reactants).

In 1913, L. Michaelis and M. Menten proposed a simple model to explain such kinetics. According to this model, the formation of a specific enzyme-substrate complex is a necessary intermediate step in catalysis.

k 1 k 3

E + S ⇄ ES → E + P

Enzyme E combines with substrate S to form an ES complex. The rate constant of this process k one . The fate of the ES complex is twofold: it can either dissociate into enzyme E and substrate S with a rate constant k 2 , or undergo further transformation, forming product P and free enzyme E, with a rate constant k 3 . It is postulated that the reaction product is not converted into the original substrate. This condition is observed at the initial stage of the reaction, while the concentration of the product is low.

The rate of catalysis is determined in stationary conditions, when the concentration of intermediate products remains constant, while the concentration of starting materials and final products changes. This occurs when the rate of formation of the ES complex is equal to the rate of its decay.

You can introduce a new constant K M - Michaelis constant(mol/l), which is equal to

Michaelis–Menten equation, which expresses the quantitative relationship between the rate of the enzymatic reaction and the concentration of the substrate, has the form

(4.2)

This equation corresponds to a plot of reaction rate versus substrate concentration. At low substrate concentrations when [S] is much lower than K M, V = V max [S] / K M, i.e. the reaction rate is directly proportional to the concentration of the substrate. At high substrate concentrations when [S] is much higher than K M, V = V max , i.e., the reaction rate is maximum and does not depend on the concentration of the substrate.

If [S] = K M, then V = V max /2.

Thus, K M equal to the substrate concentration at which the reaction rate is half the maximum.

Michaelis constant (KM) and maximum reaction rate ( V max) are important velocity characteristics at different substrate concentrations. V max is a constant value for each enzyme, which makes it possible to evaluate the effectiveness of its action.

The Michaelis constant shows the affinity of the substrate for the enzyme (in the case when k 2 >> k 3): the lower K M, the greater the affinity and the higher the reaction rate, and vice versa. Each substrate is characterized by its KM value for a given enzyme, and their values can be used to judge the substrate specificity of the enzyme. The Michaelis constant depends on the nature of the substrate, temperature, pH, ionic strength of the solution, and the presence of inhibitors.

Due to the fact that the definition V max and K M directly from the graphic dependence of Michaelis - Menten (Fig. 4.5) is ambiguous, they resort to the linearization of this equation. To do this, it is converted into such a form that it is graphically expressed as a straight line. There are several linearization methods, among which the Lineweaver-Burk and Edie-Hofstee methods are most commonly used.

transformation Lineweaver-Burke has the form

(4.3)

Build a dependency graph 1/ V = f(1/[S]) and get a straight line, the intersection of which with the y-axis gives the value 1/ V max ; the segment cut off by a straight line on the abscissa axis gives the value −1 / K M, and the tangent of the angle of inclination of the straight line to the abscissa axis is K M / V max (Fig. 4.6). This graph allows you to more accurately determine V max. As we will see below, valuable information regarding the inhibition of enzyme activity can also be extracted from this plot.

Rice. 4.6. Method for linearizing the Michaelis–Menten equation

(according to Lineweaver-Burke)

Method Edie – Hofsty is based on transforming the Michaelis–Menten equation by multiplying both sides by V max:

(4.4)

Graph in coordinates V and V/[S] is a straight line, the intersection of which with the y-axis gives the value V max, and the segment cut off by a straight line on the abscissa axis is the value V max / K M (Fig. 4.7). It makes it very easy to determine K M and V max , as well as detect possible deviations from linearity that were not detected in the previous graph.

Rice. 4.7. Method for linearizing the Michaelis–Menten equation

(according to Edie-Hofsty)

Inhibition of enzyme activity. The action of enzymes can be completely or partially suppressed by certain chemicals - inhibitors . By the nature of their action, inhibitors are divided into reversible and irreversible. This division is based on the binding strength of the inhibitor to the enzyme.

Reversible inhibitors - These are compounds that interact non-covalently with the enzyme and when they are removed, the activity of the enzyme is restored. Reversible inhibition can be competitive, non-competitive and non-competitive.

An example competitive inhibition is the action of structural analogs of the substrate, which can bind to the active center of the enzyme in a similar way as the substrate, but without turning into a product and preventing the interaction of the enzyme with the true substrate, i.e. there is competition between the substrate and the inhibitor for binding to the active center of the enzyme . As a result of the formation of enzyme-inhibitor (EI) complexes, the concentration of ES-complexes decreases and, as a result, the reaction rate decreases. In other words, a competitive inhibitor reduces the rate of catalysis by reducing the proportion of enzyme molecules that bind the substrate.

Measurement of reaction rates at different substrate concentrations makes it possible to distinguish competitive inhibition from noncompetitive inhibition. With competitive inhibition on the graph of dependence 1/ V=f(1/[S]) lines intersect the y-axis at one point 1/ V max regardless of the presence of an inhibitor, but in the presence of an inhibitor, the tangent of the slope of the straight line to the abscissa axis increases, i.e. V max does not change, while KM increases, which indicates a decrease in the affinity of the substrate for the enzyme in the presence of an inhibitor (Fig. 4.8). Therefore, at a sufficiently high concentration of the substrate under conditions of competition for the active site of the enzyme, when the substrate displaces the inhibitor from the active site, inhibition can be eliminated and the rate of the catalyzed reaction is restored. In this case, the Michaelis − Menten equation has the form

(4.5)

where [I] is the inhibitor concentration; K i is the inhibition constant.

The inhibition constant characterizes the affinity of the enzyme for the inhibitor and is the dissociation constant of the EI complex:

(4.6)

In the presence of a competitive inhibitor, the slope of the straight line to the x-axis increases by (1 + [I]/ K i).

Rice. 4.8. Competitive inhibition:

a - scheme; b - graphical expression according to Lineweaver - Burke

At non-competitive inhibition the inhibitor differs in structure from the substrate and binds not to the active, but to the allosteric site of the enzyme. This leads to a change in the conformation of the active site of the enzyme, which is accompanied by a decrease in the catalytic activity of the enzyme. Moreover, the inhibitor can bind not only to the free enzyme (E + I → EI), but also to the enzyme-substrate complex (ES + I → ESI). Both forms EI and ESI are not active. The substrate and inhibitor can be simultaneously bound by the enzyme molecule, but their binding sites do not overlap. The action of a non-competitive inhibitor is to reduce the number of enzyme turnovers, and not to reduce the proportion of substrate-bound enzyme molecules. The inhibitor does not prevent the formation of ES complexes, but inhibits the conversion of the substrate into the product. Thereby V max decreases, i.e., in the presence of an inhibitor, the intersection of the straight line with the y-axis will occur at a higher point (Fig. 4.9). To the same extent, the tangent of the angle of inclination of the straight line to the abscissa axis, equal to K M / V max I . K M in contrast to V max does not change, so non-competitive inhibition cannot be eliminated by increasing the substrate concentration.

Rice. 4.9. Noncompetitive inhibition:

a - scheme; b - graphical expression according to Lineweaver - Burke

Maximum reaction rate V max I in the presence of a noncompetitive inhibitor is described by the equation

(4.7)

In a particular case uncompetitive inhibition, when the inhibitor binds only to the ES-complex and does not bind to the free enzyme, on the dependence graph 1/ V = f(1/[S]) lines are parallel to each other and intersect the ordinate and abscissa axes at different points (Fig. 4.10).

Rice. 4.10. Uncompetitive inhibition:

a - scheme; b - graphical expression according to Lineweaver - Burke

irreversible inhibitors - these are highly reactive compounds of various chemical nature, which can interact with functionally important groups of the active center, forming strong covalent bonds. This leads to an irreversible loss of enzyme activity. In this regard, the Michaelis-Menten theory, based on the assumption that the attachment of an inhibitor to an enzyme is reversible, is not applicable in this case.

An example of irreversible inhibition is the interaction of enzymes with heavy metal ions, which are attached to the sulfhydryl groups of cysteine residues of the enzyme and form mercaptides, which are practically non-dissociating compounds, or covalent modification of the enzyme under the action of alkylating agents.

The concept of enzyme activity

In everyday biochemical practice, the amount of the enzyme is practically not estimated, but only its activity. Activity is a broader concept than quantity. It implies primarily the result of the reaction, namely the loss of the substrate or the accumulation of the product. Naturally, one cannot ignore the time that the enzyme has worked and the number of enzyme molecules. But since it is usually unrealistic to calculate the number of enzyme molecules, the amount of biological material containing the enzyme (volume or mass) is used.

Thus, when determining the activity of enzymes, three variables must be taken into account simultaneously:

the mass of the obtained product or the disappeared substrate;
time spent on reaction;
the amount of enzyme, but actually the mass or volume of biological material containing the enzyme.

To understand the relationship between these factors, a clear and simple example can be the construction of two buildings. Buildings are equated to the product of the reaction, workers are enzymes, let the team correspond to the volume of biological material. So, tasks from the 3rd grade:

A team of 10 people worked on the construction of one building, a team of 5 people worked on another building of the same kind. The construction was completed simultaneously and in full. Where is the activity of workers higher?
A team of 10 people worked on the construction of one building of 3 floors, another building of 12 floors - a team of 10 people. The construction was completed simultaneously and in full. Where is the activity of workers higher?
On the construction of one building of 5 floors, a team of 10 people worked, another of the same building - a team of 10 people. The construction of the first building took 20 days, the second was completed in 10 days. Where is the activity of workers higher?

Fundamentals of Enzyme Activity Quantification

1. Enzyme activity is expressed as the rate of accumulation of the product or the rate of loss of the substrate in terms of the amount of material containing the enzyme.

In practice, they usually use:

units of quantity of a substance - mol (and its derivatives mmol, µmol), gram (kg, mg),
units of time - minute, hour, second,
units of mass or volume - gram (kg, mg), liter (ml).

Other derivatives are also actively used - catal (mol / s), the international unit of activity (IU, Unit) corresponds to μmol / min.

Thus, enzyme activity can be expressed, for example, in mmol/s×l, g/h×l, IU/l, cat/ml, etc.

For example, it is known

2. Creation of standard conditions to be able to compare the results obtained in different laboratories - optimal pH and fixed temperature, for example, 25°C or 37°C, adherence to the incubation time of the substrate with the enzyme.

Breakdown of carbohydrates(saccharolytic activity), i.e., the ability to break down sugars and polyhydric alcohols with the formation of acid or acid and gas, is studied on Giss media that contain one or another carbohydrate and indicator. Under the action of the acid formed during the breakdown of the carbohydrate, the indicator changes the color of the medium. Therefore, these media are called "variegated series". Microbes that do not ferment this carbohydrate grow on the medium without changing it. The presence of gas is established by the formation of bubbles in media with agar or by its accumulation in a "float" on liquid media. "Float" - a narrow glass tube with a sealed end facing upwards, which is placed in a test tube with the medium before sterilization (Fig. 18).

Rice. 18. Study of the saccharolytic activity of microorganisms. I - "variegated series": a - liquid medium with carbohydrates and Andrede's indicator; b - semi-liquid medium with BP indicator: 1 - microorganisms do not ferment carbohydrate; 2 - microorganisms ferment carbohydrate with the formation of acid; 3 - microorganisms ferment carbohydrate with the formation of acid and gas; II - colonies of microorganisms that do not decompose (colorless) and decompose lactose (violet on the EMS medium - on the left, red on the Endo medium - on the right)

Milk with the growth of lactose fermenting microbes coagulates.

Proteolytic properties(i.e., the ability to break down proteins, polypeptides, etc.) is studied on media with gelatin, milk, whey, peptone. With the growth of gelatin-fermenting microbes on the gelatin medium, the medium liquefies. The nature of the liquefaction caused by different microbes is different (Fig. 19). Microbes that break down casein (milk protein) cause peptonization of milk - it takes on the appearance of whey. During the splitting of peptones, indole, hydrogen sulfide, ammonia can be released. Their formation is established using indicator pieces of paper. The filter paper is pre-impregnated with certain solutions, dried, cut into narrow strips 5-6 cm long, and after sowing the culture on the BCH, placed under the cork between it and the test tube wall. After incubation in a thermostat, the result is taken into account. Ammonia causes litmus paper to turn blue; when hydrogen sulfide is released on a paper soaked in a 20% solution of lead acetate and sodium bicarbonate, lead sulfate is formed - the paper turns black; indole causes reddening of a piece of paper soaked in a solution of oxalic acid (see Fig. 19).

Rice. 19. Proteolytic properties of microorganisms. 1 - forms of liquefaction of gelatin; II - determination of hydrogen sulfide; III - determination of indole: 1 - negative result; 2 - positive result

In addition to these media, the ability of microorganisms to degrade various nutrient substrates is determined using paper discs impregnated with certain reagents (paper indicator systems "SIB"). These disks are dipped into test tubes with the culture under study, and already after 3 hours of incubation in a thermostat at 37 ° C, the decomposition of carbohydrates, amino acids, proteins, etc. is judged by the change in the color of the disks.

Hemolytic properties (the ability to destroy red blood cells) are studied on media with blood. In this case, liquid media become transparent, and on dense media a transparent zone appears around the colony (Fig. 20). When methemoglobin is formed, the medium turns green.

Rice. 20. Hemolysis around colonies growing on blood agar

Preservation of crops

The task of storage is to maintain the viability of microorganisms and prevent their variability. To do this, it is necessary to weaken or stop the exchange in the microbial cell.

Recovery of dried crops. The tip of the ampoule is strongly heated in the flame of the burner and touched with a cotton swab slightly moistened with cold water so that microcracks form on the glass, through which air slowly seeps into the ampoule. At the same time, passing through the heated edges of the cracks, the air is sterilized.

* (With an excess of water on the swab, it can get into the ampoule and violate the sterility of the culture: it will be sucked in through the formed microcracks, since there is a vacuum in the ampoule.)

Attention! Do not forget that there is a vacuum in the sealed ampoule. If air enters it immediately through a large hole, the culture in the ampoule can be sprayed and ejected.

It is also possible to preserve cultures for a long time in liquid nitrogen (-196 ° C) in special devices.

Methods for short-term preservation of cultures are as follows: 1) subcultivation (periodic transfers to fresh media) at intervals depending on the properties of the microorganism, the medium and cultivation conditions. Cultures are stored at 4°C between reseedings; 2) preservation under a layer of oil. The culture is grown in agar in a column 5-6 cm high, poured with sterile vaseline oil (oil layer about 2 cm) and stored vertically in the refrigerator. The shelf life of different microorganisms is different, therefore, a culture is periodically sown from test tubes to check its viability; 3) storage at -20-70°C; 4) storage in sealed tubes. As needed, the stored material is sown on a fresh medium.

test questions

1. What is included in the concept of "bacteriological research"?

2. What should be the culture for such research?

3. What is a microbial colony, culture, strain, clone?

4. What is included in the concept of "cultural properties of microbes"?

Exercise

1. Study and describe several colonies. Transfer them to the agar slant and to the sector.

2. Study and describe the growth pattern - agar slant cultures. Determine the purity and morphology of the culture in the stained preparation.

3. Transfer culture from agar slant to broth and differential diagnostic media. Examine and record in the protocol the growth pattern of the culture on these media and its enzymatic properties.

Portal for the student. Self-training

CHARACTERISTICS OF SOIL ENZYMES

ENZYMATIVE PROCESSES IN SOILS

The concept of enzyme activity

Fundamentals of Enzyme Activity Quantification

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